Thomas Thum
email: thum_t@klinik.uni-wuerzburg.de

Johann Bauersachs

Bayrische Julius

Spotlight on endothelial progenitor cell inhibitors: short review

Keywords: drug therapy, endothelial progenitor cells, endothelium, nitric oxide, reactive oxygen species

Endothelial progenitor cells (EPCs) are bone-marrow-derived cells that enter the systemic circulation to replace defective or injured mature endothelial cells. EPCs also contribute to neovascularization and limit the progression of atherosclero- sis. Patients with reduced EPC levels or dysfunctional EPCs are at increased risk for coronary artery disease. Drug-mediated improvement of the mobilization, differenti- ation, function and homing of EPCs to sites of ischemia or injured endothelium may therefore be a promising novel therapeutic approach for various cardiovascular dis- eases. On the other hand, endogenous inhibitors of EPCs could also be valuable drug targets. The identification of EPC inhibitors and the development of novel drugs that can efficiently regulate production or elimination of these molecules may also be a promising approach for the future treatment of atherosclerosis. In the present review we summarize potential endogenous and exogenous inhibitors of EPCs, such as oxidized low-density lipoproteins, angiotensin II, glucose, cigarette smoke and others. Whenever possible, we also describe the underlying molecular events. Druginduced mobilization and improvement of EPC function, as well as reduction of EPC inhibitors, is likely to enhance endothelial function and reduce atherosclerotic processes.

Introduction

Endothelial progenitor cells (EPCs) are bone-marrow- derived cells that may enter the systemic circulation to replace diseased mature endothelial cells in blood ves- sels.1 Endothelial cell injury is the main stimulus for the development of atherosclerotic plaques.2 EPCs contribute significantly to endothelial function and may represent a circulating repair pool of endothelial cells.3-5 Suppression of this pool is considered to have detrimental effects on the cardiovascular system.

Patients with reduced numbers of EPCs are at increased risk for endothelial injury and, in general, for atherosclerotic plaque formation 4 Moreover, inad- equate coronary collateral development in patients with coronary heart disease is associated with reduced numbers of circulating EPCs.6 Both endothelial func- tion and damage appear to be the result of an altered balance between endothelial injury and the capacity for repair (Figure 1 ) . In a mathematical model it was demonstrated that only minor elevations in the amount of circulating EPCs could significantly delay endothelial layer defects over time.7 This is, however, only a theoretical model and does not account for EPC functional capacity, which, besides the number of EPCs, appears to be a major determinant of EPC- mediated beneficial effects.8 In general, it is assumed that increasing the amount and enhancing the function of EPCs will improve cardiovascular outcome.

Thus, much attention has been given to drugs that enhance the peripheral EPC pool, such as granulocytemacropage colony-stimulating factor,9 various HMG- CoA (3-hydroxyl-3-methylglutaryl coenzyme A) reductase inhibitors,1°,&dquo; erythropoietinl2 and, recently, angiotension-converting enzyme (ACE) inhibitors.13 3 Drug-mediated improvement of EPC mobilization and function was shown to limit atherosclerosis and restenotic processes in coronary and carotid arteries, and to enhance re-endothelialization and neovascularization after myocardial infarction, although long-term effects need to be identified. 14--16 Moreover, recent research is additionally strongly focused on the transplantation of progenitor cells 17,1 8 and, although early results are convincing, long-term outcomes and potential adverse side effects of this invasive procedure are unclear. [START_REF] Yoon | Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction[END_REF] Figure 1 Schematic overview of the potential role of EPC inhibitors on the mobilization, differentiation, homing and function of EPCs. Impairment of EPC-mediated vascular repair and direct toxic endothelial effects are likely to con- tribute to the development of endothelial dysfunction and atherosclerosis. ADMA, asymmetric dimethylarginine; AGE, advanced glycation end-products; EPC, endothelial progenitor cell; L-NAME, N( G)-nitro-L-arginine methyl ester; NO, nitric oxide; oxLDL, oxidized low-density lipoprotein; VEGF, vascular endothelial growth factor.

There is uncertainty about potential inhibitors of EPCs, although their therapeutic reduction could be an interesting way of enhancing the beneficial effects of EPCs. Understanding the molecular mechanisms of EPC mobilization, differentiation, homing, adhesion and function is a first step to developing useful drugs that may normalize disturbed EPC biology.

Endogenous inhibitors of EPCs

In general, the number of EPCs is tightly correlated with cardiac risk factors and the Framingham risk score.3,lS Certain endogenous mediators of vascular dysfunction and injury may not only adversely affect the mature endothelium of the vessel wall but diminish the number of circulating progenitor cells. .. Nitric oxide inhibitors and reactive ° ' ' oxygen species One of the most important functions of endothelial cells is the production of nitric oxide (NO) by the endothelial NO synthase (eNOS).2° Besides its well- known protective impact on vascular tone and wall homeostasis, NO is essentially involved in the mobi- lization and function of vascular progenitor cells. Indeed, Dimmler's group was the first to demonstrate that eNOS knockout mice suffer from decreased EPC mobilization. [START_REF] Aicher | Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells[END_REF] Moreover, a variety of EPC mobiliz- ing agents such as statins act, at least in part, via NO- mediated effects.14 Our group recently demonstrated that bone marrow-derived NO is correlated with enhanced EPC mobilization to the circulation after myocardial infarction (T Thum, D Fraccarollo, P Galuppo, T Tsikas, G Ertl, J Bauersachs, unpublished manuscript). Particular attention should therefore be given to potential endogenous inhibitors of NO availability that may adversely affect EPC mobiliza- tion and/or function. One interesting candidate could be the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA), which induces endothelial dysfunction22 and impairs angiogenesis in vivo. [START_REF] Jang | Angiogenesis is impaired by hypercholesterolemia -role of asymmetric dimethylarginine[END_REF] Furthermore, ADMA levels are closely correlated with coronary artery disease and acute coronary events.24,25

