Journal of Chromatographic Science 2015;53:787– 792 doi:10.1093/chromsci/bmu126 Advance Access publication September 25, 2014
Article
Quantification of Arginine and its Methylated Derivatives in Healthy Children by Liquid Chromatography-Tandem Mass Spectrometry Fernando Andrade, Marta Llarena, Sergio Lage and Luis Alda´miz-Echevarrı´ a* Division of Metabolism, Cruces University Hospital, Plaza de Cruces s/n, Barakaldo 48903, Bizkaia, Spain *Author to whom correspondence should be addressed. Email: [email protected] Received 28 July 2014; revised 28 July 2014
Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of nitric oxide synthase, which is responsible for most of the vascular nitric oxide (NO) produced. NO is an important physiological mediator of vascular tone and structure in normally functioning endothelial cells. We report the optimization of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of arginine (Arg) and its derivatives in biological samples. Chromatographic separation and mass detection were performed by reverse phase chromatography coupled with tandem mass spectrometry. For sample preparation, plasma proteins were removed by centrifugal filters. Positive electrospray ionization was performed and analytes were detected by multiple reaction monitoring. Inter- and intra-day repeatability, accuracy, recovery, and limits of detection and quantification were evaluated to validate the method. Plasma and urine levels were measured in healthy children to establish control values: 52.2–124.7 mM for Arg, 0.06 –0.16 mM for MMA, 0.42– 1.10 mM for ADMA and 0.41 – 0.96 mM for SDMA in plasma. Quantification of Arg and its methylated derivatives by LC-MS/MS can be carried out without the need of organic solvents for sample preparation, and be used as a valuable tool in research on endothelial dysfunction.
Introduction L-Arginine (Arg), the main substrate for nitric oxide (NO) synthesis, is oxidized to L-citrulline and NO by the action of endothelial nitric oxide synthase (NOS). In contrast, protein-incorporated L-Arg residues can be methylated differently with subsequent proteolysis giving rise to three types of compounds: asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA). Arg methylation is catalyzed by type I and II protein arginine methyltransferase, whose activity depends on S-adenosylmethionine as a donor of methyl groups. ADMA is produced in all body cells and competes with Arg for binding to NOS (1). Although SDMA does not inhibit NO synthesis directly, it can compete with the cationic amino acid transporter in the membrane of endothelial cells, thus accentuating Arg deficiency. Most ADMA, but not SDMA, is degraded to citrulline and dimethylamine (Figure 1) by the action of dimethylarginine dimethylaminohydrolase. This enzyme is distributed widely throughout the body, mainly in the liver and kidneys (2), and regulates ADMA levels and, therefore, NO synthesis. Although ADMA and SDMA levels could poorly reflect intracellular levels in peripheral blood mononuclear cells (3), plasma levels of ADMA in healthy subjects with no apparent cardiovascular
disease have been linked with age, blood pressure, glucose intolerance and thickness of the carotid intima – media. These findings suggest that increased ADMA levels are a marker of atherosclerotic change, because a slight change in plasma levels of ADMA is sufficient to significantly alter the intracellular levels of ADMA and thus to modify the production of NO and, in turn, contribute to the development of cardiovascular disease (4, 5). Therefore, plasma levels of ADMA have been analyzed in patients with insulin resistance (6), diabetes (7), hypercholesterolemia (8) and renal disease (9). Despite evidence that levels of this molecule are clinically relevant in young people (10), the metabolic pathway involving ADMA has been little studied. Among the studies reported to date, elevated levels of ADMA have been detected in children and adolescents with high blood pressure (11) and with chronic renal failure (12, 13). Therefore, further studies as current work are necessary to establish control values in pediatric population, in order to define if Arg-NO pathway is disturbed. There is a growing demand from clinicians and researchers for data on levels of Arg and its methylated derivatives, and thus several analytical methods have been developed, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems which give robust results, as well as allowing Arg, ADMA and SDMA to be measured in the same experiment (14). The first chromatographic methods developed, involving sample preparation by solid phase extraction, or a derivatization of Arg, ADMA and SDMA prior to chromatographic separation, used ortho-phthaldialdehyde so that the compounds produced can be quantified by a fluorescence detector (15, 16). Other methods using a tandem mass spectrometer required derivatization of samples, for example, to obtain the butyl ester derivatives (17). But nowadays, there is a great interest in simultaneous determination of Arg derivates using stable isotope dilution mass spectrometry which offers precision and accuracy (18, 19). Compared with recent publications on ADMA, we report the first method to analyze plasma samples by means of ultrafiltration, avoiding derivatization, solid phase extraction and organic solvents or acids. The use of this protein removal gives less degradation of metabolites and has not been described for ADMA and SDMA. Moreover, chromatographic separation of structural isomers is performed to enable use their optimal mass transitions for detection, which are the same for both metabolites. This separation is carried out maintaining isocratic elution with acid aqueous mobile phase that achieves suitable ADMA–SDMA chromatographic separation. The ion suppression of this method has also been evaluated. So, the aims of this study are to develop and optimize an alternative method to measure Arg and its methylated derivatives in
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Figure 1. Metabolic pathway for the formation of asymmetric dimethylarginine by protein arginine methyltransferases and proteinases, and its involvement in the inhibition of nitric oxide by competition with nitric oxide synthase for arginine. Asymmetric dimethylarginine is degradated to citrulline and amines by dimethylarginine dimethylaminohydrolase.
human fluids by LC-MS/MS, mainly simplifying sample preparation, and give reference values in pediatric population. Materials and Methods Chemicals L -Arg, ADMA, SDMA and MMA were purchased from Sigma (Madrid, Spain). Internal standards (13C6 L-Arg:HCl and D7ADMA:HCl:H2O) were obtained from Cambridge Isotope Laboratories (Andover, MA, USA), trifluoroacetic acid (TFA) and ammonium formate from Sigma-Aldrich (Madrid, Spain) and deionized water and methanol Romil-Sps from Teknokroma (Barcelona, Spain). Instrumentation and conditions Mass spectrometric analysis was performed using an Agilent Technologies 1100 series HPLC with autosampler and quaternary pump coupled to an Agilent 6410 triple quadrupole mass spectrometer with ESI ion source (both from Agilent Technologies, Madrid, Spain). The column utilized for separation was a Nucleosil C18 (4.0 150 mm) with 5-mm particles (Teknokroma) at 208C, protected by a Guard-Pak cartridge (ODS 2.0 10 mm, 5 mm; Teknokroma). The separation condition was isocratic elution with 100% of aqueous mobile phase of trifluoroacetic acid (TFA) 0.2% at 0.4 mL/min for 11 min. Sample collection Control values for Arg, ADMA, SDMA and MMA in plasma and urine were determined in 30 otherwise healthy children of both sexes, aged 6 –18 years, who underwent minor surgery in our center (hernia, phimosis, bone fractures, tympanostomy tube insertion, etc.). The study protocol was approved by the Clinical Research Ethics Committee of Cruces Hospital, and patients’ parents gave written informed consent. Blood and urine samples were taken in the morning after overnight fasting. The blood samples were immediately cooled in an ice-water bath and centrifuged (within 10 min) at 4,000 rpm for 5 min at 48C. The platelet-poor plasma and urine were aliquoted and stored at 2208C until the assay was performed, usually within a few days. 788 Andrade et al.
