Accumulation of symmetric dimethylarginine in hepatorenal syndrome

In patients with cirrhosis, nitric oxide (NO), asymmetric dimethylarginine (ADMA), and possibly symmetric dimethylarginine (SDMA) have been linked to the severity of the disease. We investigated whether plasma levels of dimethylarginines and NO are elevated in patients with hepatorenal syndrome (HRS), compared with patients with cirrhosis without renal failure (no-HRS). Plasma levels of NO, ADMA, SDMA, and l-arginine were measured in 11 patients with HRS, seven patients with no-HRS, and six healthy volunteers. SDMA concentration in HRS was higher than in no-HRS and healthy subjects (1.47 +/- 0.25 vs. 0.38 +/- 0.06 and 0.29 +/- 0.04 microM, respectively; P < 0.05). ADMA and NOx concentrations were higher in HRS and no-HRS patients than in healthy subjects (ADMA, 1.20 +/- 0.26, 1.11 +/- 0.1, and 0.53 +/- 0.06 microM, respectively; P < 0.05; NOx, 94 +/- 9.1, 95.5 +/- 9.54, and 37.67 +/- 4.62 microM, respectively; P < 0.05). In patients with HRS there was a positive correlation between serum creatinine and plasma SDMA (r2 =0.765, P < 0.001) but not between serum creatinine and ADMA or NOx. The results suggest that renal dysfunction is a main determinant of elevated SDMA concentration in HRS. Accumulation of ADMA as a result of impaired hepatic removal may be the causative factor initiating renal vasoconstriction and SDMA retention in the kidney.     

Asymmetric dimethylarginine, oxidative stress, and vascular nitric oxide synthase in essential hypertension

We reported impaired endothelium-derived relaxation factor/nitric oxide (EDRF/NO) responses and constitutive nitric oxide synthase (cNOS) activity in subcutaneous vessels dissected from patients with essential hypertension (n = 9) compared with normal controls (n = 10). We now test the hypothesis that the patients in this study have increased circulating levels of the cNOS inhibitor, asymmetric dimethylarginine (ADMA), or the lipid peroxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (HODE), which is a marker of reactive oxygen species. Patients had significantly (P < 0.001) elevated (means +/- SD) plasma levels of ADMA (P(ADMA), 766 +/- 217 vs. 393 +/- 57 nmol/l) and symmetric dimethylarginine (P(SDMA): 644 +/- 140 vs. 399 +/- 70 nmol/l) but similar levels of L-arginine accompanied by significantly (P < 0.015) increased rates of renal ADMA excretion (21 +/- 9 vs. 14 +/- 5 nmol/mumol creatinine) and decreased rates of renal ADMA clearance (18 +/- 3 vs. 28 +/- 5 ml/min). They had significantly increased plasma levels of HODE (P(HODE): 309 +/- 30 vs. 226 +/- 24 nmol/l) and renal HODE excretion (433 +/- 93 vs. 299 +/- 67 nmol/micromol creatinine). For the combined group of normal and hypertensive subjects, the individual values for plasma levels of ADMA and HODE were both significantly (P < 0.001) and inversely correlated with microvascular EDRF/NO and positively correlated with mean blood pressure. In conclusion, elevated levels of ADMA and oxidative stress in a group of hypertensive patients could contribute to the associated microvascular endothelial dysfunction and elevated blood pressure.     

Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), may be related to reduced biosynthesis of nitric oxide in diseases associated with accelerated atherosclerosis. The closely related compound symmetric dimethylarginine (SDMA) does not inhibit NOS, but may compete with arginine for cellular uptake, thereby limiting substrate availability for NOS. We report on a method for the simultaneous measurement of arginine, ADMA, and SDMA as a tool to gain insight in the role of these compounds in the regulation of NOS activity. Sample cleanup was performed by solid-phase extraction on polymeric cation-exchange columns using monomethylarginine as internal standard. After derivatization with ortho-phthaldialdehyde reagent containing 3-mercaptopropionic acid, analytes were separated by isocratic reversed-phase HPLC with fluorescence detection. The stable derivatives were separated with near baseline resolution. Using a sample volume of 0.2 ml, linear calibration curves were obtained with limits of quantification of 0.08 microM for arginine and 0.01 microM for ADMA and SDMA. Analytical recovery was 98-102%, and interassay CV was better than 3%. Plasma from healthy volunteers (n = 53) contained 94 +/- 26 microM arginine, 0.42 +/- 0.06 microM ADMA, and 0.47 +/- 0.08 microM SDMA. Due to its high precision and sensitivity this method is a valuable tool in research on the metabolism of dimethylated arginines and their role in the regulation of NOS activity.     

High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma

The balance between nitric oxide (NO) and vasoconstrictors like endothelin is essential for vascular tone and endothelial function. L-Arginine is converted to NO and L-citrulline by NO synthase (NOS). Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO formation. ADMA is degraded by dimethylamino dimethylhydrolases (DDAHs), while SDMA is exclusively eliminated by the kidney. In the present article we report a LC-tandem MS method for the simultaneous determination of arginine, ADMA, and SDMA in plasma. This method is designed for high sample throughput of only 20-mul aliquots of human or mouse plasma. The analysis time is reduced to 1.6 min by LC-tandem MS electrospray ionisation (ESI) in the positive mode. The mean plasma levels of l-arginine, ADMA, and SDMA were 74+/-19 (SD), 0.46+/-0.09, and 0.37+/-0.07 microM in healthy humans (n=85), respectively, and 44+/-14, 0.72+/-0.23, and 0.19+/-0.06 microM in C57BL/6 mice. Also, the molar ratios of arginine to ADMA were different in man and mice, i.e. 166+/-50 and 85+/-22, respectively.     

Reduced renal plasma clearance does not explain increased plasma asymmetric dimethylarginine in hypertensive subjects with mild to moderate renal insufficiency

Plasma concentrations of the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) increase already in the early stages of renal insufficiency. There is no agreement as to whether reduced renal plasma clearance (RPCL) contributes to this increase. Therefore, we investigated the relationship between estimated glomerular filtration rate (eGFR), RPCL, and plasma ADMA and SDMA in essential hypertensive patients with mild to moderate renal insufficiency. In 171 patients who underwent renal angiography, we drew blood samples from the aorta and both renal veins and measured mean renal blood flow (MRBF) using the (133)Xe washout technique. RPCL was calculated using arteriovenous concentration differences and MRBF. After correction for potential confounders, reduced eGFR was associated with higher plasma ADMA and SDMA [standardized regression coefficient (β) = -0.22 (95% confidence intervals: -0.41, -0.04) and β = -0.66 (95% confidence intervals: -0.83, -0.49), respectively]. However, eGFR was not independently associated with RPCL of ADMA. Moreover, reduced RPCL of ADMA was not associated with higher plasma ADMA. Contrary to ADMA, reduced eGFR was indeed associated with lower RPCL of SDMA [β = 0.21 (95% confidence intervals: 0.02, 0.40)]. In conclusion, our findings indicate that RPCL of ADMA is independent of renal function in hypertensive patients with mild to moderate renal insufficiency. Unlike the case for SDMA, reduced RPCL of ADMA is of minor importance for the increase in plasma ADMA in these patients, which indicates that increased plasma ADMA in this population is not a direct consequence of the kidneys failing as a plasma ADMA-regulating organ.     

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.     

Asymmetrical (ADMA) and symmetrical dimethylarginine (SDMA) as potential risk factors for cardiovascular and renal outcome in chronic kidney disease – possible candidates for paradoxical epidemiology?

