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Technical Briefs |
Medical Laboratory Bremen, D-28357 Bremen, Germany
aauthor for correspondence: fax 49-421-20727107, e-mail nikolaus.kuehn-velten{at}mlhb.de
Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are derived from posttranslational methylation of arginine residues in proteins and after proteolytic release. Whereas protein methylation appears to be important in RNA binding and protein sorting, ADMA evolved as a competitive inhibitor of nitric oxide synthase at the arginine-binding site (1)(2). At a constant arginine supply, increased ADMA concentrations decrease NO formation and impair vascular homeostasis by multiple mechanisms (3). Consequently, arginine supplementation improves small-vessel function (4). Inactivation of ADMA occurs via dimethylarginine dimethylaminohydrolase, and it has been hypothesized that this enzyme might be inhibited by homocysteine. The role of SDMA is less clear; it probably is an inhibitor of arginine transport across the cell membrane (5).
From the clinical perspective, ADMA has been established as a possible risk factor for the development of endothelial dysfunction and cardiovascular disease (6). Its plasma concentrations also predict the probability of restenosis after stent implantation. The possible contribution of high-resistance placental circulation, endothelial dysfunction, and increased ADMA concentrations to the development of preeclampsia has been investigated (7). Recently, a significant decrease in circulating ADMA was reported during hormone replacement therapy in postmenopausal women (8). Plasma concentrations of ADMA are also influenced by insulin sensitizers such as rosiglitazone, indicating a possible link of NOS activity to the metabolic syndrome (9). Thus, there is considerable demand for specific, sensitive, and rapid methods for ADMA determination in biological fluids.
Previously, several methods for ADMA and SDMA determination have been described, including gas chromatography–tandem mass spectrometry (MS/MS) (10), liquid chromatography with fluorescence detection after derivatization (5), liquid chromatography–MS/MS (11), and capillary electrophoresis–laser-induced fluorescence (12). Without derivatization, chromatographic separation is necessary even with MS/MS detection because ADMA and SDMA generate the same parent and daughter ions. Reported concentrations of ADMA cover a wide range from 
0.1 µmol/L (11)(13) to 1.5 µmol/L (9)(14). Here we describe a novel HPLC-MS/MS approach that allows a short injection interval of 6 min, attributable to separation on a short hypercarb column, and requires minimal specimen preparation.
Fresh plasma samples were obtained from EDTA-blood from apparently healthy volunteers who had given informed consent (chiefs and staff of the laboratory; 21 women and 21 men; mean age, 43.4 years; median age, 42.5 years; range, 21–73 years). To 100 µL of plasma, we added 200 µL of precipitating reagent (acetonitrile–methanol, 9+1 by volume) containing L-leucine-5,5,5-d3 (Aldrich) as the internal standard (750 nmol/L); we vortex-mixed the samples and allowed them to equilibrate for 10 min at ambient temperature. Alternatively, we used U-13C6,U-15N4-L-arginine (Cambridge Isotope Laboratories Inc.) as an internal standard (2.7 µmol/L). Tubes were then centrifuged at 12 000g for 3 min. We mixed 100 µL of the supernatant with 300 µL of distilled water and injected 10 µL of this diluted mixture into the HPLC system (Agilent 1100LC binary pump and CTC-Pal autosampler). Chromatographic separation of ADMA and SDMA was achieved by the polar retention effect of graphitic carbon on a hypercarb column [50 x 4.6 mm (i.d.); 5-µm particle size; Thermo Electron], with acetonitrile and aqueous trifluoroacetic acid (1 mL of trifluoroacetic acid/L of water) as the mobile phase with a linear gradient elution from 5% to 20% acetonitrile in aqueous trifluoroacetic acid (1 mL of trifluoroacetic acid/L of water) within 3 min at a flow rate of 0.8 mL/min. The injection interval was 6 min.