Sample preparation To measure the target compounds, 50 mL of plasma and 50 mL of each of internal standard (100 mM for 13C6-Arg and 2 mM for D7-ADMA) were mixed in an Eppendorf tube. Then, 75 mL of water were added to bring the volume to the same as that of the calibrators (225 mL) and to improve the efficiency of ultrafiltration. The resulting mixture was stirred gently, and poured into the Amicon Ultra centrifugal filters (Millipore, Billerica, MA, USA) with 10,000 nominal molecular weight limit, which were used to remove proteins from biological fluids. The filters were centrifuged for 45 min at 13,000 rpm. The filtered solution was transferred to a vial and an injection volume of 10 mL was used. In case of urine samples, the same procedure was used as described earlier, but 10 mL of urine was used and 2 mL of final filtered solution was injected.
Calibration and method validation Stock solutions were prepared in water, and were stable for at least 1 year at 48C. A solution mixture of 200 mM Arg, 2 mM ADMA, 2 mM SDMA and 1 mM MMA was used as the working solution, which was stable for 1 month at 48C or 3 months at 2358C. Calibration curves were obtained using five standards prepared in plasma sample from 0 to 3.0 mM for ADMA and SDMA; up to 1.5 mM for MMA; and up to 300 mM for Arg. Similarly, urine samples calibration curves were obtained from a range of dilutions of the stock solution in 10 mL of urine: 0–150 mM for Arg, ADMA and SDMA; and 0–37.5 mM for MMA. Internal standards were used at a concentration of 10 mM for 13C6-Arg and 20 mM for D7-ADMA. A total of 50 mL of phenol (0.1%) was added to stock and working solutions, in order to increase the stability. Since ion suppression can affect the quantitative performance of a mass detector, matrix effect has been studied because nonvolatile co-eluting matrix components reduce ion production for the analyte of interest. Peak areas of calibrator solutions and peak areas of calibrator added to plasma matrix have been compared, taking into account the area of used plasma without standard. These differences of areas between calibrators and calibrators with plasma were ,20% for Arg, ADMA, SDMA and MMA.
Results Chromatographic optimization As ADMA and SDMA are structural isomers, an ion-pair reagent is required for their separation by chromatographic methods. The usual solvents (water, methanol and acetonitrile) were combined with acetic acid, formic acid, trichloroacetic acid, TFA, ammonium formate or ammonium acetate. The best results were found with TFA at a final concentration of 0.2%. An isocratic elution with 100% of TFA as mobile phase, was used at a flow rate of 0.4 mL/min for 11 min to obtain metabolite separation (Figure 2). Between injections, the chromatographic column was washed with 10% of aqueous phase and 90% of acetonitrile at 1.5 mL/min for at least 5 min, and then, conditioned with aqueous phase for 4 min.
Tandem mass characterization of Arg derivatives Multiple reaction monitoring transitions of each compound for this measurement were m/z 175.0!70.0 for Arg, m/z
Figure 2. Typical chromatogram obtained from a human plasma by the liquid chromatography-tandem mass spectrometry method described in this paper. Separation condition was isocratic elution with trifluoroacetic acid 0.2% at 0.4 mL/min for 11 min.
189.2!70.1 for MMA, m/z 203.2!70.1 for ADMA and SDMA, m/z 181.2!74.1 for 13C6-Arg and m/z 210.1!77.0 for D7-ADMA. When precursor ion of ADMA and SDMA (m/z 203.2) is fragmented, the predominant product ion is m/z 70.1 (Figure 3), so our measurements are more sensitive using described transition for both isomers. The spray voltage was set at 3500 V, and the nebulizer gas (N2) heated to 3508C with a flow rate of 11 L/min and pressure of 40 psi. These high values in ion source were set to improve ionization efficiency of 100% aqueous mobile phase used. Each transition was monitored with 100 V fragmentator and 25 V collision energy. Electron multiplier voltage was set at 300 V. All these parameters were found and optimized in this work.
Method validation All calibration curves were linear with correlation coefficients .0.987. Data from repeatability and accuracy studies are listed in Table I. The limits of detection (S/N ¼ 3) and quantification (S/N ¼ 10) for Arg, ADMA, SDMA and MMA were measured for each analyte based upon the regression of the calibration curves in the established linear range. The parameters of Table I indicate robustness and sensitivity for clinical assessments. Several experiments for protein precipitation with 5-sulfosalicylic acid were
carried out, but the recoveries (,67% in all cases) and the correlation coefficients of calibrations (,0.850) justify the use of filtering.
Biochemical profile in plasma and urine of healthy children The proposed method was applied to analyze plasma and urine samples from healthy children, and the concentration ranges (P3 –P97) are reported in Table II.
Discussion Here, we report an alternative procedure to quantify Arg and its methylated derivatives in biological samples, providing reference values in pediatric population. Our method does not need to use organic solvents or acids for protein precipitation since the centrifugal filters efficiently remove proteins and, thus produce less degradation of metabolites. In addition, chromatographic separation of structural isomers ADMA and SDMA is performed to enable their closely spaced transitions useful for detection. In previous papers, Weaving et al. (20) established the results of fragmentation of ADMA, which are similar to those of our proposal; the precursor ion at m/z 203.2 being fragmented to yield LC-MS/MS Method for Arg Derivatives 789
Figure 3. Product ion mass spectra of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) standards in positive mode with fragmentor and collision energy optimized to fragment all precursor ion (m/z 203.2).
the predominant product ion (m/z 70.1). Several authors have published a method establishing the product ion for ADMA at m/z 46.2, but this is less sensitive than measurements based on the ion at m/z 70.1 (21). Moreover, for SDMA there is some controversy, as some authors have stated that the ion at m/z 172.0 is the best product ion for SDMA. However, our results and those of other authors (13) indicate that measurements are more sensitive using the ion at m/z 70.1 for ADMA and SDMA, because it is the main product ion when precursor ion is fragmented applying several controlled voltages (Figure 3). Notably, if the same ion transition for ADMA and SDMA has to be used, a chromatographic separation of these isomers is essential. Thus, in our method we show that the amount of TFA had greatest effect on the peak shape and separation. So, when the percentage of TFA was below 0.1%, the resolution of dimethylarginines was poor, but when it was 0.2 – 0.5%, a good chromatographic separation was achieved, despite of TFA ion suppression. Another factor of ion suppression is given by matrix effect, what was checked by 790 Andrade et al.
