Edzard Schwedhelm
email: schwedhelm@uke.uni-hamburg.de

Quantification of ADMA: analytical approaches

Keywords: asymmetric dimethylarginine, endothelial dysfunction, ELISA, gas chromatography-mass spectrometry, high-performance liquid chromatography, liquid chromatography-mass spectrometry, methylated L-arginine, nitric oxide, nitric oxide synthase Vascular Medicine 2005, 10: S89-95

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Introduction

Qualification and quantification issues had been addressed several years before Vallance and coworkers introduced asymmetric dimethylarginine (N G , N G -dimethylarginine, ADMA) as an endogenous inhibitor of nitric oxide synthase (NOS). [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF] Methylation of L-arginine residues of proteins is a well known feature of protein function and metabolism. [START_REF] Mcbride | State of the arg:protein methylation at arginine comes of age[END_REF] Monomethylation, dimethylation and trimethylation of arginine as well as of lysine are observed. Information on methylation status of proteins is obtained after (total) hydrolysis or digestion. The techniques applied for the analysis of protein methylation are ion chromatography and more recently liquid chromatography-mass spectrometry (LC-MS). [START_REF] Paik | Omega-N-methylarginine in protein[END_REF][START_REF] Nakajima | Isolation and identification of N G -monomethyl-, N G ,N G -dimethyl-, and N G ,N G Јdimethylarginine from the hydrolysate of proteins of bovine brain[END_REF][START_REF] Rappsilber | Detection of arginine dimethylated peptides by parallel precursor ion scanning mass spectrometry in positive ion mode[END_REF][START_REF] Brame | A mass spectrometry based method for distinguishing between symmetrically and asymmetrically dimethylated arginine residues[END_REF][START_REF] Gehring | Fragmentation pathways of N G -methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS[END_REF] Even though the methylation of arginine residues of proteins seems to be ubiquitous, the degree of arginine methylation is organ specific. The extent of methylation yielding dimethylarginine residues is elevated in brain, liver, lung, spleen, kidney and small intestine. [START_REF] Nakajima | Isolation and identification of N G -monomethyl-, N G ,N G -dimethyl-, and N G ,N G Јdimethylarginine from the hydrolysate of proteins of bovine brain[END_REF] The ratio of ADMA over symmetric dimethylarginine (N G ,NЈ G -dimethylarginine, SDMA) is notably increased in heart tissue (a 32-fold excess has been found). [START_REF] Nakajima | Isolation and identification of N G -monomethyl-, N G ,N G -dimethyl-, and N G ,N G Јdimethylarginine from the hydrolysate of proteins of bovine brain[END_REF] The functional consequence of such observations is currently subject to investigation. While measurement of ADMA and SDMA allows us to distinguish between the two isomers, no information is provided about which protein is methylated and at which position. Electrospray ionization (ESI) tandem mass spectrometry (tandem MS) has recently revealed that dimethylammonium (mass-to-change ratio (m/z) 46) is a specific marker ion (as well as a neutral loss of 45 Da) for ADMA in methylated proteins. [START_REF] Rappsilber | Detection of arginine dimethylated peptides by parallel precursor ion scanning mass spectrometry in positive ion mode[END_REF][START_REF] Brame | A mass spectrometry based method for distinguishing between symmetrically and asymmetrically dimethylated arginine residues[END_REF][START_REF] Gehring | Fragmentation pathways of N G -methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS[END_REF] These mass spectrometric techniques now enable us to have a substantial insight into the diverse functions of protein arginine methylation, which has been speculated to parallel the role of phosphorylation in modification of protein function. 2

High-performance liquid chromatography

The major advantage of high-performance liquid chromatography (HPLC) over ion chromatography is a reduction of analysis time from several hours to 30-60 minutes. A sensitive HPLC method for the determination of methylated arginines has been introduced by several groups using o-phthaldialdehyde (OPA) for precolumn derivatization and subsequent fluorescence (FL) detection. [START_REF] Bode-Böger | Elevated L-arginine/dimethylarginine ratio contributes to enhanced systemic NO production by dietary L-arginine in hypercholesterolemic rabbits[END_REF][START_REF] Pettersson | Determination of dimethylated arginines in human plasma by high-performance liquid chromatography[END_REF][START_REF] Chen | Determination of N(G),N(G)dimethylarginine in human plasma by high-performance liquid chromatography[END_REF] FL detection is carried out at excitation ( ex ) and emission ( em ) wavelengths of 340 and 455 nm, respectively. The crucial step of the HPLC analysis of methylated arginines is the chromatographic separation of ADMA from SDMA and to resolve both from matrix components. [START_REF] Tsikas | Determination of dimethylated arginines in human plasma by high-performance liquid chromatography[END_REF] Baseline separation of OPA derivatives of ADMA and SDMA can be achieved by means of analytical columns packed with octadecylsilica or phenyl silica, namely, reversed phase (RP) conditions. The analytical column should be at least 150 ϫ 4.6 mm. It should also be protected by a guard column packed with the same material. It is interesting that OPA derivatives of ADMA and SDMA elute on a phenylsilica column in the opposite order to octadecylsilica. To resolve analytes from matrix prior to HPLC-FL analysis, appropriate sample pretreatment has to be performed. This includes: (1) protein precipitation and (2) solid phase extraction (SPE) of biological samples. Protein precipitation can be performed with any convenient organic solvent. The organic layer has to be evaporated prior to SPE. Alternatively, protein precipitation can be accomplished by the addition of 5-sulfosalicylic acid [START_REF] Chen | Determination of N(G),N(G)dimethylarginine in human plasma by high-performance liquid chromatography[END_REF] or proteins can be separated by ultrafiltration. In some cases it might be necessary to extract lipids from samples with trifluoroethane before they are subjected to SPE. [START_REF] Bode-Böger | Elevated L-arginine/dimethylarginine ratio contributes to enhanced systemic NO production by dietary L-arginine in hypercholesterolemic rabbits[END_REF] However, in some cases it is neither necessary to extract lipids nor to precipitate proteins if SPE is applied. Thus, if not present in large amounts, proteins will be separated during the SPE procedure. To avoid clogging of the columns used for SPE, samples containing proteins should be diluted 1 ϩ 1 with deionized water. Several types of columns have been applied in SPE of ADMA; they are silica based or equipped with a polymeric stationary phase. A major advantage of polymeric material is that column preconditioning becomes unnecessary. Stationary phases are modified with weak carboxylic acid (CBA) or strong cation exchange (SCX) resins. [START_REF] Bode-Böger | Elevated L-arginine/dimethylarginine ratio contributes to enhanced systemic NO production by dietary L-arginine in hypercholesterolemic rabbits[END_REF][START_REF] Pettersson | Determination of dimethylated arginines in human plasma by high-performance liquid chromatography[END_REF][START_REF] Teerlink | Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography[END_REF] The advantage of SCX columns is better retention of the analytes. Columns with a bed of 50-100 mg can be used. In contrast, 500 mg of bed material are recommended for CBA columns. Elution of basic amino acids such as ADMA, SDMA and L-arginine from CBA columns is obtained with 1 M formic acid.

