Development of an Enzyme-Linked Immunosorbent Assay System for the Determination of Asymmetric Dimethylarginine Using a Specific Monoclonal Antibody

We produced a monoclonal antibody (mAb) against N ^G,N ^G-dimethyl-L-arginine (asymmetric dimethylarginine: ADMA), an endogenous competitive inhibitor of nitric oxide synthase (NOS), and developed an enzyme-linked immunosorbent assay (ELISA). The competitive ELISA method using the mAb determined 5 nM–100 nM ADMA, and ADMA levels in human plasma and urine were found to be 0.78 μM and 51.3 μmol/g of creatinine respectively.     

Detection and quantification of α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid: a metabolite of asymmetric dimethylarginine

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate L-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMGV. D(6)-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.     

Asymmetric dimethylarginine, an endogenous NOS inhibitor, is actively metabolized in rat erythrocytes

N(G), N(G)-Dimethyl-L-arginine (asymmetric dimethylarginine: ADMA) is an endogenous competitive inhibitor of nitric oxide synthase (NOS). Plasma ADMA concentrations have been reported to increase in connection with diseases associated with an impaired endothelial L-arginine/NO pathway. In this study, we investigated the metabolism of ADMA in circulating blood cell populations to elucidate the regulatory mechanism of elevation of plasma ADMA, a novel risk factor for cardiovascular disease. We found by RT-PCR and Western blot analyses that protein arginine methyltransferase (PRMT)1 and dimethylarginine dimethylaminohydrolase (DDAH)-1, responsible for the biosynthesis and degradation of ADMA respectively, are expressed in erythrocytes (ECs), leukocytes, and platelets. We also identified a major ADMA-containing protein in ECs as catalase, confirmed by GST-pull down assay to bind to PRMT1 in vitro. This is the first report that the ADMA-metabolizing system, including the arginine methylation of proteins and the breakdown of free ADMA, occurs in circulating blood cell-populations, and that catalase in ECs might be a potential protein targeted by PRMT1.     

Determination of NG,NG-dimethyl-L-arginine in rat plasma and dimethylarginine dimethylaminohydrolase activity in rat kidney using a monolithic silica column

A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of N(G),N(G)-dimethyl-L-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) - 140 microM (1.0 nmol per injection) using N(G)-monomethyl-L-arginine (L-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82+/-0.05 microM (n=4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.     

Accumulated endogenous NOS inhibitors,decreased NOS activity,and impaired cavernosal relaxation with ischemia

We examined whether endogenous inhibitors of nitric oxide (NO) synthesis are involved in the impaired cavernosal relaxation with ischemia in rabbits. Two weeks after cavernosal ischemia caused by partial vessel occlusion, endothelium-dependent and electrical field stimulation (EFS)-induced neurogenic NO-mediated relaxations, but not sodium nitroprusside (SNP)-induced relaxation, were significantly impaired in the isolated corpus cavernosum. The Ca(2+)-dependent NO synthase (NOS) activity and the basal and stimulated cGMP productions with carbachol or EFS were significantly decreased after ischemia. Supplementation of excess L-arginine partially recovered both of the impaired relaxations. The contents of N(G)-monomethyl-L-arginine (L-NMMA) and asymmetric N(G), N(G)-dimethyl-L-arginine (ADMA) but not L-arginine and symmetric N(G),N'(G)-dimethyl-L-arginine (SDMA) were increased in the cavernosal tissues after ischemia. Authentic L-NMMA and ADMA but not SDMA concentration dependently inhibited both relaxations without affecting the relaxation produced by SNP in the control. Excess L-arginine abolished the inhibition with L-NMMA and ADMA. These results suggest that the impaired NO-mediated cavernosal relaxations after ischemia are closely related to the decreased NOS activity and the increased accumulation of L-NMMA and ADMA.     

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive ELISA and for antigen capture in a sandwich ELISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by Western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from Egyptian or South African lineages. These monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.     