Preliminary data suggest that ADMA plasma levels are inversely correlated with circulating progenitor cells. [START_REF] Thum | Suppression of endothelial progenitor cells in human coronary artery disease by the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine[END_REF] The effects of ADMA on EPCs are currently under intense investigation. The treatment of cells with ADMA leads to increased intracellular production of reactive oxygen species (ROS) .27 ROS, in turn, may hamper the function of EPCs, although EPCs from healthy controls are equipped with anti-oxidative enzymes such as manganese superoxide dismutase. [START_REF] Dernbach | Anti-oxidative stress-associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress[END_REF] Recently, it has been shown that the endogenous release of ROS is higher in the EPCs of patients with coronary artery disease compared with healthy controls.29 Likewise, antioxidative enzymes were repressed in patients with coronary artery disease and showed markedly repressed migratory capacity.29 ROS should therefore be considered as an important endogenous inhibitor of EPCs.26

Oxidized lipoproteins

The direct toxic effects of oxidized low-density lipoproteins (oxLDL) on endothelium are well known.30,3 I Recent evidence also suggests adverse effects of oxLDL on EPCs.32 Treatment of cultured peripheral blood mononuclear cells with oxLDL reduces their differentiation into EPCs.32 Likewise, proliferation, migration and in vitro vasculogenesis were reduced in a concentration-dependent manner, demonstrating negative quantitative and qualitative effects on EPCs.33 Direct toxic effects of oxLDL include exaggerated production of ROS, enhanced expression of adhesion molecules, and downregulation of eNOS, leading to endothelial dysfunction. 30,31 Indirectly, by impairing EPC differentiation and func- tion, oxLDL probably reduces the endothelial repair pool, contributing to its pro-atherogenic effects.

Glucose and diabetes mellitus

Chronically elevated glucose levels in diabetic patients correlate with endothelial dysfunction and atherosclerosis.34,3s Tepper et al were the first to demonstrate that human EPCs from patients with type 2 diabetes exhibit impaired proliferation, adhesion and incorporation into vascular structures.36 Likewise, Loomans and colleagues determined levels of circu- lating EPCs in type 1 diabetes.37 They found that EPC numbers were reduced by 40-50%, which could explain the impaired wound angiogenesis and healing in diabetes, as well as reduced collateral formation in ischemia. Although these studies suggest increased glucose levels as an underlying cause, there is no conclusive evidence. Glucose-stimulated ROS pro- duction within EPCs may be involved. Indeed, high glucose levels stimulate ROS production via a protein kinase C-dependent activation of NADPH oxidase in endothelial cells. 38,39 Moreover, eNOS exposure to oxidants causes increased enzymatic uncoupling and generation of oxygen superoxides in diabetes.40 In addition, some of the vasculotoxic effects are medi- ated by advanced glycation end-products 41 Thus, more studies are required to determine the mechanism by which diabetes mellitus impairs EPC function.

Endostatin

Endostatin is an endogenous anti-angiogenic molecule that reduces the number and clonogenic potential of cir- culating EPCs 42 Cellular stress upregulates endothelial endostatin release43 and therefore enhanced endostatin levels may also result in impaired EPC mobilization and function, as well as progression of atherosclerosis in certain patients. Indeed, endostatin is enhanced in the pericardial fluid of patients with coronary artery disease and reduced formation of collaterals C-reactive protein As elevated levels of C-reactive protein (CRP) emerged as powerful predictors of cardiovascular disease, 45,46 their role in the biology of EPC may also be of importance. Indeed, human recombinant CRP directly inhibits EPC differentiation, survival, func- tion, angiogenesis and the response to chronic ischemia .47 These effects were attributed to a reduc- tion of eNOS expression in EPCs and could be pre- vented by pretreatment of EPCs with the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone. [START_REF] Verma | C-reactive protein attenuates endothelial progenitor cell survival, differentiation, and function: further evidence of a mechanistic link between C-reactive protein and cardiovascular disease[END_REF] Moreover, CRP additionally decreases secre- tion of arteriogenic chemokines in EPCs and may impair their vascular regenerative capacity. 48 Thus, elevated levels of CRP in patients with coronary artery disease46 could be mechanistically correlated with diminished EPC function in such patients, as reported previously.8 It is important to note that statins are able to reduce CRP levels,49 which may contribute to their well-known EPC-inducing properties.' ' I Angiotensin II Angiotensin II is the effector peptide of the renin- angiotensin system and has been implicated in the pathogenesis of atherosclerosis and coronary artery disease,. [START_REF] Schmidt-Ott | The multiple actions of angiotensin II in atherosclerosis[END_REF] Angiotensin II potentiates vascular endothe- lial growth factor (VEGF)-induced human EPC proliferation and network formation through upregulation of the VEGF receptor kinase domain-containing receptor after 24 hours of treatment.5' However, after longer periods (>7 days), angiotensin II has a significant inhibitory effect on the mitogenic activity of EPCs,.52

Moreover, angiotensin II accelerates the rate of senes- cence by diminishing telomerase activity in EPCs. [START_REF] Ferrara | Clinical applications of angiogenic growth factors and their inhibitors[END_REF] Thus, increased angiotensin II levels in various cardio- vascular diseases may increase atherosclerotic processes by enhanced senescence of circulating EPCs. Moreover, it has been reported that the treatment of patients with stable coronary artery disease with ACE inhibitors resulted in an increase of circulating EPCs. 13 3 Furthermore, in rats with myocardial infarction, we were able to demonstrate that ACE inhibition resulted in an improved matrix metalloproteinase 9-mediated mobilization of EPCs from bone marrow to the circu- lation (T Thum, D Fraccarollo, P Galuppo, T Tsikas, G Ertl, J Bauersachs, unpublished manuscript).

Exogenous inhibitors of EPCs Pharmaceutical drugs or other xenobiotics that reduce the circulating EPC pool or cause EPC dysfunction can theoretically impair endothelial function and promote atherosclerosis.

Although not investigated in this context so far, inhibitors of the VEGF protein could be such candi- dates. VEGF inhibitors were originally designed for anti-angiogenic cancer treatment and indeed early results are promising.54 However, VEGF also plays a role in the NO-mediated mobilization of progenitor cellslO,14 (and T Thum, D Fraccarollo, P Galuppo, T Tsikas, G Ertl, J Bauersachs, unpublished manuscript) and systemic inhibition may lead to impaired endothe- lial repair mechanisms.