Elution of analytes from SCX and polymeric columns is obtained with mixtures of methanol/water/ triethylamine or ammonia. Recoveries for L-arginine are crucial and protocols have to be optimized in this respect. [START_REF] Teerlink | Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography[END_REF][START_REF] Heresztyn | Determination of L-arginine and NG, NG-and NG, NGЈ-dimethyl-L-arginine in plasma by liquid chromatography as AccQ-Fluor fluorescent derivatives[END_REF] All recent protocols for HPLC determination of methylated arginines include the application of an appropriate internal standard. The internal standard has to be added prior to sample preparation (i.e. as the initial step). Several internal standards have been adopted for the simultaneous quantification of ADMA, SDMA and L-arginine: homoarginine, [START_REF] Böger | Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia[END_REF][START_REF] Pi | Improved method for simultaneous determination of L-arginine and its mono-and dimethylated metabolites in biological samples by highperformance liquid chromatography[END_REF][START_REF] Zhang | Simultaneous determination of arginine and seven metabolites in plasma by reversed-phase liquid chromatography with a time-controlled ortho-phthaldialdehyde precolumn derivatization[END_REF] N G -monomethyl-L-arginine (NMMA), [START_REF] Teerlink | Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography[END_REF][START_REF] Heresztyn | Determination of L-arginine and NG, NG-and NG, NGЈ-dimethyl-L-arginine in plasma by liquid chromatography as AccQ-Fluor fluorescent derivatives[END_REF] and N -propyl-L-arginine (N-PLA). [START_REF] Marra | High-performance liquid chromatographic assay of asymmetric dimethylarginine, symmetric dimethylarginine, and arginine in human plasma by derivatization with naphthalene-2,3dicarboxaldehyde[END_REF] A representative chromatogram of a combined standard containing arginine, homoarginine, SDMA and ADMA is depicted in Figure 1. Fluorescence was detected after SPE with CBA cartridges, online OPA derivatization, and separation on a phenyl column (250 ϫ 4.6 mm, Macherey-Nagel, Düren, Germany). [START_REF] Tsikas | Determination of dimethylated arginines in human plasma by high-performance liquid chromatography[END_REF] When applying this method for plasma samples, homoarginine was used as the internal standard. Table 1 summarizes the major characteristics of this and other methods applied. Homoarginine and NMMA, although present in only low quantities in human plasma, may be altered under pathophysiologic conditions such as renal failure. [START_REF] Marescau | Guanidino compounds in serum and urine of nondialyzed patients with chronic renal insufficiency[END_REF][START_REF] Mendes Ribeiro | Accumulation of the endogenous L-arginine analogue NGmonomethyl-L-arginine in human end-stage renal failure patients on regular haemodialysis[END_REF] N-PLA is not an endogenous compound. However, since its chromatographic properties are reported to be very similar to SDMA and ADMA, the risk of overlapping signals cannot be excluded.

A major drawback of OPA derivatives is their instability due to rapid decomposition, so on-line derivatization has to be performed. If this is not available, other derivatization agents may be used in combination or instead of OPA. In this regard, the sulfhydryl group used for the reaction with the OPA reagent is of vital importance. The use of 3-mercaptopropionic acid instead of 2-mercaptoethanol for derivatization with the OPA reagent results in more stable derivatives of L-arginine, the internal standard, ADMA and SDMA. After derivatization, the samples have to be stored at 7ЊC. Peak areas of derivatives were shown to decline by 5-10% within 20-25 hours and by approximately 35% after 72 hours. However, the relative peak area ratios were found unchanged after 72 hours (i.e. 99.4 Ϯ1.6% of the initial ratio). [START_REF] Teerlink | Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography[END_REF] Further improvement of stability was reported for naphthalene-2,3dicarboxaldehyde (NDA) derivatives. [START_REF] Marra | High-performance liquid chromatographic assay of asymmetric dimethylarginine, symmetric dimethylarginine, and arginine in human plasma by derivatization with naphthalene-2,3dicarboxaldehyde[END_REF] Owing to the different nature of the NDA derivatives, wavelengths for FL detection have to be adjusted ( ex ϭ 420 nm and em ϭ 483 nm). However, compounds have to be stored at 4ЊC after derivatization and a linear decline of fluorescence signal by 15.9, 11 and 12.9% for derivatives of arginine, SDMA and ADMA, respectively, was observed after 72 hours. [START_REF] Marra | High-performance liquid chromatographic assay of asymmetric dimethylarginine, symmetric dimethylarginine, and arginine in human plasma by derivatization with naphthalene-2,3dicarboxaldehyde[END_REF] Finally, stable derivatives, even at room temperature, are obtained with the derivatizing agent 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AccQ-Fluor TM , Waters, Milford, MA, USA). [START_REF] Heresztyn | Determination of L-arginine and NG, NG-and NG, NGЈ-dimethyl-L-arginine in plasma by liquid chromatography as AccQ-Fluor fluorescent derivatives[END_REF] FL detection is carried out at ex ϭ 250 nm and em ϭ 395 nm. Derivatives of arginine, NMMA, ADMA and SDMA are stable for at least 7 days at room temperature. Another method for the analysis of methylated arginines is the combination of on-line extraction, separation with ion-pair chromatography and postcolumn derivatization of amino acids. [START_REF] Dobashi | An automated analyzer for methylated arginines in rat plasma by highperformance liquid chromatography with post-column fluorescence reaction[END_REF] For the ion-pair chromatography, a mixture of the two ion-pair reagents sodium octanoate and cyclohexanecarboxylic acid is used. Even though the column switching protocol is quite complex and three different analytical columns are involved, the analysis of biological samples is completely automated. An equally high degree of automation can be attained by using LC-MS (i.e. liquid chromatography-mass spectrometry).

Liquid chromatography-mass spectrometry

OPA derivatives of L-arginine and its metabolites are also suitable for mass spectrometric analysis with the limitations mentioned above (stability, chromatographic conditions). OPA derivatives of ADMA and SDMA exhibit identical fragmentation patterns under positive ESI (i.e. m/z 379). L-Arginine, L-citrulline and homoarginine isoindole derivatives formed with OPA are discernable owing to different massto-charge ratios (i.e. m/z 351, 352 and 365, respectively). [START_REF] Martens-Lobenhoffer | Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation[END_REF][START_REF] Martens-Lobenhoffer | Determination of arginine and asymmetric dimethylarginine (ADMA) in human plasma by liquid chromatography/mass spectrometry with the isotope dilution technique[END_REF] The advantage of mass spectrometry is its unique specificity. Thus, the SPE step can be omitted for human plasma and urine. Only a protein precipitation for plasma samples is recommended. To add a further level of specificity, LC-tandem MS is mandatory. OPA derivatives seem to be unsuited because of their complex fragmentation pattern after MS-MS fragmentation. [START_REF] Martens-Lobenhoffer | Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation[END_REF] Derivatization of the carboxylic acid instead of the primary amino function is a feasible alternative. Derivatization can be carried out with any convenient reagent; for example, esterification or amidation can be performed. Butyl ester derivatives of ADMA and SDMA have the same parent ions (i.e. m/z 259). Nevertheless, butyl ester derivatives of ADMA and SDMA are discernable according to their major daughter ions after MS-MS fragmentation; that is, m/z 214 and 228 for ADMA and SDMA, respectively. [START_REF] Schwedhelm | LC-tandem MS method for the analysis of asymmetric dimethylarginine (ADMA)[END_REF] Thus, any preliminary chromatographic separation (e.g. LC of ADMA and SDMA) can be omitted.

Derivatization of methylated arginines with OPA reagent or 1 M HCl in butanol can be carried out on-line. However, arginine and methylated arginines have also been analysed with LC-MS and LC-tandem MS without derivatization. [START_REF] Huang | Simultaneous determination of L-arginine and its mono-and dimethylated metabolites in human plasma by high-performance liquid chromatography-mass spectrometry[END_REF][START_REF] Vishwanathan | Determination of arginine and methylated arginines in human plasma by liquid chromatography-tandem mass spectrometry[END_REF] ADMA and SDMA exhibit identical parent ions after positive atmospheric pressure chemical ionization or ESI (i.e. m/z 203). [START_REF] Huang | Simultaneous determination of L-arginine and its mono-and dimethylated metabolites in human plasma by high-performance liquid chromatography-mass spectrometry[END_REF][START_REF] Vishwanathan | Determination of arginine and methylated arginines in human plasma by liquid chromatography-tandem mass spectrometry[END_REF] L-arginine and NMMA are discernable from ADMA and SDMA owing to m/z ratios of 175 and 189 for L-arginine and NMMA, respectively. MS-MS spectra of ADMA and SDMA are very similar for the major fragments (Ն10%). Only one major fragment is exclusively formed by SDMA (i.e. m/z 172). This fragment is specific to SDMA since it contains a single methyl group bound to one guanidine nitrogen. [START_REF] Vishwanathan | Determination of arginine and methylated arginines in human plasma by liquid chromatography-tandem mass spectrometry[END_REF] To overcome this lack of a specific daughter fragment ion for ADMA, SDMA and ADMA have to be separated by chromatography prior to mass spectrometric analysis. In this context a compromise has to be found between sufficient chromatographic separation and short analysis time. Alternatively, a closer look at the minor fragments in the MS-MS spectrum of ADMA seems worth while. Indeed, dimethylammonium (m/z 46) is exclusively formed by ADMA after fragmentation (see introduction). To minimize ion suppression [START_REF] Annesley | Ion suppression in mass spectrometry[END_REF] at this low m/z ratio, a teicoplanin column can be used. [START_REF] Dalton | Measurement of asymmetric and symmetric dimethylarginines using stable isotope dilution electrospray mass spectrometry-mass spectrometry[END_REF]

