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Asymmetric dimethylarginine, smoking, and risk of coronary heart disease in apparently healthy men: prospective analysis from the population-based Monitoring of Trends and Determinants in Cardiovascular Disease/Kooperative Gesundheitsforschung in der Region Augsburg study and experimental data.

Accumulating evidence is linking asymmetric dimethylarginine (ADMA) [4] an endogenous inhibitor of all major isoforms of nitric oxide synthase (NOS), to human disease (1, 2). Increased ADMA plasma concentrations are found in various clinical settings ranging from renal failure and liver failure to atherosclerosis, hypertension, and impaired glucose tolerance (3-6). Moreover, increase of ADMA has been identified as an independent risk factor for progression of atherosclerosis, cardiovascular death, and total mortality in patients with coronary heart disease (CHD) (7-9) and renal failure (10,11) and in critically ill patients (12). In the only large prospective study that included patients with and without CHD, all smokers (29% of patients) had been excluded from the main statistical analysis, and a significantly increased risk for those in the highest tertile of plasma ADMA distribution was mainly confined to men with a previous history of CHD (7). Data are still lacking that clearly demonstrate that an increase of ADMA is also associated with death and cardiovascular events in patients without CHD or organ failure. Moreover, the underlying mechanisms responsible for the confounding effect of smoking on ADMA-associated risk remain to be elucidated. We therefore conducted a nested case-control study to assess the potential relevance of ADMA as a risk factor for coronary events in smoking and nonsmoking men without a history of CHD.

Materials and Methods


We set up this study as a prospective case-control study within the population-based Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA) Augsburg surveys, conducted in 1989/1990 (S2) and 1994/1995 (S3). The MONICA Augsburg project was part of the multinational WHO MONICA project (13,14), for which men and women from a general population of more than 280 000 inhabitants of a mixed urban/rural area were randomly invited to participate (response rates for participation in the 52 and 53 surveys were 76.9% and 74.9%, respectively). The study was approved by the local ethics committee, and all participants provided written informed consent.

Altogether, 9796 men and women ages 25 to 74 years participated in the 2 independent cross-sectional surveys (S2, 4940 participants; 53, 4856 participants). In the framework of the Kooperative Gesundheitsforschung in der Region Augsburg (KORA), vital statistics were assessed for all sampled persons in the 2 survey populations. Patients with incident acute coronary events before the 75th year of age were identified through the population-based MONICA/KORA coronary event registry (15). The median (interquartile range) follow-up time of the study population was 6.2 (3.3-7.9) years. Eighty-nine men without prevalent CHD or diabetes mellitus at baseline developed an incident acute coronary event (S2, 78 cases; 53,11 cases). For each case, we randomly selected 3 age- and survey-matched control individuals without an incident acute coronary event during follow-up from the 2 survey populations, resulting in a study sample of 356 participants (89 cases, 267 controls). We decided not to include women because of their very low event rate. We excluded from the analysis 14 men (1 case, 13 controls) with missing data for ADMA or any of the other considered variables. Therefore, the study population of the present report is based on 88 cases and 254 event-free controls ages 35 to 74 years.

The outcome variable was a combination of incident fatal or nonfatal acute myocardial infarction and sudden cardiac death. According to the MONICA manual (14), the diagnosis of a major nonfatal myocardial infarction was based on acute symptoms, cardiac enzymes, and typical electrocardiogram changes. Deaths from cardiovascular causes were validated by autopsy reports, death certificates, chart review, and information from the last treating physician.


All participants completed a standardized questionnaire, including medical history, lifestyle, and drug intake. Blood pressure, body height (m), body weight (kg), body mass index (BMI; kg/[m.sup.2]), smoking habits, and alcohol consumption (g/day) were determined as described (16). We assessed leisure-time physical activity on a 4-level graded scale for winter and summer (none, <1, 1-2, and >2 h/week) and calculated the number of years of education from the highest level of formal education completed (17). Actual hypertension was defined as systolic blood pressure [greater than or equal to]140 mm Hg and/or diastolic blood pressure [greater than or equal to]90 mm Hg, being aware of having hypertension, or taking antihypertensive medication.


We maintained human endothelial cells (EAhy 926 cells, a hybrid human cell line derived from fusion of human umbilical vein endothelial cells and A549 carcinoma cells, a kind gift of Dr. Edgell (University of North Carolina, Chapel Hill, NC) (18) in DMEM at 37[degrees]C in a humidified atmosphere containing 50 mL C[O.sub.2]/L. The cigarette smoke condensate (CSC) used in the cell culture experiments was prepared from the University of Kentucky reference cigarette 2R4F. We collected the particulate phase of smoke on a Cambridge filter pad by use of a smoking machine under standard conditions prescribed by ISO4387:2000. The smoke particulate matter was dissolved in dimethyl sulfoxide at 10 g/L and stored frozen at -80[degrees]C in separate vials. On the day of the experiment, each vial of CSC solution was opened and diluted in serum-free cell culture medium to desired concentration. Nicotine concentrations observed at CSC concentrations of 1 and 10 mg/L matched the concentration interval found in plasma of smokers. We assessed ADMA liberation, RNA expression, and cytotoxicity in 6-well plates after treatment of cells for 48 h with CSC. Control cells were treated with medium containing an equivalent amount of dimethyl sulfoxide.