Moreover, exogenous substances that affect the bioavailability of NO may additionally affect EPCs. Indeed, infusion with the NO synthase inhibitor N-nitro-L-arginine methyl ester or with ADMA resulted in a decrease of effective renal plasma flow, and an increase in renovascular resistance and blood pressure.55 It is likely that this treatment effectively reduces the eNOS activity of blood vessels and bone marrow, thus also leading to a reduction in EPC mobi- lization and function.

In addition, cigarette smoke was identified as an exogenous inhibitor of EPCs.56 Indeed, the number of circulating EPCs was reduced in chronic smokers and smoking cessation led to a rapid restoration of EPC levels. 57 This may be explained in part by the fact that smoking inhibits the release of physiological amounts of NO, which is important for EPC mobilization.21 Nicotine was found to increase the number and function of EPCs in vitro,57 as well as angiogenesis,58 so the responsible cigarette smoke constituents causing impairment of EPCs are still not known and need to be identified. ' Endogenous and exogenous inhibitors of EPCs are likely to contribute to endothelial dysfunction and atherosclerosis progression by impairment of the self-renewal capacity of endothelium. The identifica- tion of such substances is therefore of clinical importance. Therapeutic reduction of EPC inhibitors may be a promising approach for the future treatment of atherosclerosis.

Conclusion

Acknowledgment

This work was supported in part by the Ernst und Berta Grimmke-Stiftung (TT).


Stefanie M Bode-B&ouml

Fortunato Scalera

Jens Martens-Lobenhoffer

Professor Stefanie M Bode-B6ger
email: stefanie.bode-boeger@medizin.uni-magdeburg.de

S65 Asymmetric dimethylarginine (ADMA) accelerates cell senescence

Keywords: asymmetric dimethylarginine, DDAH, oxidative stress, senescence, telomerase activity

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and its accumulation has been associated with cardiovascular disease. We aimed to investigate the role of ADMA in endothelial cell senescence. Endothelial cells were cultured until the tenth passage. ADMA was replaced every 48 hours starting at the fourth passage. ADMA significantly accelerated senescence- associated β-galactosidase activity. Additionally, the shortening of telomere length was significantly speeded up and telomerase activity was significantly reduced. This effect was associated with an increase of oxidative stress: both allantoin, a marker of oxygen free radical generation, and intracellular reactive oxygen species increased significantly after ADMA treatment compared with control, whereas nitric oxide syn- thesis decreased. Furthermore, ADMA-increased oxidative stress was accompanied by a decrease in the activity of dimethylarginine dimethylaminohydrolase, the enzyme that degrades ADMA, which could be prevented by the antioxidant pyrroli- dine dithiocarbamate. Exogenous ADMA also stimulated secretion of monocyte chemotactic protein-1 and interleukin-8. Co-incubation with the methyltransferase inhibitor S -adenosylhomocysteine abolished the effects of ADMA. These data suggest that ADMA accelerates senescence, probably via increased oxygen radical formation by inhibiting nitric oxide elaboration. This study provides evidence that modest changes of intracellular ADMA levels are associated with significant effects on slowing down endothelial senescence.

Introduction

The incidence of atherosclerosis and cardiovascular disease increases dramatically with age. The links between aging and atherosclerosis are not well estab- lished. Common pathways at the cellular level have been proposed for aging and atherosclerosis. 1,2 There is accumulating evidence that telomeres and telom- erase play a role in cellular senescence. Telomeres, the physical ends of the chromosomes, are involved in the control of chromosome stability, genetic integrity and cell viability.3 Primary mammalian cells have a finite life span in tissue culture. Two sources of signals have been identified to cause the induction of senescence in cultured cells. Intrinsic signals can be induced by a critical telomere shortening, which is a consequence of telomere length reduction during each cell division ('mitotic clock').4 Cell culturing can induce stress signals that result in the expression of different cell cycle inhibitors, leading to a senescent phenotype.

Furthermore, a disturbance of the telomere-associated proteins can cause induction of senescence. Telomere shortening forces human primary cells, including endothelial cells, to stop dividing when a critical mini- mum telomere length is reached. [START_REF] Colgin | Telomere maintenance mechanisms and cellular immortalization[END_REF] The enzyme telom- erase, a ribonucleoprotein, counteracts the shortening of telomeres. Recently, it was reported that there is a higher rate of age-dependent telomere attrition in both the intima and the media of the distal compared with the proximal human abdominal aorta, accompanied by a negative correlation between telomere length and atherosclerotic grade.6 These links underscore the need for biological indicators of aging in evaluating the etiology of these age-related disorders. A novel risk factor for cardiovascular disease is asymmetric dimethylarginine (ADMA),7 an endogenous inhibitor of nitric oxide synthase (NOS). Elevations in plasma ADMA may contribute to the vascular pathophysiology observed in atherosclerosis, hypertension, hypercholesteremia and renal failure. Increased oxidative stress seems to play an important role in the pathogen- esis of these clinical conditions. Moreover, ADMA appears to be an independent predictor of cardiovascu- lar mortality.8 Recent findings suggest a significant positive correlation between age and plasma ADMA levels. 8,9 We have previously demonstrated that ADMA concentration dependently increased oxida- tive stress measured as superoxide production by cultured human endothelial cells It has been reported that cellular senescence, the limited ability of primary human cells to divide when cultured in vitro, is accelerated by increased oxidative stress 11 and is reduced by nitric oxide (NO) donor treatment.2 [START_REF] Vasa | Nitric oxide activates telomerase and delays endothelial cell senescence[END_REF] In the present study we therefore investigated if ADMA, a novel cardiovascular risk factor, influences cell senescence in cell culture and increases oxidative stress.