Internal standards in mass spectrometry

Accurate quantification in mass spectrometry is realized by stable isotope dilution of samples. Compounds labeled with stable isotopes are used as internal standards of arginine and its metabolites and added to crude samples. Labeled compounds show similar chemical properties. Nevertheless, they can be discerned from the analytes because of their higher m/z ratios in mass spectrometry. So far [ 13 C 6 ]arginine (98% [START_REF] Heresztyn | Determination of L-arginine and NG, NG-and NG, NGЈ-dimethyl-L-arginine in plasma by liquid chromatography as AccQ-Fluor fluorescent derivatives[END_REF] C isotopic purity, Cambridge Isotope Laboratories, Andover, MA, USA) and L-[guanidine- [START_REF] Pi | Improved method for simultaneous determination of L-arginine and its mono-and dimethylated metabolites in biological samples by highperformance liquid chromatography[END_REF] N 2 ]arginine (98% [START_REF] Pi | Improved method for simultaneous determination of L-arginine and its mono-and dimethylated metabolites in biological samples by highperformance liquid chromatography[END_REF] N isotopic purity, Cambridge Isotope Laboratories) have been used as internal standards for the quantification of L-arginine in LCand gas chromatography (GC)-MS. [START_REF] Martens-Lobenhoffer | Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation[END_REF][START_REF] Martens-Lobenhoffer | Determination of arginine and asymmetric dimethylarginine (ADMA) in human plasma by liquid chromatography/mass spectrometry with the isotope dilution technique[END_REF][START_REF] Tsikas | Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative[END_REF][START_REF] Albsmeier | Determination of NG, NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatographymass spectrometry[END_REF] A stable isotope labeled internal standard for the quantification of ADMA is not yet commercially available. However, an appropriate internal standard may be synthesized in situ with the samples, [START_REF] Tsikas | Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative[END_REF] or easily prepared in the laboratory. Synthesis follows the synthetic route described by Pundak and Wilchek. [START_REF] Pundak | Synthesis of guanidine-Nalkylarginines by the use of polymeric pseudoureas[END_REF] L-[3,3,4,4,5,5,-2 H 6 ]Ornithine (>98% [START_REF] Pi | Improved method for simultaneous determination of L-arginine and its mono-and dimethylated metabolites in biological samples by highperformance liquid chromatography[END_REF] N isotopic purity, Euriso-top, Saint Aubin, France) or [ 2 H 6 ]dimethylamine gas, free base (99% 2 H isotopic purity, Cambridge Isotope Laboratories) were used as labeled educts. [START_REF] Martens-Lobenhoffer | Determination of arginine and asymmetric dimethylarginine (ADMA) in human plasma by liquid chromatography/mass spectrometry with the isotope dilution technique[END_REF][START_REF] Schwedhelm | LC-tandem MS method for the analysis of asymmetric dimethylarginine (ADMA)[END_REF][START_REF] Albsmeier | Determination of NG, NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatographymass spectrometry[END_REF] An appropriate stable isotopelabeled internal standard for SDMA has not yet been synthesized because SDMA is not available via the synthetic route described by Pundak and Wilchek.

Gas chromatography-mass spectrometry

Derivatization is an indispensable prerequisite for the GC-MS analysis of amino acids. Compounds are too polar and not volatile enough in the absence of derivatization. To date, L-arginine, NMMA, SDMA and ADMA have been derivatized with fluorine compounds. This allows highly sensitive analysis with negative-ion chemical ionization (NICI). [START_REF] Tsikas | Analysis of the L-arginine/nitric oxide (NO) pathway. The unique role of mass spectrometry[END_REF] Thus, analytes are converted to their methyl ester N-pentafluoropropionyl amide derivatives and subsequently analyzed in the NICI mode with GC-MS or GC-tandem MS. [START_REF] Tsikas | Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative[END_REF][START_REF] Albsmeier | Determination of NG, NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatographymass spectrometry[END_REF] NMMA and ADMA are analyzed as their tri(N-pentafluoropropionyl) amide derivative, L-arginine as its tetra-and SDMA as its di-derivative. Thus, all analytes are discernable owing to different GC retention times and MS ions. In GC-MS the ions m/z 640 and m/z 586 are monitored in the selected ion monitoring mode for ADMA and L-arginine derivatives, respectively. The limit of detection (LOD) was determined to be 2 fmol ADMA with a signal-to-noise ratio of 11.8 Ϯ 1. [START_REF] Albsmeier | Determination of NG, NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatographymass spectrometry[END_REF] In GCtandem MS the transitions m/z 640 to m/z 378 and m/z 586 to m/z 295 are monitored in the selected reaction monitoring mode for ADMA and L-arginine derivatives, respectively. The LOD was determined to be lower in comparison to GC-MS owing to the higher selectivity of tandem MS: 1 fmol of deuterated ADMA was analysed with a signal-to-noise ratio of 343 Ϯ 30, suggesting a LOD of approximately 10 amol of ADMA. [START_REF] Tsikas | Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative[END_REF] With this very sensitive assay it was also possible to generate a daughter ion spectrum from the endogenous compound eluting from the GC column at the retention time of ADMA. This spectrum was virtually identical with authentic ADMA, irrefutably identifying the endogenous compound as ADMA. Endogenous ADMA was identified in human urine and plasma. Urine samples are derivatized without any preliminary sample purification. Plasma samples are derivatized after ultrafiltration or protein precipitation. A comparison between the urine and plasma samples from 10 individuals measured with GC-MS and GC-tandem MS revealed almost identical values for ADMA. The ratios of values obtained with GC-MS to GC-tandem MS were 1.173 Ϯ 0.097 and 1.022 Ϯ 0.034 for human urine and plasma, respectively. [START_REF] Tsikas | Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography-tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative[END_REF] To date, a limitation in GC-(tandem) MS analysis of methlyarginines is the unavailability of an appropriate internal standard for SDMA.

Basal levels of ADMA

An overview of basal plasma levels in humans measured with different assays is given in Table 1. An ELISA for the determination of ADMA has been successfully developed and is now commercially available. [START_REF] Schulze | Determination of asymmetric dimethylarginine (ADMA) using a novel ELISA assay[END_REF] Basal plasma levels obtained with this assay and with other techniques are quite similar. To obtain an overview of different assays performed in different laboratories, an interlab comparison was carried out. Nine different samples were analysed: three basal samples, three samples spiked with 1 M ADMA, and three samples spiked with 5 M ADMA. The results are given in Figure 2.

Conclusion

More than ever there is a particular need for reliable methods for the determination of ADMA and other metabolites in the L-arginine pathway. The scientific interest in ADMA and related compounds has increased quickly in the last few years. Thus, availability and rapidity are important for analytical methods in this field. Two different strategies seem to meet these criteria, at least in part that is: ELISA and mass spectrometry. Both these technologies are equal in terms of their rapidity. However, the technology required for ELISA is widely available. Nevertheless, mass spectrometry is unsurpassed in its unique selectivity. By means of mass spectrometry, it was possible to identify unambiguously ADMA on the basis of its distinct mass spectrum. Thus, any other method for the quantification of ADMA should be compared with the gold standard of mass spectrometry. This comparison should be carried out according to the rules of Bland and Altman. [START_REF] Bland | Statistical methods for assessing agreement between two methods of clinical measurement[END_REF] Nevertheless, in clinical research a single comparison may not be adequate. It is therefore indispensable to have quality controls and interlab comparisons. For the future, the primary analytical challenge in the field of ADMA will be to maintain high standards and continually perform quality control procedures.