Selected positive ion fragments, generated after electrospray ionization, were detected by the API 4000 triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex). Ion suppression (15), as checked by comparing peak areas of aqueous calibrator solutions without matrix and calibrator added to plasma matrix, was minimized by use of supernatant dilution and gradient elution. Trifluoroacetic acid is known to cause ion suppression, but it was necessary for ADMA/SDMA separation. The turbo ion spray temperature was 500 °C, and the ionization voltage was set to 3000 V. The flows of the spray, curtain, and collision gases (nitrogen) were set to 40, 10, and 4 units, respectively. The instrument was set in multiple-reaction monitoring mode, with a dwell time of 100 ms. The precursor and product ions for ADMA and SDMA were at m/z 203.0 and 70.1 (quantifier) and m/z 116.0 (qualifier), where the m/z 70.1 ion corresponds to a pyrrolinium ion as a cyclization product (2). The declustering potential and collision energy were optimized to 66 and 31 V, respectively. The precursor and product ions of the leucine internal standard were at m/z 135.0 and 89.1, with declustering potential and collision energy of 31 and 15 V. The retention times of ADMA, SDMA, and the leucine internal standard were 3.2, 3.5, and 1.5 min, respectively (Fig. 1
).
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Comparison of the two isotope-labeled internal standards revealed an advantage of leucine because the ratios of ADMA and SDMA concentrations obtained with standard addition to either samples or water were greater for labeled leucine (0.88 for ADMA and 0.92 for SDMA, respectively) than for the labeled arginine (0.80 for ADMA and 0.82 for SDMA, respectively), indicating that matrix effects become slightly more important with the latter substance.
Further validation of this novel method was therefore performed on the basis of the leucine calibrator. The limits of detection, defined as a signal-to-noise ratio of 3, were 17 nmol/L for ADMA and 14 nmol/L for SDMA; the lower limit of quantification for both compounds was 50 nmol/L; the analytically measurable range (confirmed linearity) was 50 nmol/L to 5 µmol/L; and the intraassay CVs (relative SD) in native plasma (n = 10) were 6.0% for ADMA and 6.9% for SDMA. The observed recoveries (n = 25 over a period of 6 months) in fresh plasma to which 1 µmol/L each of ADMA and SDMA had been added were 97.6% (range, 89.5–113.0%) for ADMA and 97.2% (range, 88.5–116.5%) for SDMA, corresponding to mean recoveries (value for plasma with added analyte minus value of plasma without added analyte) of 962 and 958 nmol/L, with SDs of 70 and 71 nmol/L and relative SDs of 7.3% and 7.4% for ADMA and SDMA, respectively. Recovery experiments were also conducted in a single-blind approach with similar outcomes. The addition of SDMA (3.2 µmol/L) or arginine (160 µmol/L) to native plasma samples did not affect ADMA measurements. Although these studies were performed with the API 4000 MS system, it appears possible that systems operating with a 5- to 10-fold lower sensitivity can also be used for detection.
A comparison of the concentration distributions of ADMA and SDMA in plasma samples from apparently healthy volunteers revealed that both compounds were nearly gaussian distributed, as indicated by overlapping median and mean confidence intervals and low distribution skewness. Although the distribution patterns were similar for both compounds, the statistical spread of SDMA values was greater than that of ADMA values (Table 1
).
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In conclusion, our novel method circumvents the uncertainties of derivatization procedures. It allows rapid, sensitive, and specific ADMA measurements and appears to be suitable for experimental and clinical studies on the regulation and dysregulation of NO synthesis.
References
The following articles in journals at HighWire Press have cited this article:
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  J. Martens-Lobenhoffer and S. M. Bode-Boger Fast and Efficient Determination of Arginine, Symmetric Dimethylarginine, and Asymmetric Dimethylarginine in Biological Fluids by Hydrophilic-Interaction Liquid Chromatography-Electrospray Tandem Mass Spectrometry Clin. Chem., March 1, 2006; 52(3): 488 - 493. [Abstract] [Full Text] [PDF]
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E. Schwedhelm, J. Tan-Andresen, R. Maas, U. Riederer, F. Schulze, and R. H. Boger Liquid Chromatography-Tandem Mass Spectrometry Method for the Analysis of Asymmetric Dimethylarginine in Human Plasma Clin. Chem., July 1, 2005; 51(7): 1268 - 1271. [Full Text] [PDF]
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 E. Schwedhelm Quantification of ADMA: analytical approaches Vascular Medicine, July 1, 2005; 10(1_suppl): S89 - S95. [Abstract] [PDF]
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E. Schwedhelm Quantification of ADMA: analytical approaches Vascular Medicine, May 1, 2005; 10(2_suppl): S89 - S95. [Abstract] [PDF]
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