Materials and methods

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Cell culture

Human umbilical vein endothelial cells (HUVECs, Clonetics, Germany) were cultured in endothelial basal medium until the tenth passage. ADMA, S-adenosylhomocysteine (SAH) and pyrrolidine dithiocarbamate (PDTC, Sigma, Germany) were replaced every 48 hours starting at the fourth passage. After reaching con- fluence (between 8 and 9 days) endothelial cells were trypsinized and seeded at a density of 2500 cells/cm2 using 75 cm2 flasks or six-well plates. Population dou- bling (PD) was calculated at each passage until growth arrest by using the following formula: PD = (logj~Ylog)oX)/log)o2 (Y indicates the number of cells counted at the end the passage; X is the number of cells seeded). Cumulative population doubling (CPD) was calculated as the sum of all the changes in PD. The endothelial cells were counted and the vitality was determined. Viability after trypsinization was usually >95%. HUVECs were characterized at passage 10 by specific staining for CD31 (PECAM-1 ) using the fluorescence- activated flow cytometry assay (FACS). After detach- ment with trypsin telomerase activity, telomere length, FACS analyses or dimethylarginine dimethylaminohydrolase (DDAH) activity was performed.

_-

Staining for senescence-associated °(3-galactosidase activity HUVECs were fixed and stained for senescenceassociated r3-galactosidase (SA-r3-gal) activity accord- ing to the procedure developed by Dimri et al.12 The percentage of SA-r3-gal positive cells was determined by counting the number of blue cells in 1000 cells.

Telomerase assay and telomere length Quantitative determination of telomerase activity was performed according to the manufacturer's protocol of the TeloTAGGG Telomerase PCR ELISAPLUS Kit (Roche Diagnostic GmbH, Mannheim, Germany), which is based on the telomeric repeat amplification protocol assay. Telomerase activity was measured by the polymerase chain reaction using 1.5 pLg protein.

Telomere length was analysed by the terminal restric- tion fragment length assay using a Telo TAGGG Telomere Length Assay Kit (Roche).

Measurement of nitrate and nitrite

The determination of nitrate and nitrite in cell culture supernatants was carried out in accordance with the method described by Tsikas et al. 13 DDAH activity assay DDAH activity in endothelial cells was estimated directly by measuring the amount of ADMA metabo- lized by the enzyme as described by Lin et al. 14 The ADMA level in each group was measured by highperformance liquid chromatography-mass spectrome- try as previously described by our group. 15

Detection of oxidative stress

For the determination of allantoin (a marker of oxygen free radical generation) in cell culture supernatants we adopted the method for human plasma described by Doehner et al. 16 [START_REF] Okuda | Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis[END_REF] Dihydrorhodamine 123 (DHR) was used as a marker for intracellular reactive oxygen species (ROS). The cells were incubated with gentle agitation for 20 minutes at 37°C in the presence of 10 [LM DHR 123 . The reaction was stopped by cooling on dryice for 1 minute and the subsequent addition of 500 jjd phosphate buffered solution followed by two washing steps. After final fixation with 1% paraformaldehyde the cells were analyzed by flow cytometry (Epics XL-MCL, Coulter). The level of ROS was indicated by mean fluorescence intensities (mfi) of stained probes versus negative controls.

Measurement of monocyte chemotactic protein-1 and interleukin-8

For the quantitative determination of monocyte chemotactic protein-1 (MCP-1 ) and interleukin (IL)-8 the cell supernatants were collected, centrifuged and stored at -20°C until assayed. The MCP-1 and IL-8 protein concentrations were assayed using the Quantikine human MCP-1 and IL-8 immunoassay (R&D Systems, Wiesbaden, Germany).

Statistical methods

All data were expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical significance was tested with repeated analysis of variance (ANOVA) using a least significant difference post-hoc test or ANOVA for multiple comparisons (SPSS Software 11.0). Differences were considered significant at p < 0.05.

Results

ADMA accelerates endothelial senescence by inhibiting telomerase activity

To investigate the link between endothelial cell senes- cence and ADMA, human endothelial cells were cultured until CPD 40 and incubated in the presence of different concentrations of ADMA, which was replaced every 48 hours starting from CPD 20. All concentrations of ADMA (5, 50 and 100 jjbmol/l) significantly increased SA-r3-gal activity compared with untreated cells in a dose-dependent manner (Figure 1 A) .

To examine the effect of ADMA on shortening of telomere length, genomic DNA was isolated and separated, and telomere restriction fragment length was measured. The telomere length was shortened with increasing passage in control cells (Figure 1B). The repeated addition of different concentrations of ADMA increased significantly the telomere shorten- ing compared with control cells in a dose-dependent manner.

Because the synthesis of telomeres is associated with the enzymatic activity of an RNA-protein com- plex called telomerase, we measured the relative acti- vity of telomerase in HUVECs. Telomerase activity decreased as a function of passage in control cells and was significantly further reduced in a dose-dependent manner in the ADMA treated cells (Figure 1C).

SAH-inhibited ADMA-accelerated senescence

To test whether SAH could attenuate the effect of ADMA (50 ~,mol/1) on cellular senescence, HUVECs were incubated in the presence of SAH (100 ~,mol/1) starting from CPD 20. It is interesting that a more significant decrease in SA-(3-gal activity was observed in endothelial cells treated with SAH compared with the control cells (Figure 2A), with a maximal effect at the tenth passage (CPD 40). Additionally, the shortening of telomere length was substantially delayed and the telomerase activity was significantly increased (Figures 2B and2C). The increased senescence in the presence of ADMA was completely reversed in the presence of SAH. Exposure of endothelial cells to ADMA resulted in a significant reduction of NO synthesis (CPD 40: ADMA, 2.59 ± 0.4 nmol/mg protein versus control, 4.18 ± 0.3 nmol/mg protein; p < 0.05 versus control). After SAH treatment, NO levels were increased significantly compared with control cells (CPD 40: SAH, 5.44 ± 0.3 nmol/mg protein; p < 0.05 versus control). The inhibitory effects of ADMA on endothelial NO release were abolished by SAH (CPD 40: SAH + ADMA, 4.03 ± 0.7 nmol/mg protein; p < 0.05 versus ADMA). Incubation of endothelial cells with ADMA led to a significant increase in allantoin levels compared with control (CPD 40: ADMA, 41.2 ± 1.8 nmol/mg protein versus control, 30.3 ± 1.6 nmol/mg protein; p < 0.05 versus Figure 1 Effect of different concentrations of ADMA on senescence, telomere length and telomerase activity of endothelial cells, which were incubated with ADMA (5, 50 and 100 )jLmo!/L) starting from cumulative population doubling (CPD) 20 and replaced every 48 hours (A through C): (A) endothelial senescence was detected by senescence associated j3-galactosidase (SA-(3-gal) positive cells; (B) terminal restriction fragment (TRF) lengths are plotted as a function of passage number; (C) relative telomerase activity (RTA). Each point represents the mean ± SEM of results from three experiments. *p < 0.05 vs corresponding CPD of control cells. (Figure 1A, B and C reproduced by permission of the publisher Lippincott Williams & Wilkins from: Scalera F, Borlak J, Beckmann B et al. Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senescence. Arterioscler Thromb Vase Biol 2004; 24: 1816-22.17)