Summary. Background: Asymmetrical dimethylarginine (ADMA) is an inhibitor of nitric-oxide synthase. It has been linked to atherosclerotic risk in the general population as well as in end-stage renal disease patients (ESRD), whereas symmetrical dimethylarginine (SDMA) is thought to be biological inactive. Prospective data concerning the role of both dimethylarginines are rare in patients with chronic kidney disease. Methods: 200 patients with chronic kidney disease (mean age 57.6 ± 13.0 years, 69 female, 131 male); 82 with chronic renal failure (CRF), 81 on maintenance haemodialysis (HD) and 37 renal transplant recipients (RTR) were prospectively followed for 24 months. ADMA and SDMA were measured by HPLC. The relation of plasma levels of ADMA and SDMA together with conventional risk factors for the cardiovascular and renal outcome was investigated with Cox proportional hazards model. Results: Mean serum levels of SDMA were significantly increased in all groups compared to the control group (P ≤ 0.0005), ADMA was increased only in HD and RTR (P ≤ 0.004). Forty-seven cardiovascular events (CVE) occurred during follow-up, 35 patients died, and 39 patients reached ESRD. Multivariate analysis showed diabetes (RR 3.072, P = 0.01), ESRD (RR 11.915, P < 0.0005), elevated CRP levels (RR 3.916, P < 0.0005) and surprisingly a lower ADMA level (RR 0.271, P = 0.008) as independent risk factors for CVE. Serum creatinine (RR 11.378, P = 0.001), haemoglobin (RR 0.710, P = 0.038 for an increment of 1 mmol/l), and SDMA levels (RR 1.633, P = 0.006, per 1 μmol/l increment) were predictors for the progression to ESRD. Conclusions: Data from a heterogeneous group of patients with chronic kidney disease provide evidence that conventional risk factors seem to play a more important role than elevated serum levels of ADMA or SDMA for cardiovascular events. Increasing serum SDMA concentration seems to play an additive role for the renal outcome besides serum creatinine and haemoglobin levels. Whether ADMA might possibly be a candidate for the phenomenon of “paradoxical epidemiology” in chronic kidney disease needs further investigation.     

Direct analysis of un-derivatized asymmetric dimethylarginine (ADMA) and L-arginine from plasma using mixed-mode ion-exchange liquid chromatography-tandem mass spectrometry

A high-throughput analytical method was developed for the measurement of asymmetric dimethylarginine (ADMA) and L-arginine (ARG) from plasma using LC/MS/MS. The sample preparation was simple and only required microfiltration prior to analysis. ADMA and ARG were assayed using mixed-mode ion-exchange chromatography which allowed for the retention of the un-derivatized compounds. The need for chromatographic separation of ADMA from symmetric dimethylarginine (SDMA) was avoided by using an ADMA specific product ion. As a result, the analytical method only required a total run time of 2 min. The method was validated by linearity, with r2>or=0.995 for both compounds, and accuracy, with no more than 7% deviation from the theoretical value. The estimated limit of detection and limit of quantification were suitable for clinical evaluations. The mean values of plasma ADMA and ARG taken from healthy volunteers (n=15) were 0.66+/-0.12 and 87+/-35 microM, respectively; the mean molar ratio of ARG to ADMA was 142+/-81.     

The role of asymmetric dimethylarginine in the regulation of nitric oxide level in rats with acute renal injury

Nitric oxide (NO) is synthesized from arginine (ARG) by NO synthase (NOS). Asymmetric dimethylarginine (ADMA), a competitive inhibitor of NOS, participates in the endogenous regulation of NO synthesis. The main amount of ADMA is enzymatically degraded by dimethylarginine dimethylaminohydrolase (DDAH) widely expressed in renal tissue. The aim of our study was to compare the changes in DDAH activity and ARG synthesis in kidneys, ADMA and ARG concentration in plasma and their urinary excretion under physiological conditions and in acute renal injury (ARI) induced by glycerol in rats. Urinary nitrite/nitrate excretion (NOx) was estimated as an indicator of whole-body NO synthesis. DDAH activity was decreased, ADMA excretion was increased and plasma ADMA did not change in ARI. Plasma ARG concentration, renal ARG synthesis and urinary NOx excretion were decreased. In conclusion, the diminished enzymatic hydrolysis of the NOS inhibitor ADMA and the reduced synthesis of the NOS substrate ARG might affect NO production in ARI.     