Figure 1

 1 Figure 1 Chromatogram of a combined standard containing 25 M arginine, 10 M homoarginine, 1 M symmetric dimethylarginine (SDMA), and 1 M asymmetric dimethylarginine (ADMA). Fluorescence was detected after solid phase extraction (SPE) with carboxylic acid (CBA) cartridges, online o-phthaldialdehyde (OPA) derivatization, and separation on a phenyl column (250 ϫ 4.6 mm, Macherey-Nagel, Düren, Germany). 11 Dashed line indicates gain switch of the fluorescence detector. Peak identification: 1, arginine; 2, homoarginine; 3, SDMA; 4, ADMA.

Figure 2

 2 Figure 2Interlab comparison of analysis of human blood samples for asymmetric dimethylarginine (ADMA). Nine different samples were analysed: three equal basal samples, three equal samples spiked with 1 M ADMA, and three equal samples spiked with 5 M ADMA. Methods are indicated according to samples analysed in each laboratory. HPLC, high-performance liquid chromatography; LC, liquid chromatography; GC, gas chromatography; MS, mass spectrometry; ELISA, enzyme-linked immunosorbent assay.

Table 1

 1 Summary of methods used to detect ADMA in human plasma.

	Analysis/detection		Plasma	Detection
	method	Derivative	concentration a	limit a	Reference
	HPLC
	Fluorescence	OPA	0.58 Ϯ 0.02 (n ϭ 10)	0.015	Pettersson et al 1997 9
		OPA	0.52 Ϯ 0.21	0.004 b	Tsikas et al 1998 11
		OPA	0.30 Ϯ 0.05 (n ϭ 7)	0.18 c	Pi et al 2000 15
		OPA	0.42 Ϯ 0.06 (n ϭ 53)	0.01 c	Teerlink et al 2002 12
		OPA	0.76 Ϯ 0.12 (n ϭ 35)	0.002	Zhang and Kaye 2004 16
		NDA	0.38-1.30 (range, n ϭ 50)	0.01	Marra et al 2003 17
		AccQ-Fluor	0.44 Ϯ 0.08 (n ϭ 12)	0.1 d	Heresztyn et al 2004 13
	Capillary electrophoresis
	Laser-induced
	fluorescence		0.34 Ϯ 0.02 (serum, n ϭ 5)	0.05 e	Caussé et al 2000 35
	Mass spectrometry
	GC-MS	methyl-PFP 0.60 Ϯ 0.08 (n ϭ 10)	2 fmol	Albsmeier et al 2004 30
	GC-MS/MS	methyl-PFP 0.39 Ϯ 0.06 (n ϭ 12)	10 amol	Tsikas et al 2003 29
	LC-MS (ESI)	OPA	0.45 Ϯ 0.13 (n ϭ 15)		Martens-Lobenhoffer et al 2003 22
		OPA	0.36 Ϯ 0.07 (n ϭ 47)	0.15 c	Martens-Lobenhoffer et al 2004 23
	LC-MS (APCI)	-	0.48 Ϯ 0.07 (n ϭ 40)	0.01	Huang et al 2004 25
	LC-MS/MS	-	0.12 Ϯ 0.05 (n ϭ 20)	0.005	Vishwanathan et al 2000 26
		-	0.46 (95% CI, 0.44-0.46, n ϭ 42) 0.017	Kirchherr et al 2005 18
		butyl	0.55 Ϯ 0.14 (n ϭ 22)	0.003	Schwedhelm et al 2005 24
	ELISA
			0.65 Ϯ 0.13 (n ϭ 10)	0.05 e	Schulze et al 2004 33

a Concentrations and detection limits are given in mol/l unless otherwise indicated; b determined by our laboratory; c limit of quantification; d limit of detection in plasma; e analytical sensitivity; APCI, atmospheric-pressure chemical ionisation; CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionisation; GC, gas chromatography; LC, liquid chromatography; MS, mass spectrometry; NDA, naphthalene-2,3-dicarboxaldehyde; OPA, o-phthaldialdehyde; PFP, pentafluoropropionyl.

Vascular Medicine 2005; 10: S89-95


Tom Teerlink
email: t.teerlink@vumc.nl

ADMA metabolism and clearance

Keywords: asymmetric dimethylarginine, cationic amino acid transporters, dimethylarginine dimethylaminohydrolase, methylation, nitric oxide, proteolysis Vascular Medicine 2005, 10: S73-81

The plasma concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is the resultant of many processes at cellular and organ levels. Post-translational methylation of arginine residues of proteins plays a crucial role in the regulation of their functions, which include processes such as transcription, translation and RNA splicing. Because protein methylation is irreversible, the methylation signal can be turned off only by proteolysis of the entire protein. Consequently, most methylated proteins have high turnover rates. Free ADMA, which is formed during proteolysis, is actively degraded by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH). Some ADMA escapes degradation and leaves the cell via cationic amino acid transporters. These transporters also mediate uptake of ADMA by neighboring cells or distant organs, thereby facilitating active interorgan transport. Clearance of ADMA from the plasma occurs in small part by urinary excretion, but the bulk of ADMA is degraded by intracellular DDAH, after uptake from the circulation. This review discusses the various processes involved in ADMA metabolism: protein methylation, proteolysis of methylated proteins, metabolism by DDAH, and interorgan transport. In addition, the role of the kidney and the liver in the clearance of ADMA is highlighted.

Introduction

Asymmetric dimethylarginine (ADMA) is a posttranslationally modified form of arginine that is generated in all cells during the process of protein turnover. ADMA has been found to be an independent predictor of cardiovascular morbidity and mortality [START_REF] Valkonen | Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine[END_REF][START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF] and its status as a cardiovascular risk factor is gradually gaining acceptance. Competitive inhibition of nitric oxide synthase (NOS) is the most likely mechanism for the effects of ADMA on the cardiovascular system, [START_REF] Vallance | Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis[END_REF] although nitric oxide (NO)-independent mechanisms cannot be excluded. [START_REF] Suda | Asymmetric dimethylarginine causes arteriosclerotic lesions in endothelial nitric oxide synthase-deficient mice. Involvement of reninangiotensin system and oxidative stress[END_REF] Only free ADMA, and not its protein-bound form, is capable of inhibiting NOS. Because NOS is an intracellular enzyme, the intracellular concentration of free ADMA, rather then its concentration in plasma, is relevant with respect to NOS inhibition. This review focuses on the processes that determine the intracellular concentration of free ADMA (Figure 1). There are no indications for the existence of biosynthetic routes for the direct synthesis of free ADMA by methylation of free arginine. Free ADMA is formed exclusively by the sequence of methylation of arginine residues of proteins, followed by proteolysis of these proteins. There are two pathways for the clearance of ADMA from the cell. In the first, ADMA is enzymatically degraded by dimethylarginine dimethylaminohydrolase (DDAH), which cleaves ADMA into citrulline and dimethylamine. The second pathway consists of export of ADMA from the cell to the plasma via cationic amino acid transporters (CAT) in the plasma membrane. Likewise, clearance from the plasma compartment also occurs by two routes: renal excretion and uptake by cells via CAT. The activity of CAT is thus involved in both cellular release and uptake of ADMA, and as such plays an important role in intercellular and interorgan transport of ADMA. Both the kidney and the liver seem to be important organs in the clearance of ADMA from the plasma. The interorgan transport of ADMA may have serious consequences in diseases and clinical conditions that are associated with altered ADMA metabolism. If, in a particular organ, large amounts of ADMA are formed and subsequently exported to the plasma, this may lead to uptake of ADMA by other organs, which will affect their function.

The processes of protein methylation, proteolysis, degradation of ADMA by DDAH, intracellular traffic via amino acid transporters, and metabolism of ADMA at organ level are discussed in the following sections, with special emphasis on the regulation of these processes.