Figure 2 Effect of SAH on ADMA-accelerated senes- cence. Endothelial cells were incubated with ADMA (50/l-moI!L) and SAH (100 >mol/L) starting from the fourth passage (young) and replaced every 48 hours until the tenth passage (old): (A) endothelial senescence was detected by senescence-associated j3-galactosidase (SA-(3-gal) positive cells; (B) terminal restriction fragment (TRF) lengths are plotted as a function of passage number; (C) relative telomerase activity (RTA). Each point represents the mean ± SEM of results from three experiments. *p < 0.05 vs corresponding cumulative population doubling (CPD) of control cells. control). Incubation with SAH significantly decreased allantoin formation. The stimulatory effects of ADMA on allantoin production were reversed by co- incubating the cells with SAH.

ADMA-induced senescence was associated with an increase in oxidative stress and a decrease in DDAH enzyme activity Endogenous ROS formation was measured by detec- tion of DHR during endothelial cell aging. The intra- cellular level of ROS was increased in control cells, starting at CPD 29 (Figure 3A). ADMA (5 ~,mol/1) augmented ROS formation (p < 0.05). The antioxi- dant PDTC (10 ~.~,mol/1) prevented an increase endogenous oxidative stress by ADMA.

To examine whether ADMA-induced oxidative stress is due to reduced degradation of ADMA by DDAH, DDAH activity was determined during con- tinuous passaging of endothelial cells by assessing the rate of degradation of exogenous ADMA added to the Figure 3 ADMA-inhibited NO synthesis was associated with an increase in endogenous oxidative stress and a decrease in endothelial DDAH enzyme activity.

Endothelial cells were incubated with ADMA (5 /-Lmol/L) and pyrrolidine dithiocarbamate (PDTC) (10 [Lmol/L) starting from the fourth passage (young) and added every 48 hours until the tenth passage (old): (A) endogenous reactive oxygen species formation was measured using the marker dihydrorhodamine (DHR) and the fluorescence-activated flow cytometry assay; (B) effect of ADMA and PDTC on endothelial DDAH activity. Each point represents the mean ± SEM of results from three experiments. *p < 0.05 vs corresponding cumulative population doubling (CPD) of control cells. (Figure 3B repro- duced by permission of the publisher Lippincott Williams & Wilkins from: Scalera F, Borlak J, Beckmann B et al.

Endogenous nitric oxide synthesis inhibitor asymmetric dimethyl L-arginine accelerates endothelial cell senes- cence. Arterioscler Thromb Vasc Biol 2004; 24: 1816-22. 17)

cell lysates. Concomitantly with the significant increase in ROS formation, DDAH activity signifi- cantly decreased at CPD 29 in control cells (Figure 3B). The treatment of endothelial cells with ADMA reduced DDAH enzyme activity in cell lysates compared with untreated cells (p < 0.05). PDTC enhanced DDAH activity and prevented the impairment of DDAH activity by ADMA.

Furthermore, the effect of ADMA to inhibit NO synthesis was reversed by PDTC (data not shown).

Moreover, PDTC abolished the effect of ADMA on allantoin production and endothelial senescence (data not shown).

MCP-1 and IL-8 secretion increased during ADMA-induced endothelial cell aging

The treatment of cultured endothelial cells with ADMA (50 jjLmol/1) induced a significant secretion of MCP-1 and IL-8 compared with control (MCP-1: CPD 40 ADMA, 3073 ± 67 pg/ml versus control, 2683 ± 51 pg/ml; IL-8: ADMA, 2461 ± 60 pg/ml versus control, 1968 ± 30 pg/ml; p < 0.05 versus control, respectively). When cultured endothelial cells were incubated with SAH (100 ~,mol/1), secretion of MCP-1 and IL-8 was significantly reduced compared with untreated cells (MCP-1: CPD 40 SAH, 2407 ± 67 pg/ml; IL-8: SAH, 1753 ± 58 pg/ml; p < 0.05 versus control, respectively). The stimula- tory effects of ADMA on MCP-1 and IL-8 secretion were significantly diminished by SAH (MCP-1: SAH + ADMA, 2663 ± 52 pg/ml; IL-8: SAH + ADMA, 1849 ± 36 pg/ml; p < 0.05 versus ADMA, respectively).

Discussion

ADMA accelerates endothelial aging, as demonstrated by a more rapid reduction in telomere length and a decline in telomerase activity. The effect of ADMA on accelerating endothelial senescence seems to be due to increased oxidative stress because it can be reversed by the antioxidant PDTC. SAH significantly attenuates endothelial senescence, indicating that modest changes of intracellular ADMA levels are associated with substantial effects on endothelial cell senescence.

These data provide the first evidence for a novel mechanism of endothelial cell senescence mediated by ADMA. What is the underlying mechanism by which ADMA accelerates cellular senescence? We speculate that ADMA increases oxidative stress, for example, by uncoupling NOS activity. Uncoupling of NOS catalytic activity is observed in the presence of NG- monomethyl-L-arginine (L-NMMA),1g an endogenous inhibitor of NOS. In this situation optimal electron flow within the two catalytic domains of NOS is impaired and molecular oxygen becomes the sole electron acceptor. This means that NOS generates superoxide radicals. We have shown that ADMA con- centration dependently increases superoxide production by cultured human endothelial cells.10 In the experiments presented here, the inhibition of NOS by ADMA decreases the elaboration of NO and increases endothelial oxidative stress as measured by intracellu- lar ROS and allantoin production.