comparing peak areas of aqueous calibrator solutions without matrix and calibrator added to plasma matrix. The differences between calibrators and calibrators in plasma were ,17% for Arg and ,11% for ADMA, SDMA and MMA, slightly decreasing the efficiency of formation of the desired analyte ions. So, the matrix, probably due to the presence of lipids, has little influence in electrospray ion formation, and hence, in MS response. MMA is also a potent inhibitor of NOS, although its role may be minor because of its low concentration. In some previous studies, there is a lack of quantification of MMA together with its analogues ADMA and SDMA, but here it is solved because our simultaneous determination offers satisfactory results. Besides, other procedures available for simultaneous MMA determination need solid phase extraction and derivatization (18) increasing costs and difficulty, in contrast with our method. In summary, our method requires easier sample preparation, avoiding solid phase extraction, derivatization or precipitation of proteins by organic solvents which need a subsequent
Table I Statistical Parameters of Arg and Its Methylated Derivatives Quantification by LC-MS/MS Arg Intra-day repeatability % 3.1 RSD Inter-day repeatability 7.4 % RSD Linear interval At least up to 650 mM Calibration range 0–300 (plasma), mM Calibration range 0 –150 (urine), mM Slope (SD) 0.016 (,0.001) Intercept (SD) 0.829 (0.025) 0.9895 – 0.9998 Range of correlation coefficients (R 2) Limit of quantification 1.813 (S/N ¼ 10), mM Limit of detection 0.544 (S/N ¼ 3), mM Accuracy % 2.1 Recovery % 85.4
ADMA
SDMA
MMA
3.6
3.6
11.4
2.5
1.9
12.5
At least up to 500 mM 0–3
At least up to 500 mM 0 –3
At least up to 100 mM 0–1.5
0 –150
0 –150
0 –37.5
0.473 (0.006) 0.683 (0.010) 0.005 (0.001) 0.303 (0.012) 0.650 (0.019) 0.034 (0.001) 0.9876 –0.9995 0.9899 –0.9999 0.9891 –0.9999 0.064
0.066
0.020
0.019
0.020
0.006
1.2 94.3
1.7 94.2
2.4 98.9
Table II Biochemical Profile in Plasma and Urine of Healthy Children (Median and P3 –P97)
Arg MMA ADMA SDMA Arg/ADMA
Plasma (mM)
Urine (mM)
Urine (mmol/mol Crna)
85.5 (52.2 –124.7) 0.11 (0.06 –0.16) 0.61 (0.42 –1.10) 0.49 (0.41 –0.96) 136.50 (79.5 –209.1)
48.1 0.28 89.8 73.1 0.55
4.0 (2.6 – 6.4) 0.03 (0.01 –0.06) 7.6 (4.7 – 11.8) 6.5 (4.3 – 10.2)
(19.1–87.2) (0.08 –0.72) (51.8–162.3) (36.3–125.6) (0.31 –0.69)
a
Crn, creatinine.
evaporation. In particular, we use filters which enable proteins and other compounds to be effectively removed from the matrix by ultrafiltration. In addition, the recycling of filters does not make lose their effectiveness. The results of the method validation showed satisfactory recovery, accuracy and repeatability, as well as a very wide range of linearity and low limits of detection and quantification. Moreover, we observed high repeatability (,7.4% RSD, except for MMA), accuracy (,2.5%) and recovery (.85%), as described in previous studies (22, 23). Over the past 20 years, ADMA has received an increasing interest from both clinical and basic researchers. Baseline levels of ADMA and SDMA have been measured in adult humans (24) due to its cardiovascular implications, but little has been published on these values in healthy children despite their potential relevance (10). Generally, ADMA levels in young people are higher than in healthy adults due to the immaturity of the enzyme systems responsible for the degradation of ADMA, which directly affects dimethylarginine dimethyaminohydrolase activity in children and adolescents. We provide plasma and urinary levels of ADMA in healthy children, similar to those obtained by ELISA (25) and by other MS – MS methods (12, 13). Our report in healthy children gives data of SDMA and MMA levels which can be useful to compare with levels under pathological conditions (26, 27). Moreover, SDMA levels are important in many renal diseases due to their relation with glomerular filtration rate (28), so, most papers about dimethylarginines in children revolves around chronic kidney diseases. In addition, ADMA levels have also been used to assess cardiovascular risk and
endothelial dysfunction in children under oxidative stress conditions such as hypercholesterolemia (26), diabetes (29) or an inborn error of metabolism (25). We conclude that this proposed method is an easy and accurate LC-MS/MS procedure for the simultaneous determination of Arg, ADMA, SDMA and MMA in biological fluids. The repeatability, sensitivity and specificity make this method a valuable tool in clinical and basic research on oxidative stress and endothelial dysfunction in pediatric population.
Acknowledgments This research was partially funded by the Department of Health of Basque Government and Carlos III Health Research Institute.
References 1. Bo¨ger, R.H., Sydow, K., Borlak, J., Thum, T., Lenzen, H., Schubert, B., et al.; LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethionine-dependent methyltransferases; Circulation Research, (2000); 87: 99 –105. 2. Nijveldt, R.J., Teerlink, T., van Guldener, C., Prins, H.A., van Lambalgen, A.A., Stehouwer, C.D., et al.; Handling of asymmetrical dimethylarginine and symmetrical dimethylarginine by the rat kidney under basal conditions and during endotoxaemia; Nephrology Dialysis and Transplantation, (2003); 18: 2542–2550. 3. Davids, M., Teerlink, T.; Plasma concentrations of arginine and asymmetric dimethylarginine do not reflect their intracellular concentrations in peripheral blood mononuclear cells; Metabolism, (2013); 62: 1455–1461. 4. Wilcken, D.E., Sim, A.S., Wang, J., Wang, X.L.; Asymmetric dimethylarginine (ADMA) in vascular, renal and hepatic disease and the regulatory role of L-arginine on its metabolism; Molecular Genetics and Metabolism, (2007); 91: 309– 317. 5. Cardounel, A.J., Cui, H., Samouilov, A., Johnson, W., Kearns, P., Tsai, A.L., et al.; Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function; Journal of Biological Chemistry, (2007); 282: 879– 887. 6. Stu¨hlinger, M.C., Abbasi, F., Chu, J.W., Lamendola, C., McLaughlin, T.L., Cooke, J.P., et al.; Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor; Journal of the American Medical Association, (2002); 287: 1420– 1426. 7. Tarnow, L., Hovind, P., Teerlink, T., Stehouwer, C.D., Parving, H.H.; Elevated plasma asymmetric dimethylarginine as a marker of cardiovascular morbidity in early diabetic nephropathy in type 1 diabetes; Diabetes Care, (2004); 27: 765–769. 8. Lu, T.M., Ding, Y.A., Leu, H.B., Yin, W.H., Sheu, W.H., Chu, K.M.; Effect of rosuvastatin on plasma levels of asymmetric dimethylarginine in patients with hypercholesterolemia; American Journal of Cardiology, (2004); 94: 157–161. 9. Loscalzo, J.; L-arginine and atherothrombosis; Journal of Nutrition, (2004); 134: S2798– S2800. 10. Tain, Y.L., Huang, L.T.; Asymmetric dimethylarginine: Clinical applications in pediatric medicine; Journal of the Formosan Medical Association, (2011); 110: 70 –77. 11. Goonasekera, C.D., Rees, D.D., Woolard, P., Frend, A., Shah, V., Dillon, M.J.; Nitric oxide synthase inhibitors and hypertension in children and adolescents; Journal of Hypertension, (1997); 15: 901– 909. 12. Brooks, E.R., Langman, C.B., Wang, S., Price, H.E., Hodges, A.L., Darling, L., et al.; Methylated arginine derivatives in children and adolescents with chronic kidney disease; Pediatric Nephrology, (2009); 24: 129– 134. 13. Wang, S., Vicente, F.B., Miller, A., Brooks, E.R., Price, H.E., Smith, F.A.; Measurement of arginine derivatives in pediatric patients with
LC-MS/MS Method for Arg Derivatives 791
14.