High-performance liquid chromatographic assay of asymmetric dimethylarginine,symmetric dimethylarginine,and arginine in human plasma by derivatization with naphthalene-2,3-dicarboxaldehyde

Asymmetric dimethylarginine (ADMA) is an emerging cardiovascular risk factor. Its increased levels have been hypothesized to be a cause of endothelial dysfunction in pathological conditions such as hypertension, dyslipidemia, renal failure, hyperglycemia, and hyperhomocysteinemia. It acts as a potent competitive inhibitor of nitric oxide synthase. Methods using ortho-phthaldialdehyde (OPA) as derivatization reagent are widely performed in HPLC determination of ADMA, but they produce derivatives whose fluorescence rapidly decreases during time. Moreover, these methods do not allow a clear separation of ADMA from its stereoisomer symmetric dimethylarginine (SDMA). Our work describes a new method to determine ADMA, SDMA, and arginine that uses, as derivatizing reagent, naphthalene-2,3-dicarboxaldehyde (NDA). Chromatograms with low background, showing a complete separation of ADMA and SDMA, are obtained. NDA derivatives are considerably more stable than the OPA derivatives. The calibration curves of ADMA and SDMA are linear within the range of 0.01-16.0 microM. Coefficients of variation are less than 1.7% for within day and less then 2.3% for day to day. Absolute mean recoveries from supplemented samples are between 100 and 104%. These characteristics make this method reliable and easily manageable for large routine analyses.     

Chromatographic-mass spectrometric methods for the quantification of L-arginine and its methylated metabolites in biological fluids

L-Arginine (Arg) and its methylated metabolites play a major role in the synthesis of the cell signaling molecule nitric oxide (NO). Arg serves as a substrate for the enzyme NO synthase (NOS), which produces NO, whereas monomethylarginine (L-NMMA) and asymmetric dimethylarginine (ADMA) act as competitive inhibitors of NOS. Symmetric dimethylarginine (SDMA) has virtually no inhibitory effect on NOS activity, but shares the pathway for cell entry and transport with Arg and ADMA. Accurate and reliable quantification of these substances in various biological fluids is essential for scientific research in this field. In this review, chromatographic-mass spectrometric methods for Arg and its methylated metabolites ADMA and SDMA are discussed. Mass spectrometric detection provides an intrinsic higher selectivity than detection by means of UV absorbance or fluorescence. Taking advantage of the high selectivity, approaches involving mass spectrometric detection require less laborious sample preparation and produce reliable results. A consensus emerges that the concentration values in plasma of young healthy volunteers are about 65 microM for Arg, 0.4 microM for ADMA and 0.5 microM for SDMA.     

Analysis of methylarginine metabolism in the cardiovascular system identifies the lung as a major source of ADMA

Protein arginine methylation is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). Three forms of methylarginine have been identified in eukaryotes: monomethylarginine (l-NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA), all characterized by methylation of one or both guanidine nitrogen atoms of arginine. l-NMMA and ADMA, but not SDMA, are competitive inhibitors of all nitric oxide synthase isoforms. SDMA is eliminated almost entirely by renal excretion, whereas l-NMMA and ADMA are further metabolized by dimethylarginine dimethylaminohydrolase (DDAH). To explore the interplay between methylarginine synthesis and degradation in vivo, we determined PRMT expression and DDAH activity in mouse lung, heart, liver, and kidney homogenates. In addition, we employed HPLC-based quantification of protein-incorporated and free methylarginine, combined with immunoblotting for the assessment of tissue-specific patterns of arginine methylation. The salient findings of the present investigation can be summarized as follows: 1) pulmonary expression of type I PRMTs was correlated with enhanced protein arginine methylation; 2) pulmonary ADMA degradation was undertaken by DDAH1; 3) bronchoalveolar lavage fluid and serum exhibited almost identical ADMA/SDMA ratios, and 4) kidney and liver provide complementary routes for clearance and metabolic conversion of circulating ADMA. Together, these observations suggest that methylarginine metabolism by the pulmonary system significantly contributes to circulating ADMA and SDMA levels.     