Telomere length is an important marker for cellular aging. Our data suggest that replicative senescence can be modulated by various intracellular processes. Endothelial cells are capable of synthesizing ADMA by the protein arginine methyltransferase type one. A major source of methyl groups used for various methylating reactions is S-adenosylmethionine, This methylation reaction is inhibited by SAH.19 We found that SAH attenuates endothelial senescence by increasing the activity of telomerase compared with control cells. The increased rate of endothelial senescence in the presence of ADMA was completely reversed in the presence of SAH.

The issue to be addressed is why the administration of exogenous SAH was seen to inhibit the effect of exogenous ADMA to produce its biological effects. However, it is hard to understand how an SAH- induced reduction of endogenous ADMA formation could fully counteract high concentrations of exoge- nous ADMA (50 [LM). A possible explanation would be the activation of DDAH, the enzyme that degrades ADMA.2° DDAH has a Km (Michaelis-Menten con- stant) for ADMA of about 180 [LM, so increased DDAH activity could overcome supraphysiological ADMA concentrations. This enzyme seems to be exquisitely sensitive to oxidative14,21 and nitrosative stress. 22 We observed a decrease in the formation of allantoin, reflecting the reduced generation of superoxide anions. As recently shown by Cooke's group, even modest changes in ADMA levels induced by DDAH activity can have significant effects on NO synthesis.23 We were able to show in this study that incubation with SAH significantly increases the synthesis of NO.

This finding may indicate that intracellular levels of ADMA are more important than extracellular or systemic levels and/or that there may be compartmentalization of intracellular ADMA, with much higher levels in the vicinity of NOS.

Moreover, we observed in this investigation that ADMA-increased intracellular ROS formation was accompanied by a corresponding decrease in the activity of DDAH. This finding is supported by the observation that co-incubation with PDTC, a thiol antioxidant that protects sulfhydryl groups, reduced ROS formation and consequently the impairment of DDAH activity during ADMA-accelerated senes- cence. Recently, Haendler et al reported that the onset of replicative senescence of endothelial cells is regu- lated by ROS formation. [START_REF] Haendler | Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells[END_REF] Similarly, in the present study, we found that ADMA increased oxidative stress and accelerated the rate of cellular senescence. This effect of ADMA was reversed by the antioxidant PDTC. Thus, a reduction in ADMA synthesis induced by SAH and increased DDAH activity induced by PDTC both delayed the onset of cellular senescence. We also investigated the effects of ADMA on the secretion of MCP-1 and IL-8 in endothelial cell super- natants. MCP-1 and IL-8 are major chemokines for leukocyte trafficking and have been found to be highly expressed in lesions of human atherosclerosis. [START_REF] Reape | Chemokines and atherosclerosis[END_REF] Upregulation of these proteins is induced by intra- cellular oxidative stress via the oxidant-responsive transcription factor NF-KB. 10,26 Indeed, we found that reduced NO production by ADMA was associated with increased oxidative stress and increased protein secretion of MCP-1 I and IL-8. These effects were completely reversed by SAH.

Clinical relevance

Plasma ADMA levels are positively correlated with risk factors for atherosclerosis. Miyazaki et al reported a positive correlation for ADMA with age in individu- als with no symptoms of coronary or peripheral artery disease. [START_REF] Miyazaki | Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis[END_REF] Celermajer et al reported that aging is associated with progressive endothelial dysfunction in normal humans.27 Furthermore, endothelial senescence is associated with decreases in eNOS expression and activity, as well as with augmented endothelial adhe- siveness for monocytes. These effects can be prevented by stably transfecting endothelial cells with human telomerase.28 We speculate that high plasma levels of ADMA may accelerate the loss of telomerase activity, increase cellular senescence and induce endothelial dysfunction. Our data provide a new mechanism to explain the association of plasma ADMA or endothelial dysfunction with an increased risk of cardiovascular events.


Tom Teerlink
email: t.teerlink@vumc.nl

S73 ADMA metabolism and clearance

Keywords: asymmetric dimethylarginine, cationic amino acid transporters, dimethylarginine dimethylaminohydrolase, methylation, nitric oxide, proteolysis

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 pro- teins 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 trans- porters 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 ' ~2 and its status as a cardiovascular risk factor is gradually gaining acceptance. Competitive inhibition of nitric oxide synthase (NOS) is the most likely mecha- nism for the effects of ADMA on the cardiovascular system,3 although nitric oxide (NO)-independent mechanisms cannot be excluded. 4 Only free ADMA, and not its protein-bound form, is capable of inhibit- ing 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 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 methion- ine. 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. 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 &dquo;

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.5 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 arginine- glycine-rich sequences of proteins.5 A recent pro- teomic analysis has identified more than 200 proteins that are putatively arginine methylated.6 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/B' are asymmetrically dimethylated at arginine residues in the nucleus[END_REF][START_REF] Smith | Arginine methylation of RNA helicase A determines its subcellular localization[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 synthe- sis 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, I 0 but this was not found in other studies . 11,12 2 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 homocys- teine is elevated, intracellular SAH levels may thus increase. Because SAH is a potent inhibitor of trans- methylation 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.17 It has also been shown that the production of ADMA by endothelial cells is inhibited by incubation with SAH. 18 8 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 inflamma- tory 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 autoanti- bodies specifically recognize the basic arginine-glycinerich domains of the proteins involved.] Notably, these domains are also preferred sites for the methylation of arginine residues by PRMTs.5,20 Indeed, several spliceosomal proteins have been shown to contain both ADMA and SDMA residues in their sequence.8,20

It was recently shown that autoantibodies to dsDNA cross-react with the arginine-glycine-rich domain of heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2).2 ~ In the presence of anti-dsDNA, methy- lation of hnRNP A2 by PRMTI was increased in a dose-dependent manner to 3.5 times the control level .2 Anti-dsDNA antibodies may therefore cause increased ADMA production by upregulating the dimethylation of arginine residues by PRMT 1. 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,22 but traditional Framingham risk factors fail fully to account for this excess of cardiovascular events.23 In addition, the endothelial dysfunction associated with SLE cannot be explained by classic risk factors.24 We have recently assessed the associa- tion between ADMA and cardiovascular disease in a cross-sectional study in 107 patients with SLE.25 The prevalence of cardiovascular disease increased across tertiles of plasma ADMA concentrations (p = 0.023). Most importantly, we observed a significant positive association between plasma ADMA concentrations and titres of anti-dsDNA autoantibodies (r = 0.43; p < 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 cells26 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). bodies 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.