Development and characterization of monoclonal antibodies against Rift Valley fever virus nucleocapsid protein generated by DNA immunization

This paper describes the generation of monoclonal antibodies directed to immunogenic nucleoprotein N epitopes of Rift Valley fever virus (RVFV), and their application in diagnostics, both for antibody detection in competitive eLISA and for antigen capture in a sandwich eLISA. Monoclonal antibodies (mAbs) were generated after DNA immunization of Balb/c mice and characterized by western blot, ELISA and cell immunostaining assays. At least three different immunorelevant epitopes were defined by mAb competition assays. Interestingly, two of the mAbs generated were able to distinguish between RVFV strains from egyptian or South African lineages. these monoclonal antibodies constitute useful tools for diagnosis, especially for the detection of serum anti-RVFV antibodies from a broad range of species by means of competitive ELISA.Key words: RVF, nucleoprotein, competitive ELISA, RVFV lineages     

Role of the human erythrocyte in generation and storage of asymmetric dimethylarginine

Proteolytic activity in whole blood may lead to release of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). We investigated the role of the human erythrocyte in storage and generation of ADMA in healthy controls (n = 36) and critically ill patients (n = 38). Both free and total (sum of free and protein-incorporated) ADMA were measured. Upon incubation of intact erythrocytes with extracellular ADMA (0 to 40 μmol/l), equilibrium between intra- and extracellular ADMA was reached within 3 h. Compared with controls, patients had significantly higher basal concentrations of ADMA in plasma (0.88 ± 0.75 vs. 0.41 ± 0.07 μmol/l) and erythrocytes (1.28 ± 0.55 vs. 0.57 ± 0.14 μmol/l). Intracellular and plasma ADMA were significantly correlated in the patient group only (r = 0.834). Upon lysis, followed by incubation at 37°C for 2 h, free ADMA increased sevenfold (to 8.60 ± 3.61 μmol/l in patients and 3.90 ± 0.78 μmol/l in controls). In lysates of controls, free ADMA increased further to 9.85 ± 1.35 μmol/l after 18 h. Total ADMA was 15.43 ± 2.44 μmol/l and did not change during incubation. The increase of free ADMA during incubation corresponded to substantial release of ADMA from the erythrocytic protein-incorporated pool (21.9 ± 4.6% at 2 h and 60.8 ± 7.6% at 18 h). ADMA was released from proteins other than hemoglobin, which only occurred after complete lysis and was blocked by combined inhibition of proteasomal and protease activity. Neither intact nor lysed erythrocytes mediated degradation of free ADMA. We conclude that intact erythrocytes play an important role in storage of ADMA, whereas upon erythrocyte lysis large amounts of free ADMA are generated by proteolysis of methylated proteins, which may affect plasma levels in hemolysis-associated diseases.     

Asymmetric dimethylarginine attenuates serum starvation-induced apoptosis via suppression of the Fas (APO-1/CD95)/JNK (SAPK) pathway

Asymmetric dimethylarginine (ADMA) is synthesized by protein arginine methyltransferases during methylation of protein arginine residues and released into blood upon proteolysis. Higher concentrations of ADMA in blood have been observed in patients with metabolic diseases and certain cancers. However, the role of ADMA in colon cancer has not been well investigated. ADMA serum levels in human patients diagnosed with colon cancer were found to be higher than those present in healthy subjects. ADMA treatment of LoVo cells, a human colon adenocarcinoma cell line, attenuated serum starvation-induced apoptosis and suppressed the activation of the Fas (APO-1/CD95)/JNK (SAPK) (c-Jun N terminal protein kinase/stress-activated protein kinase)pathway. ADMA also suppressed the activation of JNK triggered by death receptor ligand anti-Fas mAb and exogenous C₂-ceramide. Moreover, we demonstrated that ADMA pretreatment protected LoVo cells from doxorubicin hydrochloride-induced cell death and activation of the Fas/JNK pathway. In summary, our results suggest that the elevated ADMA in colon cancer patients may contribute to the blocking of apoptosis of cancer cells in response to stress and chemotherapy.     