Comparison of HPLC method and commercial ELISA assay for asymmetric dimethylarginine (ADMA) determination in human serum

The performance of a new ELISA assay kit (DLD Diagnostika GmbH, Hamburg, Germany) for the determination of asymmetric dimethylarginine (ADMA) was evaluated against a reversed phase HPLC method. ADMA concentrations of 55 serum samples were measured with both methods. The intra-assay CV for ADMA-ELISA was 19% (n=10). Inter-assay CVs for ADMA-ELISA were 9% for kit control 1 (0.410+/-0.037 microM) and 14% for kit control 2 (1.174+/-0.165 microM). The intra- and inter-assay CVs for HPLC assay for ADMA were 2.5% (0.586+/-0.015 microM) and 4.2% (0.664+/-0.028 microM), respectively. There was no correlation between these two methods (R(2)=0.0972). The effect of storage conditions of the samples on ADMA concentrations was investigated by HPLC. ADMA concentration was stable after four freezing and thawing cycles. Overall, the HPLC method offered better sensitivity, selectivity and, very importantly, simultaneous determination of ADMA, SDMA, l-homoarginine and l-arginine.     

Simultaneous gas chromatography-tandem mass spectrometry quantification of symmetric and asymmetric dimethylarginine in human urine

Previously, we demonstrated the utility of a gas chromatography–tandem mass spectrometry (GC–MS/MS) method for the quantitative determination of asymmetric dimethylarginine (ADMA) in biological samples. Here we report the extension of this method to symmetric dimethylarginine (SDMA) in human urine. SDMA and ADMA were simultaneously quantitated in urine by using their in situ prepared trideuteromethyl esters as internal standards. The GC–MS/MS method was validated for SDMA and ADMA in spot urine samples of 19 healthy adults. In these samples, the creatinine-corrected excretion rate was 3.23 ± 0.63 μmol/mmol for SDMA and 3.14 ± 0.98 μmol/mmol for ADMA.     

Free fatty acids do not acutely increase asymmetrical dimethylarginine concentrations.

Concentrations of asymmetrical dimethylarginine (ADMA) and free fatty acids (FFAs) are elevated in insulin resistance which is associated with impaired vascular function. We hypothesized that FFAs could alter vascular tone by affecting ADMA concentrations. Plasma FFA levels were increased in seventeen healthy male volunteers by Intralipid/heparin infusion; hemodynamic and biochemical parameters were measured after 90 minutes. Plasma collected before and during Intralipid/heparin or equivalent synthetic FFAs was incubated with human umbilical vein endothelial cells (HUVECs) in vitro. Intralipid/heparin infusion resulted in an approximately seven-fold increase in plasma FFA levels to 1861 +/- 139 micromol/l, which was paralleled by increased systemic blood pressure and forearm blood flow. Intralipid/heparin did not affect ADMA (baseline mean 0.59 [95 % confidence interval [CI]: 0.54; 0.64] and 0.56 [CI: 0.51; 0.59] after 90 minutes), but slightly decreased SDMA (from 0.76, [CI: 0.70; 0.83] to 0.71 [CI: 0.64; 0.74], p < 0.05), and had no effect on ADMA/SDMA ratio. There was no correlation between ADMA and FFA concentrations or forearm blood flow. Incubation of HUVECs with FFA-rich plasma or synthetic FFAs induced an ADMA release after 24 hours, but not after 90 minutes. Acutely increased FFA levels caused hemodynamic effects but did not affect ADMA. Prolonged elevation of FFA levels might influence vascular function by increasing ADMA levels.     

