Determination of dimethylated arginines in human plasma by high-performance liquid chromatography

doi:10.1016/S0378-4347(96)00525-7

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 692, Issue 2, 9 May 1997, Pages 257-262

https://doi.org/10.1016/S0378-4347(96)00525-7 Get rights and content

Abstract

Using high-performance liquid chromatography (HPLC) with multigradient elution, $N^{G},N^{G} - dimethyl- l -arginine$ (asymmetric-DMA, ADMA) and $N^{G},N^{G} - dimethyl- l -arginine$ (symmetric-DMA, SDMA) can be separated from human plasma samples. The dimethylarginine compounds in plasma, after extraction with a cation-exchange column, are converted to fluorescent derivatives with o-phthaldialdehyde (OPA) in an alkaline medium and the derivatives are separated simultaneously within 50 min on a reversed-phase column (Ultracarb 3 ODS(20)). The recoveries of ADMA and SDMA are over 80% and the method permits quantitative determination of dimethylated arginines at concentrations as low as 0.1 μmol/l in human plasma.

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Cited by (109)

L-arginine biosensors: A comprehensive review
2017, Biochemistry and Biophysics Reports
Arginine has been considered as the most potent nutraceutics discovered ever, due to its powerful healing property, and it's been known to scientists as the Miracle Molecule. Arginine detection in fermented food products is necessary because, high level of arginine in foods forms ethyl carbamate (EC) during the fermentation process. Therefore, L-arginine detection in fermented food products is very important as a control measure for quality of fermented foods, food supplements and beverages including wine. In clinical analysis arginine detection is important due to their enormous inherent versatility in various metabolic pathways, topmost in the synthesis of Nitric oxide (NO) and tumor growth. A number of methods are being used for arginine detection, but biosensors technique holds prime position due to rapid response, high sensitivity and high specificity. However, there are many problems still to be addressed, including selectivity, real time analysis and interference of urea presence in the sample. In the present review we aim to emphasize the significant role of arginine in human physiology and foods. A small attempt has been made to discuss the various techniques used for development of arginine biosensor and how these techniques affect their performance. The choice of transducers for arginine biosensor ranges from optical, pH sensing, ammonia gas sensing, ammonium ion-selective, conductometric and amperometric electrodes because ammonia is formed as a final product.
Simultaneous determination of homocysteine and asymmetric dimethylarginine in human urine by liquid chromatography-tandem mass spectrometry
2013, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
The various analytical methods for HCY determinations were enzyme and immunoassay [11], HPLC with ultraviolet (UV) [12], fluorescence [13], or electrochemical detection (ECD) [14], capillary electrophoresis with fluorescence detection [15], gas chromatography–mass spectrometry (GC–MS) [16,17] and liquid chromatography–tandem mass spectrometry (LC–MS/MS) [18–20]. Quantification of ADMA was made by several methods, ELISA [21], HPLC [22], LC coupled with fluorescence [23], capillary electrophoresis [24], gas chromatography–mass spectrometry (GC–MS) [25] and liquid chromatography–tandem mass spectrometry (LC–MS/MS) [26–28]. The existing analytical techniques such as immunoassays need expensive reagents and GC/MS analysis requires tedious time-consuming extraction and derivatization steps.
Increased circulating concentrations of homocysteine (HCY) and asymmetric dimethylarginine (ADMA) are associated with vascular disease and vascular risk factors. HCY has been shown to inhibit the activity of endothelial dimethylaminohydrolase (DDAH), causing the accumulation of ADMA and the inhibition of nitric oxide synthesis. The concentrations of HCY and ADMA in biological fluids are used in the clinical diagnosis of cardiovascular diseases and this necessitates the development of a rapid and sensitive method for simultaneous determination of HCY and ADMA. A rapid, simple and sensitive method for simultaneous determination of HCY and ADMA by liquid chromatography–tandem mass spectrometry (LC–MS/MS) coupled with electro spray ionization (ESI) in human urine was reported here. The methodology designed here was used to estimate these molecules in urine samples collected from patients reported to Cardiology Department of our hospital. Chromatographic separation was performed on Atlantis HILIC silica (100 mm × 2.1 mm, 5 μm, Waters). Positive multiple reactions monitoring (MRM) mode was chosen for quantification of each analyte and cystamine dihydrochloride (CYA) was used as the internal standard (IS) for the assay. The intra-assay precision and accuracy were in the range of 2.4–4.8 and −1.8% to 3.1%, respectively. The inter-assay precision and accuracy were in the range of 3.0–4.2% and −1.2% to 3.2%, respectively. The recoveries were between 94.9% and 101.4%. Our approach is simple, rapid and could be extended to routine urine assay.