Protein methylation

Methylation of the amino acids lysine, histidine and arginine in proteins is a specific form of posttranslational modification used by cells to expand the functional repertoire of their proteome. The terminal guanidino group of arginine can be dimethylated by protein arginine methyltransferases (PRMTs), of which two classes exist. [START_REF] Gary | RNA and protein interactions modulated by protein arginine methylation[END_REF] Both classes of PRMT catalyze the monomethylation of arginine but, when a second methyl group is attached to monomethylarginine, the reaction product is PRMT dependent. Type 1 PRMTs catalyze the formation of ADMA, whereas type 2 PRMTs lead to the formation of symmetric dimethylarginine (SDMA). Both types of PRMT preferentially methylate arginine located in arginineglycine-rich sequences of proteins. [START_REF] Gary | RNA and protein interactions modulated by protein arginine methylation[END_REF] A recent proteomic analysis has identified more than 200 proteins that are putatively arginine methylated. [START_REF] Boisvert | A proteomic analysis of arginine-methylated protein complexes[END_REF] Many of these have strong interactions with nucleic acids and are involved in processes such as splicing of pre-mRNA, transcription, protein translation, and signal transduction. [START_REF] Gary | RNA and protein interactions modulated by protein arginine methylation[END_REF][START_REF] Boisvert | A proteomic analysis of arginine-methylated protein complexes[END_REF][START_REF] Bachand | PRMT3 is a ribosomal protein methyltransferase that affects the cellular levels of ribosomal subunits[END_REF][START_REF] Miranda | Spliceosome Sm proteins D1, D3, and B/BЈ are asymmetrically dimethylated at arginine residues in the nucleus[END_REF] Arginine methylation is also involved in targeting proteins for nuclear import. 9

Regulation of methylation by S-adenosylmethionine and S-adenosylhomocysteine

All PRMTs use S-adenosylmethionine (SAM), which is synthesized from methionine and ATP, as the methyl group donor. After transfer of its methyl group, SAM is converted into S-adenosylhomocysteine (SAH). SAH is enzymatically converted into homocysteine, which is either metabolized in the trans-sulfuration route or remethylated to methionine. Because synthesis of ADMA requires the transfer of two methyl groups, two equivalents of homocysteine are formed as a byproduct. Several groups have investigated the link between the metabolic pathways of ADMA and homocysteine. A positive association between plasma levels of homocysteine and ADMA has been reported, [START_REF] Holven | Folic acid treatment reduces elevated plasma levels of asymmetric dimethylarginine in hyperhomocysteinaemic subjects[END_REF] but this was not found in other studies. [START_REF] Dierkes | Fenofibrate increases the L-arginine:ADMA ratio by increase of L-arginine concentration but has no effect on ADMA concentration[END_REF][START_REF] Jonasson | Hyperhomocysteinaemia is not associated with increased levels of asymmetric dimethylarginine in patients with ischaemic heart disease[END_REF] Oral methionine loading can be used to induce acute hyperhomocysteinemia. It is interesting that we and other investigators have observed a significant increase in plasma ADMA concentration after a methionine-loading test. [START_REF] Böger | Elevation of asymmetrical dimethylarginine may mediate endothelial dysfunction during experimental hyperhomocyst(e)inaemia in humans[END_REF][START_REF] Stühlinger | Endothelial dysfunction induced by hyperhomocyst(e)inemia. Role of asymmetric dimethylarginine[END_REF][START_REF] Wanby | Asymmetric dimethylarginine and total homocysteine in plasma after oral methionine loading[END_REF] It is important to note that the enzymatic hydrolysis of SAH to homocysteine is reversible, with equilibrium dynamics that strongly favor SAH synthesis rather than hydrolysis. In situations where homocysteine is elevated, intracellular SAH levels may thus increase. Because SAH is a potent inhibitor of transmethylation reactions, its accumulation may lead to hypomethylation of macromolecules. [START_REF] Yi | Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation[END_REF] In accordance with this mechanism, DNA hypomethylation has been observed in hyperhomocysteinemic patients with end-stage renal disease. [START_REF] Ingrosso | Folate treatment and unbalanced methylation and changes of allelic expression induced by hyperhomocysteinaemia in patients with uraemia[END_REF] It has also been shown that the production of ADMA by endothelial cells is inhibited by incubation with SAH. [START_REF] Böger | LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethioninedependent methyltransferases[END_REF] Whether endogenous levels of SAM and SAH play an important role in the regulation of synthesis of ADMA is not clear at present, but this certainly deserves further investigation.

Stimulation of protein methylation by autoantibodies

Recent reports suggest a link between the occurrence of autoantibodies and protein methylation. Systemic lupus erythematosus (SLE) is a systemic inflammatory disease characterized by the presence of a host of autoantibodies, many of which recognize antigens from the cell nucleus. These autoantibodies play an important role in the pathogenesis and activity of SLE. Common antigenic targets include double-stranded DNA (dsDNA) and several ribonucleoproteins, including the spliceosomal complex. The autoantibodies specifically recognize the basic arginine-glycinerich domains of the proteins involved. [START_REF] Mcclain | Anti-Sm autoantibodies in systemic lupus target highly basic surface structures of complexed spliceosomal autoantigens[END_REF] Notably, these domains are also preferred sites for the methylation of arginine residues by PRMTs. [START_REF] Gary | RNA and protein interactions modulated by protein arginine methylation[END_REF][START_REF] Zieve | The anti-Sm immune response in autoimmunity and cell biology[END_REF] Indeed, several Arginine residues of proteins are methylated by protein arginine methyltransferases (PRMT). In this transmethylation process, S-adenosylmethionine (SAM), which serves as donor of the methyl group, is converted into S-adenosylhomocysteine (SAH). SAH is hydrolyzed to homocysteine, which can be remethylated to methionine. Free ADMA is formed on proteolysis of methylated proteins. Most ADMA is hydrolyzed by intracellular dimethylarginine dimethylaminohydrolase (DDAH) into citrulline and dimethylamine. Some ADMA is exported from the cell by cationic amino acid transporters (CAT). A small fraction of circulating ADMA is excreted by the kidneys, but most ADMA is taken up by other cells or organs and degraded by DDAH.

spliceosomal proteins have been shown to contain both ADMA and SDMA residues in their sequence. [START_REF] Miranda | Spliceosome Sm proteins D1, D3, and B/BЈ are asymmetrically dimethylated at arginine residues in the nucleus[END_REF][START_REF] Zieve | The anti-Sm immune response in autoimmunity and cell biology[END_REF] It was recently shown that autoantibodies to dsDNA cross-react with the arginine-glycine-rich domain of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2). [START_REF] Sun | Autoantibodies to dsDNA cross-react with the arginine-glycine-rich domain of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and promote methylation of hnRNP A2[END_REF] In the presence of anti-dsDNA, methylation of hnRNP A2 by PRMT1 was increased in a dose-dependent manner to 3.5 times the control level. [START_REF] Sun | Autoantibodies to dsDNA cross-react with the arginine-glycine-rich domain of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) and promote methylation of hnRNP A2[END_REF] Anti-dsDNA antibodies may therefore cause increased ADMA production by upregulating the dimethylation of arginine residues by PRMT1.

This mechanism of excess ADMA production possibly plays a role in the pathology of rheumatoid diseases such as SLE. Cardiovascular disease is an important cause of morbidity and mortality in patients with SLE, [START_REF] Manzi | Age-specific incidence rates of myocardial infarction and angina in women with systemic lupus erythematosus: comparison with the Framingham Study[END_REF] but traditional Framingham risk factors fail fully to account for this excess of cardiovascular events. [START_REF] Esdaile | Traditional Framingham risk factors fail to fully account for accelerated atherosclerosis in systemic lupus erythematosus[END_REF] In addition, the endothelial dysfunction associated with SLE cannot be explained by classic risk factors. [START_REF] El-Magadmi | Systemic lupus erythematosus: an independent risk factor for endothelial dysfunction in women[END_REF] We have recently assessed the association between ADMA and cardiovascular disease in a cross-sectional study in 107 patients with SLE. [START_REF] Bultink | Elevated plasma levels of asymmetric dimethylarginine are associated with cardiovascular events, disease activity and organ damage in patients with systemic lupus erythematosus[END_REF] The prevalence of cardiovascular disease increased across tertiles of plasma ADMA concentrations (p ϭ 0.023). Most importantly, we observed a significant positive association between plasma ADMA concentrations and titres of anti-dsDNA autoantibodies (r ϭ 0.43; p Ͻ 0.001), corroborating the in vitro data described above. Anti-dsDNA autoantibodies have also been shown to stimulate the expression of pro-inflammatory cytokines by mononuclear cells [START_REF] Sun | Monoclonal anti-doublestranded DNA autoantibody stimulates the expression and release of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha from normal human mononuclear cells involving in the lupus pathogenesis[END_REF] and we hypothesize that these antibodies play a dual role in the pathology of SLE by augmenting the inflammatory reaction and stimulating ADMA production (Figure 2).