A syngeneic monoclonal anti-idiotypic antibody with internal image properties restricted to a single aldosterone-binding system

Immunization with a murine anti-aldosterone mAb (AAC) resulted in the isolation of a syngeneic monoclonal anti-idiotypic antibody, LH9G4. LH9G4 bound to Fab fragments of AAC and was affinity-purified on an AAC column. LH9G4 inhibited the binding of aldosterone to AAC in a dose-dependent manner with an apparent dissociation constant of 0.5 nM as determined by competitive inhibition assays in ELISA and RIA. LH9G4 and aldosterone have similar relative affinities for AAC. Kinetic studies and Scatchard plot analysis support a reversible and reciprocal competitive inhibition mechanism between LH9G4 and aldosterone for the paratope of AAC. The possibility of a steric hindrance mechanism was eliminated. No cross-reactivity was seen with six other murine anti-aldosterone mAb, with a rabbit polyclonal antibody or with aldosterone receptor. The anti-idiotypic antibody, defined as a "restricted" internal image of aldosterone, is apparently directed at a private idiotope present in the paratope of AAC but not in binding sites of other aldosterone-binding proteins. Biophysical considerations involving characteristics of nonbonded attractive forces can explain these findings. An advantage of the one-step auto-anti-idiotypic procedure for the generation of Ab2-beta or internal image antibodies is discussed.     

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.     

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.     

Asymmetric dimethylarginine (ADMA)--a modulator of nociception in opiate tolerance and addiction?

Nitric oxide (NO) is generated from l-arginine by NO synthases, of which three forms have been identified: endothelial, inducible and neuronal (eNOS, iNOS and nNOS, respectively). The l-arginine metabolite asymmetric dimethylarginine (ADMA) is a potent, noncompetitive inhibitor of nNOS, while its congener N(G)-monomethyl-l-arginine (l-NMMA) is a less potent, competitive inhibitor. In rat neurons large amounts of ADMA are found, suggesting its importance in modulating neuronal activity. Humans generate approximately 300mumol ( approximately 60mg) ADMA per day. It is released from myelin basic proteins that are highly expressed in neuronal tissue. ADMA is mainly degraded by the action of the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which exists in two isoforms. DDAH1 is highly expressed in brain, suggesting specific function in this area. The presence of nNOS and DDAH1 in brain suggests that ADMA may have specific CNS activity and be more than an unregulated metabolite. Increased NO production-either prior to or concurrently with opioid administration-results in an enhanced rate and extent of development of tolerance to morphine in mice. NO produces an alteration in the mu-opioid receptor that increases constitutive receptor activity. It thereby reduces the ability of a selective mu-opioid agonist to activate the mu-opioid receptor; these in vitro molecular effects occur in a time course consistent with the in vivo development of antinociceptive tolerance in mice. Amongst many other synthetic NOS inhibitors of varying specificity, 7-nitroindazole (7-NI) has been shown to have a high affinity (IC(50) 0.71 microM) to nNOS. Selective blockade of nNOS by 7-NI attenuated morphine withdrawal in opiate dependent rats, suggesting nNOS as a viable target for development of pharmacotherapies. We hypothesize that, by inhibiting nNOS and reducing NO levels, ADMA may decrease mu-opiate receptor constitutive activity, resulting in alteration of the analgesic dose-response curve of morphine.     

Asymmetric dimethylarginine upregulates LOX-1 in activated macrophages: role in foam cell formation

Intimal infiltration by monocytes and accumulation of lipids represent a critical step in the formation of fatty streaks during atherogenesis. Because elevated plasma levels of asymmetric dimethylarginine (ADMA), a potent nitric oxide (NO) synthase (NOS) inhibitor, are prevalent in diverse cardiovascular diseases, the goal of this study was to examine the contribution of NO deficiency to macrophage lipid accumulation. Inhibition of NO synthesis in PMA-primed human monocytic leukemia HL-60 cells resulted in a twofold increase in expression of the receptor for oxidized LDL (OxLDL), termed the lectin-like OxLDL receptor (LOX-1). Blockade of inducible NOS in activated macrophages resulted in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-OxLDL accumulation and imparted macrophages with a foamy appearance as detected with oil-red O lipid staining. ADMA (15 microM) or N(G)-nitro-l-arginine methyl ester (l-NAME, 300 microM), both of which suppress inducible NOS activity, increased oil-red staining 1.9- and 2.8-fold, respectively. Macrophages treated with ADMA or l-NAME showed a 2.4-fold increase in accumulation of DiI-OxLDL. To examine the role of LOX-1 in this process, we used small interfering RNA (siRNA) duplex-mediated LOX-1 gene silencing. LOX-1 expression was suppressed twofold by siRNA as shown by Western blot analysis. This suppression was associated with a two- to fourfold decrease in DiI-OxLDL uptake as identified by fluorescence microscopy and decreased oil-red O staining by activated macrophages. In conclusion, accumulation of ADMA (a competitive inhibitor of NOS) in patients with chronic renal failure may be responsible for upregulation of LOX-1 receptor and increased OxLDL uptake, thus contributing to lipidosis and foam cell formation. The data illustrate an additional nonendothelial mode of antiatherogenic action of NO: prevention of LOX-1 induction and lipid accumulation by macrophages.     