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 para- digm has been challenged by the identification of an enzyme that can demethyliminate monomethylated arginine.27 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. 2829 It should be noted that PAD4 does not seem to be able to deiminate ADMA29 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 com- petitive 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 pro- teins 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. 30

Individual proteins are destroyed at widely differing rates, with half-life times ranging from several minutes to many years. 31 It is very likely that the turnover of methylated proteins occurs at a high rate.

In contrast to other post-translational protein modifi- cations 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 37°C 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 accumu- lated 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 con- vincingly that the proteolytic machinery of the cell has the capacity to degrade methylated proteins at a very high rate.. , ~ -. : ' .

-_. ~.~ _ : . :v ; &dquo; &dquo; Figure 3 Rapid in vitro proteolysis of methylated proteins. Rat kidney was homogenized in saline and incubated at 37°C. 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 argi- nine (i.e. the sum of free and protein-bound amino acids) was determined after acid hydrolysis (closed circles). Analysis of ADMA was performed after incuba- tion in the absence (panel A) and presence (panel B) of a large excess monomethylarginine, which inhibits degradation of ADMA by dimethylarginine dimethylaminohydrolase (DDAH).

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 mis- folded and damaged proteins, whose accumulation would otherwise interfere with normal cellular function.3 ~ ,32 The 26S proteasome is a large multiprotein complex, consisting of a barrel-shaped core with com- bined 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 recog- nition 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.33,34 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.3s,36 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.37 Final degradation of these peptides into individual amino acids thus requires the adjunctive action of intracellular endoand 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. 38,[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 40-42 A small part of ADMA is cleared by urinary excretion, but it has been estimated that more than 80% is metabolized by DDAH.3° The very high Km 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.43 On the other, ADMA acts as an endogenous inhibitor of angiogenesis,44 and it has been shown that overexpression of DDAH may pro- mote expression of vascular endothelial growth factor and induce tumor neovascularization. 45,46 Apparently, because neither very high nor very low ADMA levels are desirable, ADMA concentrations must be kept between certain limits. The Km of DDAH of approximately 180 pLmol/1,38 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 illus- trated in Figure 3A, which shows that at low ADMA concentrations DDAH activity is also very low, lead- ing to a rise in ADMA levels during rapid proteolysis.

As the concentration of ADMA increases, DDAH activity also goes up, thereby preventing the accumu- lation of ADMA to very high levels. The high Km 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. 47

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. 48-52 It is interesting to note that inhibi- tion of endothelial NOS by elevated ADMA levels may lead to uncoupling of NOS, resulting in a shift from NO production to superoxide production. 53,54 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.ss.s6 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.57 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.58 In addition, a negative association between ADMA and endogenous estrogen levels in women with coronary heart disease has been described. 59 We have studied the ADMA lowering effect of estrogens in the setting of hormone replacement therapy in postmenopausal women.60,61 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. 60,61 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 blood- stream. Transport of basic amino acids such as arginine, lysine and omithine across the cell mem- brane is facilitated by functionally distinct transport systems, principally systems y+ and y+L. System y+ belongs to a family of CAT that exclusively trans- ports cationic amino acids, whereas system y+L transports both cationic and neutral amino acids.62 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 y+ and y+L systems is involved in NO synthesis in endothelial cells,63 whereas arginine uptake by platelets is exclusively mediated by system y+L.64 Altered expression of CAT has been implicated in several clinical conditions, including congestive heart failure, chronic and acute renal failure, and septic shock. 65-67 It has been shown that system y+ 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 y+ 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 inter- esting that both endothelial NOS and system y+ are localized to caveolae in the plasma membrane.69 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.2>~° 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.71 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.72-74 In the experimental design, arteriovenous concentration differences and blood flow through the organ were measured, allow- ing the calculation of fractional extraction and net organ fluxes. Arteriovenous concentration differences were determined by a high-performance liquid chro- matography method with high precision and sensiti- vity75 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 extrac- tion 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.73, [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.76

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.77 We have per- formed 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 sub- group with prolonged postoperative hepatic injury.78

In a prospective study in patients undergoing liver Figure [START_REF] Suda | Asymmetric dimethylarginine causes arteriosclerotic lesions in endothelial nitric oxide synthase-deficient mice. Involvement of reninangiotensin system and oxidative stress[END_REF] 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 micros- pheres 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. transplantation, preoperative ADMA concentrations were elevated, especially in those with chronic hepatic failure.79 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.g°>gl [START_REF] Valkonen | Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine[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.82 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.

. '~J,.. , : = -.

Figure 2

 2 Figure 2 Scheme depicting the dual role of autoanti-

Acknowledgments

-v. ~.. The author gratefully acknowledges the skillful labo- ratory 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.


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

S89-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

Methylated L-arginine analogs are involved in nitric oxide synthase activity regulation. Methods based on high-performance liquid chromatography with fluorescence, capillary electrophoresis, or ion exchange chromatography with absorbance detection were first applied for the quantitative determination of N -monomethyl-L-arginine (NMMA), asymmetric dimethylarginine (ADMA) and sym- metric dimethylarginine (SDMA) in human blood and urine. These assays revealed elevated circulating levels of ADMA in various diseases and gave accumulating evidence of the usefulness of ADMA as a cardiovascular risk factor. However, the methods used are hampered by the fact that NMMA, ADMA and SDMA can be dis- tinguished from L-arginine only by means of chromatographic separation. This has promoted the development of alternatives that involve mass spectrometry (MS) technology. Today, various MS-based approaches such as liquid chromatography (LC)-MS, LC-MS/MS, gas chromatography (GC)-MS, and GC-MS/MS are available. L-arginine and its analogs have been subjected to LC-MS analysis with and without further derivatization to their o-phthaldialdehyde derivatives. For these methods, labelled L-arginine was used as the internal standard. The first MS-based method that distinguishes NMMA, ADMA, SDMA and L-arginine by mass-to-charge (m/z)- ratio has been reported by Tsikas et al. This GC-MS approach has been further improved by Albsmeier et al by introducing labelled ADMA as an internal standard. As an alternative to existing methods, a commercially available ELISA kit has recently been developed and validated.