Reversibility of arginine methylation

The methylation of proteins is generally considered to be irreversible because demethylating enzymes in eukaryotic cells have been elusive. Recently, this paradigm has been challenged by the identification of an enzyme that can demethyliminate monomethylated arginine. [START_REF] Zhang | Molecular biology: no exception to reversibility[END_REF] Two research groups have reported that peptidylarginine deiminase 4 (PAD4), which has a nuclear localization, is able to convert the monomethylated arginine present in histone proteins to citrulline by hydrolytic cleavage of the monomethylamine group. [START_REF] Wang | Human PAD4 regulates histone arginine methylation levels via demethylimination[END_REF][START_REF] Cuthbert | Histone deimination antagonizes arginine methylation[END_REF] It should be noted that PAD4 does not seem to be able to deiminate ADMA [START_REF] Cuthbert | Histone deimination antagonizes arginine methylation[END_REF] but, as monomethylarginine is an intermediate product in the synthesis of ADMA, its deimination by PAD4 interferes with the synthesis of ADMA. In a strict sense, this deimination reaction is not a reversal of arginine methylation because it produces citrulline instead of arginine.

The reaction described above may be considered an exception to the rule. In general, protein methylation is a one-way process and methylated arginine residues remain an integral part of the protein until it is degraded by proteolysis.

Proteolysis of methylated proteins

Proteolysis of methylated proteins is the process that leads directly to the liberation of free ADMA, which, in contrast to protein-bound ADMA, serves as a competitive inhibitor of NOS. Protein synthesis and proteolysis are the anabolic and catabolic counterparts of protein turnover, respectively. Under physiological conditions, where no net protein synthesis or degradation occurs, both aspects are in balance. Proteolysis ultimately results in the complete degradation of proteins down to the level of free amino acids, which are re-utilized for de novo synthesis of proteins. Amino acids that have been modified by post-translational processes cannot be re-utilized for protein synthesis and are either excreted or metabolized. Whole-body protein turnover has been estimated at 300 g/day. [START_REF] Achan | Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase[END_REF] Individual proteins are destroyed at widely differing rates, with half-life times ranging from several minutes to many years. [START_REF] Glickman | The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction[END_REF] It is very likely that the turnover of methylated proteins occurs at a high rate. In contrast to other post-translational protein modifications that are reversible, such as phosphorylation, arginine methylation is virtually irreversible. This implies that, if arginine methylation is considered as a switch that turns on a particular function of a protein, the only means to switch off that function is by degrading the methylated protein and replacing it by a newly synthesized unmethylated copy.

Rapid in vitro proteolysis of methylated proteins

We have obtained experimental in vitro evidence that proteins containing methylated arginine are degraded at a very high rate. Incubation of a homogenate of rat kidney at 37ЊC resulted in a very rapid release of free ADMA, which was subsequently degraded by DDAH (Figure 3A). If ADMA degradation was prevented by blocking the activity of DDAH, free ADMA accumulated to a much higher concentration. Comparison with total ADMA (i.e. the sum of free and proteinbound ADMA, measured after acid hydrolysis) revealed that 66% of the protein-bound ADMA was released after 60 minutes (Figure 3B). The amount of protein-bound SDMA was approximately six-fold lower in comparison with protein-bound ADMA, but, in agreement with the data on ADMA, proteolysis resulted in the liberation of more than 60% of SDMA after 60 minutes (Figure 3C). In contrast, the release of protein-bound arginine occurred at a much lower rate, and was less than 8% at the end of the incubation period (Figure 3D). Although the conditions used in this experiment were unphysiological, it shows convincingly that the proteolytic machinery of the cell has the capacity to degrade methylated proteins at a very high rate.

Potential role of the proteasome

We can only speculate on the mechanisms involved in the accelerated proteolysis of proteins containing methylated arginine residues. Most intracellular proteins are degraded by the proteasome, which serves as a quality-control system by rapidly degrading misfolded and damaged proteins, whose accumulation would otherwise interfere with normal cellular function. [START_REF] Glickman | The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction[END_REF][START_REF] Goldberg | Protein degradation and protection against misfolded or damaged proteins[END_REF] The 26S proteasome is a large multiprotein complex, consisting of a barrel-shaped core with combined tryptic, chymotryptic and caspase-like protease activities (the 20S proteasome), which is capped at the ends by 19S regulatory complexes. In contrast to simple proteolytic enzymes, which cut their substrate at a single spot in a scissor-like fashion, the proteasome acts more like a shredder that in a single pass cuts a protein molecule into a large number of peptides. Proteins destined for degradation by the proteasome are conjugated to ubiquitine, which serves as a recognition signal for the regulatory subunits of the 26S proteasome. However, there are strong indications that proteins damaged by oxidative processes do not require ubiquitination, but are directly degraded by the 20S proteasome. [START_REF] Shringarpure | Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome[END_REF][START_REF] Orlowski | Ubiquitin-independent proteolytic functions of the proteasome[END_REF] Oxidation of proteins leads to destabilization of their tertiary structure, resulting in exposure of hydrophobic patches that are normally buried in the interior of the protein. It has been suggested that exposure of these hydrophobic sequences may trigger proteasomal degradation. [START_REF] Giulivi | Exposure of hydrophobic moieties promotes the selective degradation of hydrogen peroxide-modified hemoglobin by the multicatalytic proteinase complex, proteasome[END_REF][START_REF] Mehlhase | Proteolytic response to oxidative stress in mammalian cells[END_REF] It is tempting to speculate that methylation of arginine residues, which also increases the hydrophobicity of the protein surface, functions as a similar signal. The proteasomal machinery is very effective in cleaving proteins into peptides of approximately 3-22 amino acids in length, with a mean length of 6-9 residues. [START_REF] Kisselev | The sizes of peptides generated from protein by mammalian 26 and 20 S proteasomes. Implications for understanding the degradative mechanism and antigen presentation[END_REF] Final degradation of these peptides into individual amino acids thus requires the adjunctive action of intracellular endo-and exopeptidases. Whether peptidases with specificity for methylated arginine residues exist remains to be established.

Degradation of ADMA by DDAH

Intracellular ADMA is metabolized to citrulline and dimethylamine, a reaction catalyzed by DDAH. [START_REF] Ogawa | Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney[END_REF][START_REF] Macallister | Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase[END_REF] Two isoforms of DDAH have been identified that are widely expressed in rat and human tissues. [START_REF] Kimoto | Detection of NG,NG-dimethylarginine dimethylaminohydrolase in the nitric oxide-generating systems of rats using monoclonal antibody[END_REF][START_REF] Tran | Chromosomal localization, gene structure, and expression pattern of DDAH1: comparison with DDAH2 and implications for evolutionary origins[END_REF][START_REF] Leiper | Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases[END_REF] A small part of ADMA is cleared by urinary excretion, but it has been estimated that more than 80% is metabolized by DDAH. [START_REF] Achan | Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase[END_REF] The very high K m value (Michaelis-Menten constant) of DDAH and its sensitivity to oxidative stress are two peculiar features of this enzyme that will be discussed in the following sections.