15.
16.
17.
18.
19.
20.
21.
chronic kidney disease using high-performance liquid chromatography-tandem mass spectrometry; Clinical Chemistry and Laboratory Medicine, (2007); 45: 1305– 1312. Pecchini, P., Malberti, F., Mieth, M., Quinn, R., Tripepi, G., Mallamaci, F., et al.; Measuring asymmetric dimethylarginine (ADMA) in CKD: a comparison between enzyme-linked immunosorbent assay and liquid chromatography-electrospray tandem mass spectrometry; Journal of Nephrology, (2012); 25: 1016– 1022. Blackwell, S., O’Reilly, D.S., Talwar, D.K.; HPLC analysis of asymmetric dimethylarginine (ADMA) and related arginine metabolites in human plasma using a novel non-endogenous internal standard; Clinica Chimica Acta, (2009); 401: 14– 19. Martens-Lobenhoffer, J., Krug, O., Bode-Bo¨ger, S.M.; Determination of arginine and asymmetric dimethylarginine (ADMA) in human plasma by liquid chromatography/mass spectrometry with the isotope dilution technique; Journal of Mass Spectrometry, (2004); 39: 1287–1294. Schwedhelm, E., Maas, R., Tan-Andresen, J., Schulze, F., Riederer, U., Bo¨ger, R.H.; High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma; Journal of Chromatography B, (2007); 851: 211– 219. Davids, M., Swieringa, E., Palm, F., Smith, D.E., Smulders, Y.M., Scheffer, P.G.; Simultaneous determination of asymmetric and symmetric dimethylarginine, L-monomethylarginine, L-arginine, and L-homoarginine in biological samples using stable isotope dilution liquid chromatography tandem mass spectrometry; Journal of Chromatography B, (2012); 900: 38 –47. Martens-Lobenhoffer, J., Bode-Bo¨ger, S.M.; Quantification of Larginine, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma: a step improvement in precision by stable isotope dilution mass spectrometry; Journal of Chromatography B, (2012); 904: 140–143. Weaving, G., Rocks, B.F., Bailey, M.P., Titheradge, M.A.; Arginine and methylated arginines in human plasma and urine measured by tandem mass spectrometry without the need for chromatography or sample derivatisation; Journal of Chromatography B, (2008); 874: 27–32. Martens-Lobenhoffer, J., Schwedhelm, E., Tsikas, D.; Quantification of arginine and its mono- and dimethylated analogs NMMA, ADMA and SDMA in biological fluids by LC-MS/MS: Is LC superfluous? Journal of Chromatography B, (2009); 877: 3261–3266.
792 Andrade et al.
22. Bishop, M.J., Crow, B., Norton, D., Paliakov, E., George, J., Bralley, J.A.; Direct analysis of un-derivatized asymmetric dimethylarginine (ADMA) and L-arginine from plasma using mixed-mode ionexchange liquid chromatography-tandem mass spectrometry; Journal of Chromatography B, (2007); 859: 164–169. 23. Gervasoni, J., Bonelli, F., Zuppi, C., Zappacosta, B., Mordente, A., Calvani, R.; Determination of asymmetric dimethyl arginine in human serum by liquid chromatography-tandem mass spectrometry: clinical application in hypertensive subjects; Clinical Chemistry and Laboratory Medicine, (2011); 49: 2109– 2115. 24. El-Khoury, J.M., Bunch, D.R., Reineks, E., Jackson, R., Steinle, R., Wang, S.; A simple and fast liquid chromatography-tandem mass spectrometry method for measurement of underivatized L-arginine, symmetric dimethylarginine, and asymmetric dimethylarginine and establishment of the reference ranges; Analytical and Bioanalytical Chemistry, (2012); 402: 771– 779. 25. Huemer, M., Simma, B., Mayr, D., Mo¨slinger, D., Mu¨hl, A., Schmid, I., et al.; Free asymmetric dimethylarginine (ADMA) is low in children and adolescents with classical phenylketonuria (PKU); Journal of Inherited Metabolic Disease, (2012); 35: 817– 821. 26. Jehlicˇ ka, P., Stozˇ icky´ , F., Mayer, O., Jr, Varvarˇ ovska´ , J., Racek, J., Trefil, L., et al.; Asymmetric dimethylarginine and the effect of folate substitution in children with familial hypercholesterolemia and diabetes mellitus type 1; Physiological Research, (2009); 58: 179 – 184. 27. Di Gangi, I.M., Chiandetti, L., Gucciardi, A., Moret, V., Naturale, M., Giordano, G.; Simultaneous quantitative determination of N(G), N(G)-dimethyl-L-arginine or asymmetric dimethylarginine and related pathway’s metabolites in biological fluids by ultrahigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry; Analytica Chimica Acta, (2010); 677: 140– 148. 28. Marcovecchio, M.L., Dalton, R.N., Turner, C., Prevost, A.T., Widmer, B., Amin, R., et al.; Symmetric dimethylarginine, an endogenous marker of glomerular filtration rate, and the risk for microalbuminuria in young people with type 1 diabetes; Archives of Disease in Childhood, (2010); 95: 119– 124. 29. Heilman, K., Zilmer, M., Zilmer, K., Kool, P., Tillmann, V.; Elevated plasma adiponectin and decreased plasma homocysteine and asymmetric dimethylarginine in children with type 1 diabetes; Journal of Clinical Laboratory Investigation, (2009); 69: 85– 91.