Increased asymmetric dimethylarginine (ADMA) dimethylaminohydrolase (DDAH) activity in childhood hypercholesterolemia type II

Asymmetric dimethylarginine (ADMA) systemic concentrations are elevated in hypercholesterolemic adults and contribute to nitric oxide (NO) dependent endothelial dysfunction. Decreased activity of the key ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase (DDAH) may be involved. Yet, the ADMA/DDAH/NO pathway has not been investigated in childhood hypercholesterolemia. We studied 64 children with hypercholesterolemia type II (HCh-II) and 54 normocholesterolemic (NCh) children (mean ± SD; age, years: 11.1 ± 3.5 vs. 11.9 ± 4.6). Plasma and urine ADMA was measured by GC-MS/MS. Dimethylamine (DMA), the ADMA metabolite, creatinine, nitrite and nitrate in urine were measured by GC-MS. The DMA/ADMA molar ratio in urine was calculated to estimate whole body DDAH activity. ADMA plasma concentration (mean ± SD; nM: 571 ± 85 vs. 542 ± 110, P = 0.17) and ADMA urinary excretion rate (mean ± SD: 7.1 ± 2 versus 7.2 ± 3 μmol/mmol creatinine, P = 0.6) were similar in HCh-II and NCh children. Both DMA excretion rate [median (25th-75th percentile): 56.3 (46.4-109.1) vs. 45.2 (22.2-65.5) μmol/mmol creatinine, P = 0.0004] and DMA/ADMA molar ratio [median (25th-75th percentile): 9.2 (6.0-16.3) vs. 5.4 (3.8-9.4), P = 0.0004] were slightly but statistically significantly increased in HCh-II children compared to NCh children. Plasma and urinary nitrite and nitrate were similar in both groups. In HCh-II whole body DDAH activity is elevated as compared to NCh. HCh-II children treated with drugs for hypercholesterolemia had lower plasma ADMA levels than untreated HCh-II or NCh children, presumably via increased DDAH activity. Differences between treated and untreated HCh-II children were not due to differences in age. In conclusion, HCh-II children do not have elevated ADMA plasma levels, largely due to an apparent increase in DDAH activity. While this would tend to limit development of endothelial dysfunction, it is not clear whether this might be medication-induced or represent a primary change in HCh-II children.     

HPLC analysis of asymmetric dimethylarginine,symmetric dimethylarginine,homoarginine and arginine in small plasma volumes using a Gemini-NX column at high pH

There is increasing recognition of the clinical importance of endogenous nitric oxide synthase inhibitors in critical illness. This has highlighted the need for an accurate high performance liquid chromatography (HPLC) method for detection of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in small volumes of blood. Here, the validation of an accurate, precise HPLC method for the determination of ADMA, SDMA, homoarginine and arginine concentrations in plasma is described. Solid phase extraction is followed by derivatisation with AccQ-Fluor™ and reversed phase separation on a Gemini-NX column at pH 9. Simultaneous detection by both UV–vis and fluorescence detectors affords extra validation. This solid phase extraction method gives absolute recoveries of more than 85% for ADMA and SDMA and relative recoveries of 102% for ADMA and 101% for SDMA. The intra-assay relative standard deviations are 2.1% and 2.3% for ADMA and SDMA, respectively, with inter-assay relative standard deviations of 2.7% and 3.1%, respectively. Advantages of this method include improved recovery of all analytes using isopropanol in the solid phase extraction; sharp, well-resolved chromatographic peaks using a high pH mobile phase; a non-endogenous internal standard, n-propyl l-arginine; and accurate and precise determination of methylated arginine concentrations from only 100 μL of plasma.     