Total RNA was extracted from EAhy 296 cells by RNAzol (Wak-Chemie) and reverse-transcribed (Superscript II, Invitrogen) by use of random hexamers. We quantified mRNA expression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2 by use of the Applied Biosystems ABI Prism 7900 HT system (TagMan). We carried out TagMan reactions in 384-well plates according to the manufacturer's instructions (Applied Biosystems) using premade probes for DDAH1 (Probe Hs00201707 m1 generating an amplicon of 77 by from the NM 012137.2 transcript) and DDHA2 (probe Hs00203889 m1 generating an amplicon of 85 by from the NM 013974.1 transcript) and glyceraldehyde-3-phosphate dehydrogenase as an endogenous control (probe Hs99999905 m1 generating an amplicon of 122 by from the M33197.1 transcript). We performed relative quantification of gene expression using the OOCT method as described in the user guide for the ABI Prism 7900 HT system.


We collected a nonfasting venous blood sample from all participants in a supine resting position. We measured total serum cholesterol (TC) and HDL cholesterol (HDL-C) by routine enzymatic methods. Samples for measurement of C-reactive protein (CRP) and ADMA were stored at -70[degrees]C until analysis. We measured serum CRP concentrations with a high-sensitivity immunoradiometric assay (range, 0.05 to 10 mg/L) as described (19). In cell culture supernatants, we measured ADMA and Larginine by ultrasensitive liquid chromatography-tandem mass spectrometry as previously validated and described in detail (20). We measured plasma concentrations of ADMA by use of a commercial ELISA reagent set (DLD) (21). The lower limit of detection for the ADMA ELISA was <0.05 [micro]mol/L; intra- and interassay CVs were 4.5% and 8.3%, respectively. Cross-reactivities with SDMA and L-arginine were 1.2% and <0.02%, respectively. The correlation coefficient of the plasma ADMA values obtained by ELISA and liquid chromatography-tandem mass spectrometry (n = 29) was 0.984.

We measured oxidized LDL by use of a competitive ELISA (Mercodia). We measured lactate dehydrogenase (LDH) by use of a commercial reagent set (cytotoxicity detection reagent set; Roche Diagnostics) and protein concentration by use of the Bradford protein assay (BioRad). We estimated glomerular filtration rate (GFR) according to the abbreviated Modification of Diet in Renal Disease (MDRD) formula (22).

All analyses were run in a blinded fashion.


We computed means or proportions for baseline demographic and clinical characteristics for men with and without an incident coronary event. The distribution of CRP concentration was markedly skewed, and we therefore calculated the geometric mean (geometric SD) from the log-transformed data. We tested differences in mean values of continuous variables for statistical significance by t-test and differences in proportions by [chi square] test. We assessed associations among continuous variables by Pearson correlation coefficient r.

We used Cox proportional hazards analysis to assess the independent risk for the occurrence of a 1st coronary event. Study participants were grouped into tertiles of plasma ADMA concentrations, and the risk was calculated relative to the bottom tertile. Results are presented as hazard ratios (HRs) together with their 95% confidence interval (CI). First, crude HRs were calculated (model 1). Results were then adjusted for age (continuous) and survey (S2 or S3; model 2) and for age (continuous), survey (S2, S3), education years (<12, [greater than or equal to]12 years), smoking status (yes, no), alcohol consumption (0 g/d, 1-39.9 g/d, [greater than or equal to]40 g/d), obesity (BMI <30.0 kg/[m.sup.2], [greater than or equal to]BMI[greater than or equal to]30.0 kg/[m.sup.2]), physical activity (inactive, active), actual hypertension (no, yes), TC/HDL-C ratio (continuous), and GFR (continuous; model 3). To further evaluate the interaction of ADMA and smoking status with regard to incident coronary events, we calculated adjusted HRs according to smoking status and tertile of ADMA, with never-smoking and low ADMA tertile as reference. For a test of trend, we coded ADMA tertiles with their median values and repeated the Cox regression described above. Moreover, to test for possible modifications of the ADMA effect on a coronary event by risk factors, we included interaction terms of ADMA tertiles and the parameter under concern in the multivariate Cox regression models. Cell culture data were compared by ANOVA followed by Dunnett multiple comparison test.