Article
Quantification of Arginine and its Methylated Derivatives in Healthy Children by Liquid Chromatography-Tandem Mass Spectrometry Fernando Andrade, Marta Llarena, Sergio Lage and Luis Alda´miz-Echevarrı´ a* Division of Metabolism, Cruces University Hospital, Plaza de Cruces s/n, Barakaldo 48903, Bizkaia, Spain *Author to whom correspondence should be addressed. Email: [email protected] Received 28 July 2014; revised 28 July 2014
Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of nitric oxide synthase, which is responsible for most of the vascular nitric oxide (NO) produced. NO is an important physiological mediator of vascular tone and structure in normally functioning endothelial cells. We report the optimization of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of arginine (Arg) and its derivatives in biological samples. Chromatographic separation and mass detection were performed by reverse phase chromatography coupled with tandem mass spectrometry. For sample preparation, plasma proteins were removed by centrifugal filters. Positive electrospray ionization was performed and analytes were detected by multiple reaction monitoring. Inter- and intra-day repeatability, accuracy, recovery, and limits of detection and quantification were evaluated to validate the method. Plasma and urine levels were measured in healthy children to establish control values: 52.2–124.7 mM for Arg, 0.06 –0.16 mM for MMA, 0.42– 1.10 mM for ADMA and 0.41 – 0.96 mM for SDMA in plasma. Quantification of Arg and its methylated derivatives by LC-MS/MS can be carried out without the need of organic solvents for sample preparation, and be used as a valuable tool in research on endothelial dysfunction.
Introduction L-Arginine (Arg), the main substrate for nitric oxide (NO) synthesis, is oxidized to L-citrulline and NO by the action of endothelial nitric oxide synthase (NOS). In contrast, protein-incorporated L-Arg residues can be methylated differently with subsequent proteolysis giving rise to three types of compounds: asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA). Arg methylation is catalyzed by type I and II protein arginine methyltransferase, whose activity depends on S-adenosylmethionine as a donor of methyl groups. ADMA is produced in all body cells and competes with Arg for binding to NOS (1). Although SDMA does not inhibit NO synthesis directly, it can compete with the cationic amino acid transporter in the membrane of endothelial cells, thus accentuating Arg deficiency. Most ADMA, but not SDMA, is degraded to citrulline and dimethylamine (Figure 1) by the action of dimethylarginine dimethylaminohydrolase. This enzyme is distributed widely throughout the body, mainly in the liver and kidneys (2), and regulates ADMA levels and, therefore, NO synthesis. Although ADMA and SDMA levels could poorly reflect intracellular levels in peripheral blood mononuclear cells (3), plasma levels of ADMA in healthy subjects with no apparent cardiovascular
disease have been linked with age, blood pressure, glucose intolerance and thickness of the carotid intima – media. These findings suggest that increased ADMA levels are a marker of atherosclerotic change, because a slight change in plasma levels of ADMA is sufficient to significantly alter the intracellular levels of ADMA and thus to modify the production of NO and, in turn, contribute to the development of cardiovascular disease (4, 5). Therefore, plasma levels of ADMA have been analyzed in patients with insulin resistance (6), diabetes (7), hypercholesterolemia (8) and renal disease (9). Despite evidence that levels of this molecule are clinically relevant in young people (10), the metabolic pathway involving ADMA has been little studied. Among the studies reported to date, elevated levels of ADMA have been detected in children and adolescents with high blood pressure (11) and with chronic renal failure (12, 13). Therefore, further studies as current work are necessary to establish control values in pediatric population, in order to define if Arg-NO pathway is disturbed. There is a growing demand from clinicians and researchers for data on levels of Arg and its methylated derivatives, and thus several analytical methods have been developed, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems which give robust results, as well as allowing Arg, ADMA and SDMA to be measured in the same experiment (14). The first chromatographic methods developed, involving sample preparation by solid phase extraction, or a derivatization of Arg, ADMA and SDMA prior to chromatographic separation, used ortho-phthaldialdehyde so that the compounds produced can be quantified by a fluorescence detector (15, 16). Other methods using a tandem mass spectrometer required derivatization of samples, for example, to obtain the butyl ester derivatives (17). But nowadays, there is a great interest in simultaneous determination of Arg derivates using stable isotope dilution mass spectrometry which offers precision and accuracy (18, 19). Compared with recent publications on ADMA, we report the first method to analyze plasma samples by means of ultrafiltration, avoiding derivatization, solid phase extraction and organic solvents or acids. The use of this protein removal gives less degradation of metabolites and has not been described for ADMA and SDMA. Moreover, chromatographic separation of structural isomers is performed to enable use their optimal mass transitions for detection, which are the same for both metabolites. This separation is carried out maintaining isocratic elution with acid aqueous mobile phase that achieves suitable ADMA–SDMA chromatographic separation. The ion suppression of this method has also been evaluated. So, the aims of this study are to develop and optimize an alternative method to measure Arg and its methylated derivatives in
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Figure 1. Metabolic pathway for the formation of asymmetric dimethylarginine by protein arginine methyltransferases and proteinases, and its involvement in the inhibition of nitric oxide by competition with nitric oxide synthase for arginine. Asymmetric dimethylarginine is degradated to citrulline and amines by dimethylarginine dimethylaminohydrolase.
human fluids by LC-MS/MS, mainly simplifying sample preparation, and give reference values in pediatric population. Materials and Methods Chemicals L -Arg, ADMA, SDMA and MMA were purchased from Sigma (Madrid, Spain). Internal standards (13C6 L-Arg:HCl and D7ADMA:HCl:H2O) were obtained from Cambridge Isotope Laboratories (Andover, MA, USA), trifluoroacetic acid (TFA) and ammonium formate from Sigma-Aldrich (Madrid, Spain) and deionized water and methanol Romil-Sps from Teknokroma (Barcelona, Spain). Instrumentation and conditions Mass spectrometric analysis was performed using an Agilent Technologies 1100 series HPLC with autosampler and quaternary pump coupled to an Agilent 6410 triple quadrupole mass spectrometer with ESI ion source (both from Agilent Technologies, Madrid, Spain). The column utilized for separation was a Nucleosil C18 (4.0 150 mm) with 5-mm particles (Teknokroma) at 208C, protected by a Guard-Pak cartridge (ODS 2.0 10 mm, 5 mm; Teknokroma). The separation condition was isocratic elution with 100% of aqueous mobile phase of trifluoroacetic acid (TFA) 0.2% at 0.4 mL/min for 11 min. Sample collection Control values for Arg, ADMA, SDMA and MMA in plasma and urine were determined in 30 otherwise healthy children of both sexes, aged 6 –18 years, who underwent minor surgery in our center (hernia, phimosis, bone fractures, tympanostomy tube insertion, etc.). The study protocol was approved by the Clinical Research Ethics Committee of Cruces Hospital, and patients’ parents gave written informed consent. Blood and urine samples were taken in the morning after overnight fasting. The blood samples were immediately cooled in an ice-water bath and centrifuged (within 10 min) at 4,000 rpm for 5 min at 48C. The platelet-poor plasma and urine were aliquoted and stored at 2208C until the assay was performed, usually within a few days. 788 Andrade et al.