Simultaneous bioanalysis of l-arginine, l-citrulline, and dimethylarginines by LC-MS/MS
2011, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
l-Arginine (ARG) is converted to nitric oxide (NO) and l-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography–mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (¹⁵N₄-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples.
Concentrations of unlabeled ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. ¹³C₆-ARG, D₄-CIT and D₇-ADMA were used as internal standards for ARG and ¹⁵N₄-ARG, CIT, and dimethylarginines, respectively.
The calibration curves of ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to ¹⁵N₄-ARG, which allowed the observation of generation of ¹⁵N₃-CIT and ¹⁵N₃-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats.
An LC–MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.
Simultaneous quantitative determination of N<sup>G</sup>,N<sup>G</sup>-dimethyl-l-arginine or asymmetric dimethylarginine and related pathway's metabolites in biological fluids by ultrahigh-performance liquid chromatography/electrospray ionization-tandem mass spectrometry
2010, Analytica Chimica Acta
Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide (NO) formation inhibitor, has emerged as a promising biomarker of NO-associated endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. The interest in potentially fundamental role of this metabolite, in basic and clinical research, led to the development of numerous analytical methods for the quantitative determination of ADMA and dimethylarginines in biological systems, notably plasma, serum and urine.
The aim of this work was to present a simple, fast and accurate UPLC–tandem-MS-based method for the simultaneous determination and quantification of arginine, ADMA, SDMA, NMMA, homo-arginine and citrulline. This method is designed for high sample throughput of only 10 μL of human plasma, serum or urine.
The analysis time is reduced to 1.9 min by an ultrahigh-performance liquid chromatography run coupled with electrospray ionization (ESI) in the positive mode tandem mass spectrometry detection.
The method was validated in plasma, serum and urine. Correlation coefficients (r²) of the calibration curves in all matrices considered ranged from 0.9810 to 0.9993. Inter- and intra-assay precision, accuracy, recovery and carry-over were evaluated for validation. The LOD was 0.01 μM for all compounds in water, plasma and serum and 0.1 μM in urine. The LOQ was 0.05 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit in water, plasma and serum; while in urine was 0.1 μM for ADMA, SDMA, NMMA and H-Arg and 0.5 μM for Arg and Cit.
The precision was ranged from 1% to 15% expressed as CV% and the accuracy (bias %) was <±7% for all added concentrations with the exception of NMMA (−10%).
ADMA mean plasma levels, measured in healthy adults and newborns, were in accord with literature data published: (M ± SD) 0.56 ± 0.10 μM and 0.84 ± 0.21 μM, respectively, showing that ADMA levels in plasma decreased with age. In serum we have similar data (0.54 ± 0.18 μM and 1.14 ± 0.36 μM), while in neonatal urine ADMA was 11.98 ± 7.13 μmol mmol⁻¹ creatinine.
Data from calibration curves and method validation reveal that the method is accurate and precise. The fast run time, the feasibility of high sample throughput and the small amount of sample required make this method very suitable for routine analysis in the clinical setting.
HPLC analysis of asymmetric dimethylarginine (ADMA) and related arginine metabolites in human plasma using a novel non-endogenous internal standard
2009, Clinica Chimica Acta
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthesis which has been implicated in the endothelial dysfunction. Methods for ADMA measurement often yield widely differing results, and few methods simultaneously offer satisfactory accuracy and precision. We describe a fully validated HPLC method for analysis of arginine and its methylated derivatives in human plasma using a novel internal standard.
Arginine and related metabolites are extracted from plasma by solid phase extraction (SPE), derivatised with ortho-phthaldialdehyde and separated by isocratic reverse phase chromatography. Monoethylarginine (MEA), which is not endogenously present in human plasma was used as internal standard. SPE and chromatographic procedures are optimised and recovery, precision, linearity and sensitivity of the assay established. The suitability and performance of MEA is compared with that of monomethylarginine (MMA), the internal standard most commonly used in HPLC methods.