Kinetic properties of DDAH

There is abundant evidence that the activity of DDAH plays a critical role in regulating intracellular ADMA levels. On the one hand, impairment of DDAH activity, with subsequent elevated ADMA levels and reduced production of nitric oxide, can accelerate atherosclerosis. [START_REF] Dayoub | Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis. Genetic and physiological evidence[END_REF] On the other, ADMA acts as an endogenous inhibitor of angiogenesis, [START_REF] Cooke | NO and angiogenesis[END_REF] and it has been shown that overexpression of DDAH may promote expression of vascular endothelial growth factor and induce tumor neovascularization. [START_REF] Smith | Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype[END_REF][START_REF] Kostourou | Effects of overexpression of dimethylarginine dimethylaminohydrolase on tumor angiogenesis assessed by susceptibility magnetic resonance imaging[END_REF] Apparently, because neither very high nor very low ADMA levels are desirable, ADMA concentrations must be kept between certain limits. The K m of DDAH of approximately 180 mol/l, 38 much higher than normal intracellular concentrations, may endow the enzyme with the properties to accomplish this task. It ensures that under normal conditions the enzyme works in the linear part of the substrate-velocity curve, which means that the rate of ADMA degradation is roughly proportional to its concentration. This is illustrated in Figure 3A, which shows that at low ADMA concentrations DDAH activity is also very low, leading to a rise in ADMA levels during rapid proteolysis. As the concentration of ADMA increases, DDAH activity also goes up, thereby preventing the accumulation of ADMA to very high levels. The high K m value of DDAH may thus provide buffering of ADMA concentrations, ensuring the presence of sufficient amounts of ADMA to allow its function as a regulator of NOS activity, while at the same time serving as a safety valve by rapidly degrading ADMA under conditions of excess supply.

DDAH and oxidative stress

DDAH is very sensitive to oxidative stress because the active site of the enzyme contains a critical sulfhydryl group that is required for its catalytic activity. [START_REF] Murray-Rust | Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylaminohydrolase[END_REF] Consequently, pathologic stimuli that induce oxidative stress in endothelial cells, such as inflammatory cytokines, hyperhomocysteinemia, hyperglycemia, infectious agents and oxidized low-density lipoprotein (LDL), may reduce DDAH activity and allow ADMA to accumulate. [START_REF] Ito | Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase[END_REF][START_REF] Leiper | S-nitrosylation of dimethylarginine dimethylaminohydrolase regulates enzyme activity: further interactions between nitric oxide synthase and dimethylarginine dimethylaminohydrolase[END_REF][START_REF] Lin | Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase[END_REF][START_REF] Stühlinger | Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine[END_REF][START_REF] Cooke | Asymmetrical dimethylarginine: the über marker?[END_REF] It is interesting to note that inhibition of endothelial NOS by elevated ADMA levels may lead to uncoupling of NOS, resulting in a shift from NO production to superoxide production. [START_REF] Böger | An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes[END_REF][START_REF] Sydow | ADMA and oxidative stress[END_REF] The fact that ADMA itself may induce oxidative stress suggests that under some pathologic conditions the DDAH-ADMA-NOS system may be precipitated into a vicious circle.

The effect of therapy with anti-oxidant vitamins on ADMA levels has not been studied thoroughly, but the results of two small studies suggest that vitamins C and E have no effect. [START_REF] Engler | Antioxidant vitamins C and E improve endothelial function in children with hyperlipidemia. Endothelial Assessment of Risk from Lipids in Youth (EARLY) trial[END_REF][START_REF] Saran | Impact of vitamin E on plasma asymmetric dimethylarginine (ADMA) in chronic kidney disease (CKD): a pilot study[END_REF] In contrast, estrogens, which also have anti-oxidant properties, seem promising. Several in vitro and in vivo studies on the effect of estrogen on ADMA metabolism have been conducted. Exposure of human and murine endothelial cells to estradiol resulted in an increase in the activity of DDAH and was accompanied by a reduced release of ADMA. [START_REF] Holden | Estrogen stimulates dimethylarginine dimethylaminohydrolase activity and the metabolism of asymmetric dimethylarginine[END_REF] Injection of rats with LDL increased serum levels of ADMA and reduced the endotheliumdependent relaxation of aortic rings. Both effects were attenuated by pretreatment with estradiol. [START_REF] Dai | 17beta-Estradiol preserves endothelial function by reduction of the endogenous nitric oxide synthase inhibitor level[END_REF] In addition, a negative association between ADMA and endogenous estrogen levels in women with coronary heart disease has been described. [START_REF] Li | Effect of endogenous estrogen on endothelial function in women with coronary heart disease and its mechanism[END_REF] We have studied the ADMA lowering effect of estrogens in the setting of hormone replacement therapy in postmenopausal women. [START_REF] Post | Effect of hormone replacement therapy on plasma levels of the cardiovascular risk factor asymmetric dimethylarginine: a randomized, placebo-controlled 12-week study in healthy early postmenopausal women[END_REF][START_REF] Teerlink | Oestrogen replacement therapy lowers plasma levels of asymmetrical dimethylarginine in healthy postmenopausal women[END_REF] Both conjugated estrogen and estradiol reduced plasma ADMA concentrations to a similar extent, but the effect of estradiol was found to be strongly enhanced if administered in combination with the synthetic progestogen trimegestone. [START_REF] Post | Effect of hormone replacement therapy on plasma levels of the cardiovascular risk factor asymmetric dimethylarginine: a randomized, placebo-controlled 12-week study in healthy early postmenopausal women[END_REF][START_REF] Teerlink | Oestrogen replacement therapy lowers plasma levels of asymmetrical dimethylarginine in healthy postmenopausal women[END_REF] It is striking that this combination of hormones lowered not only ADMA but also arginine levels. It is tempting to speculate that arginine levels were reduced by an increased consumption of arginine by NOS, resulting from diminished inhibition of NOS by ADMA.

Role of amino acid transporters

Both the generation of free ADMA by proteolysis of methylated proteins and its degradation by DDAH are intracellular processes. Most of the ADMA is probably rapidly degraded in the cytosol of the same cell where it was made. However, a small part may escape degradation and leave the cell to enter the bloodstream. Transport of basic amino acids such as arginine, lysine and ornithine across the cell membrane is facilitated by functionally distinct transport systems, principally systems yϩ and yϩL. System yϩ belongs to a family of CAT that exclusively transports cationic amino acids, whereas system yϩL transports both cationic and neutral amino acids. [START_REF] Mann | Regulation of amino acid and glucose transporters in endothelial and smooth muscle cells[END_REF] These transporters mediate the bidirectional transport of cationic amino acids, thus supporting important metabolic functions such as protein synthesis, NO production, polyamine biosynthesis, and interorgan amino acid flow. Arginine transport through both yϩ and yϩL systems is involved in NO synthesis in endothelial cells, [START_REF] Arancibia-Garavilla | Nitric oxide synthesis requires activity of the cationic and neutral amino acid transport system y ϩ L in human umbilical vein endothelium[END_REF] whereas arginine uptake by platelets is exclusively mediated by system yϩL. [START_REF] Mendes Ribeiro | Identification of system y ϩ L as the high-affinity transporter for L-arginine in human platelets: upregulation of L-arginine influx in uraemia[END_REF] Altered expression of CAT has been implicated in several clinical conditions, including congestive heart failure, chronic and acute renal failure, and septic shock. [START_REF] Reade | Increased cationic amino acid flux through a newly expressed transporter in cells overproducing nitric oxide from patients with septic shock[END_REF][START_REF] Mendes Ribeiro | Abnormalities in L-arginine transport and nitric oxide biosynthesis in chronic renal and heart failure[END_REF][START_REF] Kaye | In vivo and in vitro evidence for impaired arginine transport in human heart failure[END_REF] It has been shown that system yϩ is capable of transporting both ADMA and SDMA across the cell membrane. [START_REF] Closs | Interference of L-arginine analogues with L-arginine transport mediated by the yϩ carrier hCAT-2B[END_REF] Changes in the expression of CAT may therefore not only influence the interorgan transport and cellular uptake of arginine, but also of ADMA and SDMA. In addition, both methylated arginines may compete with arginine for cellular uptake. It is interesting that both endothelial NOS and system yϩ are localized to caveolae in the plasma membrane. [START_REF] Mcdonald | A caveolar complex between the cationic amino acid transporter 1 and endothelial nitric-oxide synthase may explain the 'arginine paradox[END_REF] This colocalization ensures an efficient supply of plasma arginine to NOS, but it may also provide plasma ADMA with direct access to NOS.