Sample preparation To measure the target compounds, 50 mL of plasma and 50 mL of each of internal standard (100 mM for 13C6-Arg and 2 mM for D7-ADMA) were mixed in an Eppendorf tube. Then, 75 mL of water were added to bring the volume to the same as that of the calibrators (225 mL) and to improve the efficiency of ultrafiltration. The resulting mixture was stirred gently, and poured into the Amicon Ultra centrifugal filters (Millipore, Billerica, MA, USA) with 10,000 nominal molecular weight limit, which were used to remove proteins from biological fluids. The filters were centrifuged for 45 min at 13,000 rpm. The filtered solution was transferred to a vial and an injection volume of 10 mL was used. In case of urine samples, the same procedure was used as described earlier, but 10 mL of urine was used and 2 mL of final filtered solution was injected.
Calibration and method validation Stock solutions were prepared in water, and were stable for at least 1 year at 48C. A solution mixture of 200 mM Arg, 2 mM ADMA, 2 mM SDMA and 1 mM MMA was used as the working solution, which was stable for 1 month at 48C or 3 months at 2358C. Calibration curves were obtained using five standards prepared in plasma sample from 0 to 3.0 mM for ADMA and SDMA; up to 1.5 mM for MMA; and up to 300 mM for Arg. Similarly, urine samples calibration curves were obtained from a range of dilutions of the stock solution in 10 mL of urine: 0–150 mM for Arg, ADMA and SDMA; and 0–37.5 mM for MMA. Internal standards were used at a concentration of 10 mM for 13C6-Arg and 20 mM for D7-ADMA. A total of 50 mL of phenol (0.1%) was added to stock and working solutions, in order to increase the stability. Since ion suppression can affect the quantitative performance of a mass detector, matrix effect has been studied because nonvolatile co-eluting matrix components reduce ion production for the analyte of interest. Peak areas of calibrator solutions and peak areas of calibrator added to plasma matrix have been compared, taking into account the area of used plasma without standard. These differences of areas between calibrators and calibrators with plasma were ,20% for Arg, ADMA, SDMA and MMA.
Results Chromatographic optimization As ADMA and SDMA are structural isomers, an ion-pair reagent is required for their separation by chromatographic methods. The usual solvents (water, methanol and acetonitrile) were combined with acetic acid, formic acid, trichloroacetic acid, TFA, ammonium formate or ammonium acetate. The best results were found with TFA at a final concentration of 0.2%. An isocratic elution with 100% of TFA as mobile phase, was used at a flow rate of 0.4 mL/min for 11 min to obtain metabolite separation (Figure 2). Between injections, the chromatographic column was washed with 10% of aqueous phase and 90% of acetonitrile at 1.5 mL/min for at least 5 min, and then, conditioned with aqueous phase for 4 min.
Tandem mass characterization of Arg derivatives Multiple reaction monitoring transitions of each compound for this measurement were m/z 175.0!70.0 for Arg, m/z
Figure 2. Typical chromatogram obtained from a human plasma by the liquid chromatography-tandem mass spectrometry method described in this paper. Separation condition was isocratic elution with trifluoroacetic acid 0.2% at 0.4 mL/min for 11 min.
189.2!70.1 for MMA, m/z 203.2!70.1 for ADMA and SDMA, m/z 181.2!74.1 for 13C6-Arg and m/z 210.1!77.0 for D7-ADMA. When precursor ion of ADMA and SDMA (m/z 203.2) is fragmented, the predominant product ion is m/z 70.1 (Figure 3), so our measurements are more sensitive using described transition for both isomers. The spray voltage was set at 3500 V, and the nebulizer gas (N2) heated to 3508C with a flow rate of 11 L/min and pressure of 40 psi. These high values in ion source were set to improve ionization efficiency of 100% aqueous mobile phase used. Each transition was monitored with 100 V fragmentator and 25 V collision energy. Electron multiplier voltage was set at 300 V. All these parameters were found and optimized in this work.
Method validation All calibration curves were linear with correlation coefficients .0.987. Data from repeatability and accuracy studies are listed in Table I. The limits of detection (S/N ¼ 3) and quantification (S/N ¼ 10) for Arg, ADMA, SDMA and MMA were measured for each analyte based upon the regression of the calibration curves in the established linear range. The parameters of Table I indicate robustness and sensitivity for clinical assessments. Several experiments for protein precipitation with 5-sulfosalicylic acid were
carried out, but the recoveries (,67% in all cases) and the correlation coefficients of calibrations (,0.850) justify the use of filtering.
Biochemical profile in plasma and urine of healthy children The proposed method was applied to analyze plasma and urine samples from healthy children, and the concentration ranges (P3 –P97) are reported in Table II.
Discussion Here, we report an alternative procedure to quantify Arg and its methylated derivatives in biological samples, providing reference values in pediatric population. Our method does not need to use organic solvents or acids for protein precipitation since the centrifugal filters efficiently remove proteins and, thus produce less degradation of metabolites. In addition, chromatographic separation of structural isomers ADMA and SDMA is performed to enable their closely spaced transitions useful for detection. In previous papers, Weaving et al. (20) established the results of fragmentation of ADMA, which are similar to those of our proposal; the precursor ion at m/z 203.2 being fragmented to yield LC-MS/MS Method for Arg Derivatives 789
Figure 3. Product ion mass spectra of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) standards in positive mode with fragmentor and collision energy optimized to fragment all precursor ion (m/z 203.2).
the predominant product ion (m/z 70.1). Several authors have published a method establishing the product ion for ADMA at m/z 46.2, but this is less sensitive than measurements based on the ion at m/z 70.1 (21). Moreover, for SDMA there is some controversy, as some authors have stated that the ion at m/z 172.0 is the best product ion for SDMA. However, our results and those of other authors (13) indicate that measurements are more sensitive using the ion at m/z 70.1 for ADMA and SDMA, because it is the main product ion when precursor ion is fragmented applying several controlled voltages (Figure 3). Notably, if the same ion transition for ADMA and SDMA has to be used, a chromatographic separation of these isomers is essential. Thus, in our method we show that the amount of TFA had greatest effect on the peak shape and separation. So, when the percentage of TFA was below 0.1%, the resolution of dimethylarginines was poor, but when it was 0.2 – 0.5%, a good chromatographic separation was achieved, despite of TFA ion suppression. Another factor of ion suppression is given by matrix effect, what was checked by 790 Andrade et al.