SPE yields high and reproducible recoveries (> 90%). The analytes of interest are chromatographically well resolved. The method has high sensitivity (LOD, 0.01 μmol/L for arginine and 0.001 μmol/L for ADMA, SDMA and homoarginine) and good precision (CV, 2.5% for ADMA). The data obtained with the internal standards MEA and MMA is comparable in terms of assay precision and population reference intervals.
We describe an optimised isocratic HPLC method for the simultaneous measurement of arginine and related metabolites in plasma which exhibits satisfactory precision and is suitable for routine use. Its main advantage over other published HPLC methods is the use of the novel internal standard, MEA, which unlike other commonly used internal standards is not inherent in human plasma.
Effect of early L-arginine therapy on intrauterine growth restriction in preeclampsia. A randomized controlled trial in Latin-American women
2009, Progresos en Obstetricia y Ginecologia
Evaluar el beneficio de la administración temprana de L-arginina en la preeclampsia en el riesgo relativo del crecimiento fetal.
Se aleatorizó a 100 mujeres con preeclampsia a recibir L-arginina o placebo hasta el día del parto. Se comparó a 96 infantes nacidos de gestantes preeclámpticas (50 con tratamiento y 46 sin tratamiento) y con 50 infantes nacidos de gestantes sanas para evaluar el riesgo relativo de crecimiento intrauterino restringido (CIR) y el efecto de L-arginina en éste. El peso al nacer relacionado con la edad gestacional se comparó con curvas de crecimiento respectivas. Los infantes más pequeños del percentil 10 fueron clasificados como CIR. Se utilizaron las pruebas de la U de Mann-Whitney, ANOVA de la χ² para evaluar las diferencias estadísticas significativas (p < 0,05) entre los grupos.
No hubo diferencias entre los grupos con preeclampsia antes del estudio. La preeclampsia se asoció en el 21% de los casos al CIR. El riesgo de CIR fue 5 veces más alto en infantes nacidos de preeclámpticas sin terapia de L-arginina comparado con los controles (riesgo relativo [RR] = 5,0; intervalo de confianza [IC], 1,5-16,2) y 2 veces más alto en infantes nacidos de preeclámpticas en tratamiento con L-arginina (RR = 2,0; IC del 95%, 1,9-7,6). Los estudios del perfil biofísico fetal y la puntuación en la prueba de Apgar al nacer demostraron mejoras estadísticamente significativas con el uso de L-arginina en la preeclampsia (p < 0,05).
El crecimiento fetal mejora significativamente con la terapia de L-arginina administrada de forma temprana en gestantes con preeclampsia.
To assess the benefit of early L-arginine administration in preeclampsia on the relative risk to fetal growth.
One-hundred women with preeclampsia were randomized to receive either L-arginine or placebo until the day of delivery. To evaluate the relative risk of intrauterine growth restriction (IUGR) and the effect of L-arginine on this process, 96 live singleton infants of women with preeclampsia (50 with treatment and 46 without treatment) were compared; these infants were also compared with a further 50 control infants of healthy women. Gestational age-related birth weight was compared using standard growth curves. Infants smaller than the 10^th percentile were classified as IUGR. The Mann-Witney U-test, ANOVA, and chi-square test were used to evaluate statistically significant differences (P<.05) between the groups.
No significant differences were found between the groups with preeclampsia before randomization. Preeclampsia was associated with a 21% reduction in birth weight. The risk of IUGR was five times higher in infants born after preeclampsia without L-arginine therapy than in control pregnancies (RR = 5.0; 95%IC: 1.5-16.2) and was two times higher in infants born after preeclampsia with L-arginine therapy (RR = 2.0; 95% CI: 1.9-7.6). The fetal biophysical profile and Apgar score were significantly more favorable in the L-arginine group (P<.05).
Fetal growth markedly improves with early L-arginine therapy in women with preeclampsia.

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Regular paperDetermination of dimethylated arginines in human plasma by high-performance liquid chromatography

Abstract

Hypertension

Nature

Biochem. Biophys. Res. Commun.

Pharmacol. Rev.

Lancet

Biochem. Chromatogr.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Chromatogr.

Biochem. Biophys. Res. Commun.

Regular paper
Determination of dimethylated arginines in human plasma by high-performance liquid chromatography