Rapid determination of Phytophthora infestans sporangia using a surface plasmon resonance immunosensor

Phytophthora infestans is the cause of late blight disease in potato and is an economically important pathogen worldwide. Early disease detection is important to implement disease control measures. In this study a surface plasmon resonance (SPR) immunosensor for detection of P. infestans sporangia is presented. The specificity of an existing mouse monoclonal antibody (phyt/G1470 mAb) against P. infestans was investigated in plate-trapped antigen ELISA and in subtractive inhibition ELISA. No or only limited cross-reactivity was observed against representatives having air-borne spores from Ascomycetes, Deuteromycetes as well as Basidiomycetes. phyt/G1470 mAb was incorporated in a subtractive inhibition SPR assay, consisting of a pre-incubation of mAb and sporangia, a centrifugation step to remove sporangia-bound phyt/G1470 mAb and quantification of remaining phyt/G1470 mAb by SPR. Good intra- and interday assay variability was observed and the assay had a detection limit of 2.2x10(6) sporangia/ml. Analysis time was 75 min, which is superior to existing P. infestans detection methods.     

An enzyme hydrolyzing methylated inhibitors of nitric oxide synthase is present in circulating human red blood cells.

N(G),N(G)-dimethyl-L-arginine (asymmetric dimethylarginine or ADMA) and N(G)-monomethyl-L-arginine (L-NMMA) are post-translationally synthesized amino acids of nuclear proteins. Upon release during protein turnover, they are not used in protein synthesis, but are excreted or metabolized by dimethylarginine dimethylaminohydrolase (DDAH) found in many tissues. DDAH is present in monocytic and polynuclear cells of blood, but no report has appeared of its presence in red blood cells (RBCs). Because methylated arginines can inhibit nitric oxide synthase (NOS) and elevations are reported in several diseases, we explored whether RBCs express this enzyme. DDAH is present in RBCs as supported by hydrolysis of both ADMA and L-NMMA, but not symmetric dimethylarginine, and by immunoprecipitation/Westem blot using a specific monoclonal antibody to human DDAH. In a pilot study of end-stage renal disease (ESRD) patients, RBC DDAH activity with ADMA as substrate correlated inversely with age (p = 0.005) and enzyme activities were higher in patients with greater diastolic blood pressure drops during hemodialysis (p = 0.02). Similar correlations were found with white cell DDAH activity. Thus, human RBCs can hydrolyze methylated arginines. These findings indicate the RBC could be used to assess the status of DDAH in various disease states.     

Trichinella spiralis: influence of an immunodominant, carbohydrate-associated determinant on the host antibody response repertoire.