comparing peak areas of aqueous calibrator solutions without matrix and calibrator added to plasma matrix. The differences between calibrators and calibrators in plasma were ,17% for Arg and ,11% for ADMA, SDMA and MMA, slightly decreasing the efficiency of formation of the desired analyte ions. So, the matrix, probably due to the presence of lipids, has little influence in electrospray ion formation, and hence, in MS response. MMA is also a potent inhibitor of NOS, although its role may be minor because of its low concentration. In some previous studies, there is a lack of quantification of MMA together with its analogues ADMA and SDMA, but here it is solved because our simultaneous determination offers satisfactory results. Besides, other procedures available for simultaneous MMA determination need solid phase extraction and derivatization (18) increasing costs and difficulty, in contrast with our method. In summary, our method requires easier sample preparation, avoiding solid phase extraction, derivatization or precipitation of proteins by organic solvents which need a subsequent
Table I Statistical Parameters of Arg and Its Methylated Derivatives Quantification by LC-MS/MS Arg Intra-day repeatability % 3.1 RSD Inter-day repeatability 7.4 % RSD Linear interval At least up to 650 mM Calibration range 0–300 (plasma), mM Calibration range 0 –150 (urine), mM Slope (SD) 0.016 (,0.001) Intercept (SD) 0.829 (0.025) 0.9895 – 0.9998 Range of correlation coefficients (R 2) Limit of quantification 1.813 (S/N ¼ 10), mM Limit of detection 0.544 (S/N ¼ 3), mM Accuracy % 2.1 Recovery % 85.4
ADMA
SDMA
MMA
3.6
3.6
11.4
2.5
1.9
12.5
At least up to 500 mM 0–3
At least up to 500 mM 0 –3
At least up to 100 mM 0–1.5
0 –150
0 –150
0 –37.5
0.473 (0.006) 0.683 (0.010) 0.005 (0.001) 0.303 (0.012) 0.650 (0.019) 0.034 (0.001) 0.9876 –0.9995 0.9899 –0.9999 0.9891 –0.9999 0.064
0.066
0.020
0.019
0.020
0.006
1.2 94.3
1.7 94.2
2.4 98.9
Table II Biochemical Profile in Plasma and Urine of Healthy Children (Median and P3 –P97)
Arg MMA ADMA SDMA Arg/ADMA
Plasma (mM)
Urine (mM)
Urine (mmol/mol Crna)
85.5 (52.2 –124.7) 0.11 (0.06 –0.16) 0.61 (0.42 –1.10) 0.49 (0.41 –0.96) 136.50 (79.5 –209.1)
48.1 0.28 89.8 73.1 0.55
4.0 (2.6 – 6.4) 0.03 (0.01 –0.06) 7.6 (4.7 – 11.8) 6.5 (4.3 – 10.2)
(19.1–87.2) (0.08 –0.72) (51.8–162.3) (36.3–125.6) (0.31 –0.69)
a
Crn, creatinine.
evaporation. In particular, we use filters which enable proteins and other compounds to be effectively removed from the matrix by ultrafiltration. In addition, the recycling of filters does not make lose their effectiveness. The results of the method validation showed satisfactory recovery, accuracy and repeatability, as well as a very wide range of linearity and low limits of detection and quantification. Moreover, we observed high repeatability (,7.4% RSD, except for MMA), accuracy (,2.5%) and recovery (.85%), as described in previous studies (22, 23). Over the past 20 years, ADMA has received an increasing interest from both clinical and basic researchers. Baseline levels of ADMA and SDMA have been measured in adult humans (24) due to its cardiovascular implications, but little has been published on these values in healthy children despite their potential relevance (10). Generally, ADMA levels in young people are higher than in healthy adults due to the immaturity of the enzyme systems responsible for the degradation of ADMA, which directly affects dimethylarginine dimethyaminohydrolase activity in children and adolescents. We provide plasma and urinary levels of ADMA in healthy children, similar to those obtained by ELISA (25) and by other MS – MS methods (12, 13). Our report in healthy children gives data of SDMA and MMA levels which can be useful to compare with levels under pathological conditions (26, 27). Moreover, SDMA levels are important in many renal diseases due to their relation with glomerular filtration rate (28), so, most papers about dimethylarginines in children revolves around chronic kidney diseases. In addition, ADMA levels have also been used to assess cardiovascular risk and
endothelial dysfunction in children under oxidative stress conditions such as hypercholesterolemia (26), diabetes (29) or an inborn error of metabolism (25). We conclude that this proposed method is an easy and accurate LC-MS/MS procedure for the simultaneous determination of Arg, ADMA, SDMA and MMA in biological fluids. The repeatability, sensitivity and specificity make this method a valuable tool in clinical and basic research on oxidative stress and endothelial dysfunction in pediatric population.
Acknowledgments This research was partially funded by the Department of Health of Basque Government and Carlos III Health Research Institute.
References 1. Bo¨ger, R.H., Sydow, K., Borlak, J., Thum, T., Lenzen, H., Schubert, B., et al.; LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethionine-dependent methyltransferases; Circulation Research, (2000); 87: 99 –105. 2. Nijveldt, R.J., Teerlink, T., van Guldener, C., Prins, H.A., van Lambalgen, A.A., Stehouwer, C.D., et al.; Handling of asymmetrical dimethylarginine and symmetrical dimethylarginine by the rat kidney under basal conditions and during endotoxaemia; Nephrology Dialysis and Transplantation, (2003); 18: 2542–2550. 3. Davids, M., Teerlink, T.; Plasma concentrations of arginine and asymmetric dimethylarginine do not reflect their intracellular concentrations in peripheral blood mononuclear cells; Metabolism, (2013); 62: 1455–1461. 4. Wilcken, D.E., Sim, A.S., Wang, J., Wang, X.L.; Asymmetric dimethylarginine (ADMA) in vascular, renal and hepatic disease and the regulatory role of L-arginine on its metabolism; Molecular Genetics and Metabolism, (2007); 91: 309– 317. 5. Cardounel, A.J., Cui, H., Samouilov, A., Johnson, W., Kearns, P., Tsai, A.L., et al.; Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function; Journal of Biological Chemistry, (2007); 282: 879– 887. 6. Stu¨hlinger, M.C., Abbasi, F., Chu, J.W., Lamendola, C., McLaughlin, T.L., Cooke, J.P., et al.; Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor; Journal of the American Medical Association, (2002); 287: 1420– 1426. 7. Tarnow, L., Hovind, P., Teerlink, T., Stehouwer, C.D., Parving, H.H.; Elevated plasma asymmetric dimethylarginine as a marker of cardiovascular morbidity in early diabetic nephropathy in type 1 diabetes; Diabetes Care, (2004); 27: 765–769. 8. Lu, T.M., Ding, Y.A., Leu, H.B., Yin, W.H., Sheu, W.H., Chu, K.M.; Effect of rosuvastatin on plasma levels of asymmetric dimethylarginine in patients with hypercholesterolemia; American Journal of Cardiology, (2004); 94: 157–161. 9. Loscalzo, J.; L-arginine and atherothrombosis; Journal of Nutrition, (2004); 134: S2798– S2800. 10. Tain, Y.L., Huang, L.T.; Asymmetric dimethylarginine: Clinical applications in pediatric medicine; Journal of the Formosan Medical Association, (2011); 110: 70 –77. 11. Goonasekera, C.D., Rees, D.D., Woolard, P., Frend, A., Shah, V., Dillon, M.J.; Nitric oxide synthase inhibitors and hypertension in children and adolescents; Journal of Hypertension, (1997); 15: 901– 909. 12. Brooks, E.R., Langman, C.B., Wang, S., Price, H.E., Hodges, A.L., Darling, L., et al.; Methylated arginine derivatives in children and adolescents with chronic kidney disease; Pediatric Nephrology, (2009); 24: 129– 134. 13. Wang, S., Vicente, F.B., Miller, A., Brooks, E.R., Price, H.E., Smith, F.A.; Measurement of arginine derivatives in pediatric patients with
LC-MS/MS Method for Arg Derivatives 791
14.