Introduction

Qualification and quantification issues had been addressed several years before Vallance and coworkers introduced asymmetric dimethylarginine (NG, NG-dimethylarginine, ADMA) as an endogenous inhibitor of nitric oxide synthase (NOS).' 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 methyla- tion are ion chromatography and more recently liquid chromatography-mass spectrometry (LC-MS).3-7 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'dimethylarginine from the hydrolysate of proteins of bovine brain[END_REF] The ratio of ADMA over symmetric dimethylarginine (N~,N'G-dimethylarginine, SDMA) is notably increased in heart tissue (a 32-fold excess has been found).4 The functional consequence of such observa- tions 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.5-T hese 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 intro- duced by several groups using o-phthaldialdehyde (OPA) for precolumn derivatization and subsequent fluorescence (FL) detection.8-10 FL detection is carried out at excitation (À ex) and emission (Xlm) 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 compo- nents.&dquo; Baseline separation of OPA derivatives of ADMA and SDMA can be achieved by means of ana- lytical columns packed with octadecylsilica or phenyl silica, namely, reversed phase (RP) conditions. The analytical column should be at least 150 X 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: [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF] 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 acidl° 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.8 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+1 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 col- umn preconditioning becomes unnecessary. Stationary phases are modified with weak carboxylic acid (CBA) or strong cation exchange (SCX) resins. 8,9,12 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 recom- mended 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. 12,13 All recent protocols for HPLC deter- mination 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,14-16 NG-monomethyl-L-arginine (NMMA),12,13 and N°-propyl-L-arginine (N-PLA).17 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 sepa- ration on a phenyl column (250 X 4.6 mm, Macherey-Nagel, Duren, Germany).11 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.19,20 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 over- lapping signals cannot be excluded.

A major drawback of OPA derivatives is their insta- bility due to rapid decomposition, so on-line derivati- zation has to be performed. If this is not available, other derivatization agents may be used in combina- tion 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 7°C. 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 ±1.6% of the initial ratio).12 Further improvement of stability was reported for naphthalene-2,3dicarboxaldehyde (NDA) derivatives.17 Owing to the different nature of the NDA derivatives, wavelengths for FL detection have to be adjusted (~eX = 420 nm and Xem = 483 nm). However, compounds have to be stored at 4°C after derivatization and a linear decline of fluorescence signal by 15.9, 11 and 12.9% for derivatives of arginine, SDMA and ADMA, respec- tively, was observed after 72 hours.17 Finally, stable derivatives, even at room temperature, are obtained with the derivatizing agent 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AccQ-FluorTM, Waters, Milford, MA, USA).13 FL detection is carried Figure 1 Chromatogram of a combined standard containing 25 ~,M arginine, 10 [LM homoarginine, 1 J-LM symmetric dimethylarginine (SDMA), and 1 VLM 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 x 4.6 mm, Macherey-Nagel, Duren, Germany),11 Dashed line indicates gain switch of the fluorescence detector. Peak identification: 1, arginine; 2, homoarginine; 3, SDMA; 4, ADMA.

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

aconcentrations and detection limits are given in )jLmot/! unless otherwise indicated; bdetermined by our laboratory; climit of quantification; dlimit of detection in plasma; eanalytical sensitivity; APCI, atmospheric-pressure chemical ionisation; CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionisation; GC, gas chromatography; liquid chromatography; MS, mass spectrometry; NDA, naphthalene-2,3-dicarboxaldehyde; OPA, o-phthaldialdehyde; PFP, pentafluoropropionyl.

out at ~eX = 250 nm and ~em = 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 post- column derivatization of amino acids.21 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, chromato- graphic 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 mass- to-charge ratios (i.e. m/z 351, 352 and 365, respectively).22,23 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 manda- tory. OPA derivatives seem to be unsuited because of their complex fragmentation pattern after MS-MS fragmentation.22 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, respec- tively.24 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 HCI 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.25,26 ADMA and SDMA exhibit identical parent ions after positive atmospheric pressure chemical ionization or ESI (i.e. m/z 203).2s,26

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 (2:: 10%). Only one major fragment is exclu- sively 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.26 To over- come 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 suppression27 at this low m/z ratio, a teicoplanin column can be used. 28

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 C6]arginine (98% 13C isotopic purity, Cambridge Isotope Laboratories, Andover, MA, USA) and L-[guanidine-

15 N2]arginine (98% 15N isotopic purity, Cambridge Isotope Laboratories) have been used as internal standards for the quantification of L-arginine in LC- and gas chromatography (GC)-MS.22,23,29,30 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,29 or easily prepared in the laboratory. Synthesis follows the synthetic route described by Pundak and Wilchek.3'

L-[3,3,4,4,5,5,-2H6]Ornithine (>98% 15N isotopic purity, Euriso-top, Saint Aubin, France) or

[2H6]dimethylamine gas, free base (99% 2H isotopic purity, Cambridge Isotope Laboratories) were used as labeled educts. 23,24,30 An appropriate stable isotope- labeled 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).32

Thus, analytes are converted to their methyl ester N-pentafluoropropionyl amide derivatives and subse- quently analyzed in the NICI mode with GC-MS or GC-tandem MS.29,30 NMMA and ADMA are ana- lyzed 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 ± 1.30 In GC- tandem 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 deriva- tives, 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 ± 30, suggesting a LOD of approximately 10 amol of ADMA.29 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 with- out 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 ± 0.097 and 1.022 ± 0.034 for human urine and plasma, respectively.29 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 Figure 2 Interlab 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 (jbM ADMA, and three equal samples spiked with 5 )Jb!B/) 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.. ~ '. -available.33 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 RM ADMA, and three samples spiked with 5 VLM 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, avail- ability and rapidity are important for analytical meth- ods 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 selec- tivity. 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 compar- ison should be carried out according to the rules of Bland and Altman.36 Nevertheless, in clinical research a single comparison may not be adequate. It is there- fore indispensable to have quality controls and inter- lab 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.

,