Host antibody responses to the G2.1 epitope, a carbohydrate-associated determinant shared by several Trichinella spiralis glycoproteins, were examined by competitive inhibition enzyme-linked immunosorbent assay (ELISA). The G2.1 epitope dominated the AKR/J mouse antibody response whether the antigens were injected or introduced through infection, as determined by the serum blocking ability of a G2.1 epitope-specific monoclonal antibody (mAb). Serum T. spiralis-binding activity from several other infected mouse strains was blocked 22 to 86% by the G2.1 epitope-specific mAb. In addition to mice, the G2.1 epitope evoked powerful antibody responses in four other species. The binding activity of Trichinella-reactive antibodies from infected rats and pigs was inhibited 56 and 34%, respectively, by the mAb. Greater than 48% of the T. spiralis serum-binding activity from 4/5 infected humans was G2.1-specific. Most of the rabbit antibody response induced by injection of a previously characterized 43-kDa antigen was also directed to the G2.1 determinant. The specificities of 10 T. spiralis-reactive mAb were examined, and 7 reacted with the immunodominant epitope. Finally, of nine helminth species examined, only T. spiralis and T. pseudospiralis extracts efficiently blocked G2.1-specific antibody binding to solid-phase antigens. These results suggest that the responses to G2.1 epitope may play an important role during infection.     

Identification of a Highly Conserved Surface on Tat Variants

Extracellular Tat is suspected to protect HIV-1-infected cells from cellular immunity. Seropositive patients are unable to produce neutralizing antibodies against Tat, and Tat is still secreted under antiviral treatment. In mice, the Tat OYI vaccine candidate generates neutralizing antibodies such as the mAb 7G12. A peptide called MIMOOX was designed from fragments of Tat OYI identified as the possible binding site for mAb 7G12. MIMOOX was chemically synthesized, and its structure was stabilized with a disulfide bridge. Circular dichroism spectra showed that MIMOOX had mainly β turns but no α helix as Tat OYI. MIMOOX was recognized by mAb 7G12 in ELISA only in reduced conditions. Moreover, a competitive recognition assay with mAb 7G12 between MIMOOX and Tat variants showed that MIMOOX mimics a highly conserved surface in Tat variants. Rat immunizations with MIMOOX induce antibodies recognizing Tat variants from the main HIV-1 subtypes and confirm the Tat OYI vaccine approach.     

ADMA-- 一个新的心血管疾病危险因子

非对称性二甲基精氨酸(asymmetric dimethylarginine,ADMA)为内源性一氧化氮合酶(endogenous Nitric-Oxide Synthase,eNOS)竞争性抑制剂,可抑制NOS的活性,减少一氧化氮(nitric oxide,NO)的合成,致使内皮功能障碍。近年来研究认为ADMA是冠心病的独立预测因子,与心功能不全及心血管疾病死亡率明显相关。本文将针对ADMA与心血管疾病的相关性进行综述。     

Unilateral nephrectomy causes an abrupt increase in inflammatory mediators and a simultaneous decrease in plasma ADMA: a study in living kidney donors

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases (NOS). Reducing inducible NOS activity in acute inflammation seems to be desirable. In vitro data show that ADMA increases in response to inflammatory mediators, yet the effect of acute inflammation in vivo is scarcely studied. The aim of the study was to evaluate ADMA plasma levels before, during, and after the acute (nonbacterial) inflammatory-like state. Plasma ADMA, l-arginine, C-reactive protein, and IL-6 were determined in 24 healthy subjects undergoing living related kidney donation before as well as 1, 6, 12, 24, 72, and 168 h thereafter. Six hours after nephrectomy, ADMA levels decreased compared with baseline (0.488 ± 0.075 vs. 0.560 ± 0.060 μmol/l, P < 0.05). This difference became even more marked 24 h after the operation (0.478 ± 0.083 μmol/l, P < 0.01 vs. baseline), when the proinflammatory cytokine IL-6 peaked. Seven days after unilateral nephrectomy, ADMA levels were elevated above baseline (0.63 ± 0.05 μmol/l, P < 0.001 vs. baseline). l-Arginine levels decreased already 1 h after nephrectomy (97.5 ± 22.5 μmol/l, P < 0.01 vs. baseline) and paralleled the change in ADMA thereafter. At the end of the observation period when inflammation markers were regressing, l-arginine levels were significantly elevated above baseline (160.6 ± 25.1 μmol/l, P < 0.001 vs. baseline). In summary, this is the first study showing that both ADMA and l-arginine decrease temporarily after unilateral nephrectomy coinciding with the increase in inflammatory mediators. The l-arginine/ADMA ratio, a surrogate for NO production capacity, was only altered for <24 h.