15.
16.
17.
18.
19.
20.
21.
chronic kidney disease using high-performance liquid chromatography-tandem mass spectrometry; Clinical Chemistry and Laboratory Medicine, (2007); 45: 1305– 1312. Pecchini, P., Malberti, F., Mieth, M., Quinn, R., Tripepi, G., Mallamaci, F., et al.; Measuring asymmetric dimethylarginine (ADMA) in CKD: a comparison between enzyme-linked immunosorbent assay and liquid chromatography-electrospray tandem mass spectrometry; Journal of Nephrology, (2012); 25: 1016– 1022. Blackwell, S., O’Reilly, D.S., Talwar, D.K.; HPLC analysis of asymmetric dimethylarginine (ADMA) and related arginine metabolites in human plasma using a novel non-endogenous internal standard; Clinica Chimica Acta, (2009); 401: 14– 19. Martens-Lobenhoffer, J., Krug, O., Bode-Bo¨ger, S.M.; Determination of arginine and asymmetric dimethylarginine (ADMA) in human plasma by liquid chromatography/mass spectrometry with the isotope dilution technique; Journal of Mass Spectrometry, (2004); 39: 1287–1294. Schwedhelm, E., Maas, R., Tan-Andresen, J., Schulze, F., Riederer, U., Bo¨ger, R.H.; High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma; Journal of Chromatography B, (2007); 851: 211– 219. Davids, M., Swieringa, E., Palm, F., Smith, D.E., Smulders, Y.M., Scheffer, P.G.; Simultaneous determination of asymmetric and symmetric dimethylarginine, L-monomethylarginine, L-arginine, and L-homoarginine in biological samples using stable isotope dilution liquid chromatography tandem mass spectrometry; Journal of Chromatography B, (2012); 900: 38 –47. Martens-Lobenhoffer, J., Bode-Bo¨ger, S.M.; Quantification of Larginine, asymmetric dimethylarginine and symmetric dimethylarginine in human plasma: a step improvement in precision by stable isotope dilution mass spectrometry; Journal of Chromatography B, (2012); 904: 140–143. Weaving, G., Rocks, B.F., Bailey, M.P., Titheradge, M.A.; Arginine and methylated arginines in human plasma and urine measured by tandem mass spectrometry without the need for chromatography or sample derivatisation; Journal of Chromatography B, (2008); 874: 27–32. Martens-Lobenhoffer, J., Schwedhelm, E., Tsikas, D.; Quantification of arginine and its mono- and dimethylated analogs NMMA, ADMA and SDMA in biological fluids by LC-MS/MS: Is LC superfluous? Journal of Chromatography B, (2009); 877: 3261–3266.
792 Andrade et al.
22. Bishop, M.J., Crow, B., Norton, D., Paliakov, E., George, J., Bralley, J.A.; Direct analysis of un-derivatized asymmetric dimethylarginine (ADMA) and L-arginine from plasma using mixed-mode ionexchange liquid chromatography-tandem mass spectrometry; Journal of Chromatography B, (2007); 859: 164–169. 23. Gervasoni, J., Bonelli, F., Zuppi, C., Zappacosta, B., Mordente, A., Calvani, R.; Determination of asymmetric dimethyl arginine in human serum by liquid chromatography-tandem mass spectrometry: clinical application in hypertensive subjects; Clinical Chemistry and Laboratory Medicine, (2011); 49: 2109– 2115. 24. El-Khoury, J.M., Bunch, D.R., Reineks, E., Jackson, R., Steinle, R., Wang, S.; A simple and fast liquid chromatography-tandem mass spectrometry method for measurement of underivatized L-arginine, symmetric dimethylarginine, and asymmetric dimethylarginine and establishment of the reference ranges; Analytical and Bioanalytical Chemistry, (2012); 402: 771– 779. 25. Huemer, M., Simma, B., Mayr, D., Mo¨slinger, D., Mu¨hl, A., Schmid, I., et al.; Free asymmetric dimethylarginine (ADMA) is low in children and adolescents with classical phenylketonuria (PKU); Journal of Inherited Metabolic Disease, (2012); 35: 817– 821. 26. Jehlicˇ ka, P., Stozˇ icky´ , F., Mayer, O., Jr, Varvarˇ ovska´ , J., Racek, J., Trefil, L., et al.; Asymmetric dimethylarginine and the effect of folate substitution in children with familial hypercholesterolemia and diabetes mellitus type 1; Physiological Research, (2009); 58: 179 – 184. 27. Di Gangi, I.M., Chiandetti, L., Gucciardi, A., Moret, V., Naturale, M., Giordano, G.; Simultaneous quantitative determination of N(G), N(G)-dimethyl-L-arginine or asymmetric dimethylarginine and related pathway’s metabolites in biological fluids by ultrahigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry; Analytica Chimica Acta, (2010); 677: 140– 148. 28. Marcovecchio, M.L., Dalton, R.N., Turner, C., Prevost, A.T., Widmer, B., Amin, R., et al.; Symmetric dimethylarginine, an endogenous marker of glomerular filtration rate, and the risk for microalbuminuria in young people with type 1 diabetes; Archives of Disease in Childhood, (2010); 95: 119– 124. 29. Heilman, K., Zilmer, M., Zilmer, K., Kool, P., Tillmann, V.; Elevated plasma adiponectin and decreased plasma homocysteine and asymmetric dimethylarginine in children with type 1 diabetes; Journal of Clinical Laboratory Investigation, (2009); 69: 85– 91.