Metabolism of ADMA at organ level

Several studies have shown that ADMA accumulates in renal failure and is associated with cardiovascular mortality in end-stage renal disease. [START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF][START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF] The human kidney is responsible for urinary excretion of both ADMA and SDMA but, in addition, the kidneys clear a substantial amount of ADMA by metabolic degradation, probably by DDAH. [START_REF] Nijveldt | Net renal extraction of asymmetrical (ADMA) and symmetrical (SDMA) dimethylarginine in fasting humans[END_REF] Apparently, the kidney plays an important role in the metabolism of ADMA, but less information on the role of other organs in the clearance of ADMA is available. In recent years our group has studied the metabolism of both ADMA and SDMA at organ level in the rat. [START_REF] Nijveldt | The liver is an important organ in the metabolism of asymmetrical dimethylarginine (ADMA)[END_REF][START_REF] Nijveldt | Handling of asymmetrical dimethylarginine and symmetrical dimethylarginine by the rat kidney under basal conditions and during endotoxaemia[END_REF][START_REF] Nijveldt | Gut and liver handling of asymmetric and symmetric dimethylarginine in the rat under basal conditions and during endotoxemia[END_REF] In the experimental design, arteriovenous concentration differences and blood flow through the organ were measured, allowing the calculation of fractional extraction and net organ fluxes. Arteriovenous concentration differences were determined by a high-performance liquid chromatography method with high precision and sensitivity [START_REF] Teerlink | Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography[END_REF] and blood flow through individual organs was measured using radioactive microspheres. Data on renal and hepatic clearance of ADMA and SDMA in the rat are summarized in Figure 4. Fractional extraction of ADMA (i.e. the percentage that is cleared from the plasma) is slightly higher in the kidney than in the liver (Figure 4A). However, because total plasma flow through the liver is higher than through the kidney, the liver clears more ADMA from the circulation than the kidney, as evidenced by the higher organ flux of the liver (Figure 4B). Comparable data for SDMA show that SDMA is cleared by the kidney, whereas extraction by the liver is negligible. It can be concluded that the liver plays an important role in the metabolism of ADMA by taking up large amounts from the systemic circulation. Endotoxemia, induced by the administration of lipopolysaccharide, resulted in a lower systemic ADMA concentration, in combination with a reduced uptake of ADMA by the kidney and an increased uptake by the liver. [START_REF] Nijveldt | Handling of asymmetrical dimethylarginine and symmetrical dimethylarginine by the rat kidney under basal conditions and during endotoxaemia[END_REF][START_REF] Nijveldt | Gut and liver handling of asymmetric and symmetric dimethylarginine in the rat under basal conditions and during endotoxemia[END_REF] Mittermayer et al have studied the effect of endotoxemia on plasma ADMA levels in humans. No effect on ADMA or SDMA levels was observed, but a decreased arginine level and arginine/ADMA ratio were found. [START_REF] Mittermayer | Acute Escherichia coli endotoxaemia decreases the plasma L-arginine/asymmetrical dimethylarginine ratio in humans[END_REF] The potential role of the liver in the metabolism of ADMA was already recognized in 1977 by Carnegie et al, who described increased urinary excretion of ADMA in patients with liver disease. [START_REF] Carnegie | Urinary excretion of methylarginine in human disease[END_REF] We have performed several clinical studies that confirm the essential role of the liver in the elimination of ADMA in humans. In patients undergoing major liver resection, ADMA levels were significantly elevated in a subgroup with prolonged postoperative hepatic injury. [START_REF] Nijveldt | Elevation of asymmetric dimethylarginine (ADMA) in patients developing hepatic failure after major hepatectomy[END_REF] In a prospective study in patients undergoing liver transplantation, preoperative ADMA concentrations were elevated, especially in those with chronic hepatic failure. [START_REF] Siroen | The transplanted liver graft is capable of clearing asymmetric dimethylarginine[END_REF] After transplantation, ADMA concentration decreased very rapidly but, in patients who experienced acute rejection, ADMA concentrations were higher during the first postoperative month compared with nonrejectors. These results are in line with reports by other investigators on elevated plasma levels of ADMA in patients with end-stage liver disease and alcoholic cirrhosis. [START_REF] Tsikas | Elevated plasma and urine levels of ADMA and 15(S)-8-iso-PGF2alpha in end-stage liver disease[END_REF][START_REF] Lluch | Plasma concentrations of nitric oxide and asymmetric dimethylarginine in human alcoholic cirrhosis[END_REF] In a population of critically ill patients with clinical evidence of dysfunction of two or more organs, plasma ADMA concentrations were elevated and significantly related to hepatic function. [START_REF] Nijveldt | Asymmetrical dimethylarginine (ADMA) in critically ill patients: high plasma ADMA concentration is an independent risk factor of ICU mortality[END_REF] Moreover, plasma ADMA concentration emerged as a strong and independent risk factor for mortality in these patients. Based on these findings we hypothesize that the accumulation of ADMA is a causative factor in the development of multiple organ failure by interfering with important physiological functions of NO production. 83

Conclusions

The sequence of events leading to the generation of free ADMA (i.e. methylation of arginine residues by PRMTs and proteolysis) is not well characterized. Often, the formation of ADMA is regarded as an inevitable byproduct of protein turnover. This seems an oversimplification because both methylation and the breakdown of proteins are highly regulated processes. The crucial role of DDAH in the degradation of ADMA and its extreme sensitivity to oxidative stress are well documented. The bulk of ADMA is metabolized by DDAH, but this degradation may occur in a different cell or organ from the one in which ADMA is formed. CAT play an important role in trafficking of ADMA between cells and organs. The kidney and the liver play a significant role as sinks for excess ADMA. The interorgan transport of ADMA may have serious consequences in disease states. Overproduction or defective clearance of ADMA in a single organ may lead to its accumulation in other organs, thereby impairing their function.

Figure 1

 1 Figure 1 Schematic overview of the metabolism and clearance of asymmetric dimethylarginine (ADMA).Arginine residues of proteins are methylated by protein arginine methyltransferases (PRMT). In this transmethylation process, S-adenosylmethionine (SAM), which serves as donor of the methyl group, is converted into S-adenosylhomocysteine (SAH). SAH is hydrolyzed to homocysteine, which can be remethylated to methionine. Free ADMA is formed on proteolysis of methylated proteins. Most ADMA is hydrolyzed by intracellular dimethylarginine dimethylaminohydrolase (DDAH) into citrulline and dimethylamine. Some ADMA is exported from the cell by cationic amino acid transporters (CAT). A small fraction of circulating ADMA is excreted by the kidneys, but most ADMA is taken up by other cells or organs and degraded by DDAH.

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Figure 2

 2 Figure 2 Scheme depicting the dual role of autoantibodies against double-stranded DNA (dsDNA) in systemic lupus erythematosus (SLE). These autoantibodies increase the production of asymmetric dimethylarginine (ADMA) by stimulating the asymmetric dimethylation of arginine residues in arginine-glycine-rich domains of ribonucleoproteins. Elevated ADMA levels accelerate atherosclerosis by inhibiting nitric oxide (NO) synthesis. In addition, methylation of ribonucleoproteins leads to enhanced expression of inflammatory cytokines, thereby exacerbating SLE disease activity and atherogenesis.

Figure 3

 3 Figure 3 Rapid in vitro proteolysis of methylated proteins. Rat kidney was homogenized in saline and incubated at 37ЊC. At specified times, an aliquot was analyzed for free asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and arginine (open triangles). The total amount of ADMA, SDMA, and arginine (i.e. the sum of free and protein-bound amino acids) was determined after acid hydrolysis (closed circles). Analysis of ADMA was performed after incubation in the absence (panel A) and presence (panel B) of a large excess monomethylarginine, which inhibits degradation of ADMA by dimethylarginine dimethylaminohydrolase (DDAH).

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Figure 4

 4 Figure 4 Renal and hepatic clearance of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in the rat. Blood flow through the organs was measured by injection of radiolabeled microspheres and concentrations of ADMA and SDMA were determined in the renal, hepatic and portal veins, and in the aorta. From these measurements the fractional extraction (panel A) and organ flux (panel B) for ADMA (open bars) and SDMA (filled bars) were calculated. Error bars indicate standard error of the mean.

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Acknowledgments

The author gratefully acknowledges the skillful laboratory assistance of Sigrid de Jong (Department of Clinical Chemistry) and the collaboration of Irene Bultink and Alexandre Voskuyl (Department of Rheumatology) and Robert Nijveldt, Michiel Siroen and Paul van Leeuwen (Department of Surgery) of the VU University Medical Center.