HPLC analysis of ADMA and other methylated L-arginine analogs in biological fluids

Post-translational methylation of arginine residues in proteins leads to generation of N(G)-monomethylarginine (MMA) and both symmetric and asymmetric dimethylarginine (SDMA and ADMA), that are released into the cytosol upon proteolysis. Both MMA and ADMA are inhibitors of nitric oxide synthase and especially elevated levels of ADMA are associated with endothelial dysfunction and cardiovascular disease. Plasma concentrations of ADMA and SDMA are very low, typically between 0.3 and 0.8 microM, making their quantification by HPLC an analytical challenge. Sample preparation usually involves a cleanup step by solid-phase extraction on cation-exchange columns followed by derivatization of amino acids into fluorescent adducts. Because ADMA and SDMA concentrations in healthy subjects show a very narrow distribution, with a between-subject variability of 13% for ADMA and 19% for SDMA, very low imprecision is an essential assay feature. Procedures for sample cleanup, derivatization, and chromatographic separation of arginine and its methylated analogs are the main topics of this review. In addition, important aspects of method validation, pre-analytical factors, and reference values are discussed.     

Contribution of whole blood to the control of plasma asymmetrical dimethylarginine

The endogenous nitric oxide (NO) synthase (NOS) inhibitor asymmetrical dimethylarginine (ADMA) is elevated in many patients and may contribute to the initiation and progression of their disease. While some mechanistic pathways have been identified, tissue-specific contributions to ADMA control remain unclear. We sought to determine if whole blood (WB) could participate in ADMA control ex vivo. Anesthetized male Sprague-Dawley rats underwent exsanguinations, and WB preparations were incubated at 37 degrees C for 5 h. ADMA and symmetrical dimethylarginine were analyzed by high-pressure liquid chromatography. Incubation of lysed red blood cell (RBC) supernatant yielded a significant decrease in ADMA that was blocked by 4124W, a synthetic inhibitor of dimethylarginine dimethylaminohydrolase, the only reported enzyme to hydrolyze ADMA. Hydrolysis of ADMA was diminished by addition of physiologically relevant concentrations of zinc (i.e., 20 microM). Conversely, when rat WB or WB supernatant was incubated at 37 degrees C, it liberated quantities of free ADMA (1-2 microM) that in vivo would likely have pathological consequences. Addition of arginine methyltransferase inhibitors to these incubations did not reduce ADMA release, indicating no dominant role for active protein methylation during these incubations. This ADMA liberation was significantly reduced by addition of protease inhibitors, indicating a dependence on peptide bond hydrolysis. Total ADMA (protein incorporated plus free) was determined by acid hydrolysis and found to be 43.18 +/- 4.79 microM in WB with approximately 95% of this in RBCs. These ex vivo data demonstrate the potential of blood to control the NO-NOS system by modulating free ADMA.     

A stable-isotope based technique for the determination of dimethylarginine dimethylaminohydrolase (DDAH) activity in mouse tissue

The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for the hydrolysis of asymmetric dimethylarginine (ADMA) to L-citrulline and dimethylamine. DDAH is currently investigated as a promising target for therapeutic interventions, as ADMA has been found to be elevated in cardiovascular disease. In many tissues continuous endogenous formation of ADMA and L-citrulline poses considerable limitations to the presently used assays for DDAH activity, which are commonly based on the measurement of ADMA or L-citrulline. We therefore developed a stable-isotope-based assay suitable for 96-well plates to determine DDAH activity. Using deuterium-labeled ADMA ([(2)H(6)]-ADMA) as substrate and double stable-isotope labeled ADMA ([(13)C(5)-(2)H(6)]-ADMA) as internal standard we were able to simultaneously determine formation and metabolism of ADMA in renal and liver tissue of mice by LC-tandem MS. Endogenous formation of ADMA could largely be abolished by addition of protease inhibitors, while metabolism of [(2)H(6)]-ADMA was not significantly altered. The intra-assay coefficient of variation for the determination of endogenous ADMA and [(2)H(6)]-ADMA was 2.4% and 4.8% in renal and liver tissue, respectively. The inter-assay coefficient of variation for DDAH activity based on degradation of [(2)H(6)]-ADMA determined in separate samples from the same organs was determined to be 8.9% and 10% for mouse kidney and liver, respectively. The present DDAH activity assay allows for the first time to simultaneously determine DDAH activity and endogenous formation of ADMA, SDMA, and L-arginine in tissue.