All significance tests were 2-tailed, and probability values <0.05 were considered statistically significant. All analyses were performed with the Statistical Analysis System (version 8.2, SAS Institute) and Prism4 (GraphPad Software).



Baseline characteristics of cases and controls are shown in Table 1. The median (interquartile range) time to a 1st coronary event was 2.9 (1.2-5.1) years. Men who experienced an event were more frequently smokers or had hypertension compared with controls. Likewise, significantly higher concentrations of CRP, TC (and TC/HDL-C ratio), and oxidized LDL were measured in cases compared with controls, whereas the difference in BMI reached only borderline significance. Cases and controls did not differ significantly in age (matching variable), educational level, physical activity, alcohol intake, diastolic blood pressure, or HDL-C. Mean (SD) plasma ADMA concentrations in participants who experienced an event and in controls were also similar: 0.80 (0.22) and 0.79 (0.21) [micro]mol/L, P = 0.72.


Correlations between ADMA and cardiovascular risk factors are shown in Table 2. ADMA and age were positively correlated (r = 0.28, P <0.001 in controls and r = 0.41, P <0.001 in cases), whereas there was an inverse correlation of ADMA and GFR (r = -0.25, P <0.001 in controls and r = -0.326, P = 0.002 in cases). We observed no statistically significant correlations of ADMA with BMI or with systolic or diastolic blood pressure, TC, HDL-C, oxidized LDL, or CRP. In both groups (cases and controls), differences in ADMA concentrations between smokers and nonsmokers were observed: mean (SD) ADMA concentrations among cases were 0.69 (0.17) and 0.87 (0.23), respectively (P <0.001), and among controls, 0.74 (0.20) and 0.80 (0.22), respectively (P = 0.067).


To assess the risk of an incident coronary event according to baseline concentrations of ADMA, we calculated Cox proportional hazard models (Table 3). For this purpose, ADMA concentrations were categorized into tertiles (0.36-0.70, 0.71-0.85, and 0.86-2.30 [micro]mol/L). Median ADMA plasma concentrations in the low, intermediate, and high ADMA tertiles were 0.61, 0.77, and 0.97 [micro]mol/L, respectively.

When all 342 men were included, irrespective of smoking status, the trend toward a higher HR for an incident coronary event in the intermediate and the top tertile vs the bottom tertile of the ADMA distribution did not reach statistical significance in any of the Cox regression models (Table 3). In the fully adjusted model (3) the HR for the top ADMA tertile was 1.35 (95% CI 0.78-2.33; P = 0.282). In the same model, the relative risk of a future coronary event was 2.00 (95% CI 1.27-3.16; P = 0.003) for smokers compared with nonsmokers. By contrast, analysis of ADMA-associated risk for smokers and nonsmokers separately revealed striking differences (Table 3). The adjusted relative risk comparing the top tertile to the bottom tertile of the ADMA distribution was 0.48 (95% CI 0.161.46; P = 0.198) for smokers, but it was 2.40 (95% CI 1.14-5.08; P = 0.021) for nonsmokers. Smoking was the only risk factor with a significant interaction with ADMA values (P = 0.031). In further analysis, adjusting for CRP (n = 316), the HRs were similar, with HRs (95% CIs) of 0.48 (0.14-1.66) for smokers and 2.35 (1.10-5.03) for nonsmokers.

To explore the interaction of ADMA and smoking in more detail, we assessed HRs after grouping the participants according to smoking status and tertile of ADMA (Fig. 1). Using as reference nonsmoking men with plasma ADMA in the lowest tertile (n = 77), the complex interaction of smoking and ADMA with respect to coronary risk became even more evident. In accordance with the initial analysis in nonsmoking men, the risk of suffering a coronary event increased with tertiles of ADMA [HR 1.63 (0.77-3.46) and 2.37 (1.14-4.95), respectively, for men in the 2nd (n = 88) and 3rd (n = 91) ADMA tertiles]. By contrast, the coronary risk in the middle and top ADMA tertiles did not further increase in current smokers [HR 2.25 (0.87-7.79) in the 2nd ADMA tertile (n = 24) and HR 2.36 (0.81-6.86) in the 3rd ADMA tertile (n = 17)]. Smoking status was associated with the highest risk in patients with low ADMA concentrations (n = 45) [HR 4.49 (2.08-9.68)].


Incubation of EAhy 926 cells for 48 h with 1.0 and 10.0 mg/L CSC resulted in declines of the ADMA concentration in the cell culture medium (normalized to cellular protein) by 28.2% and 24.8%, respectively (Fig. 2A), whereas very low concentrations of CSC had no effect. The ADMA/L-arginine ratio declined with increasing concentrations (1.0 and 10.0 mg/L) of CSC, by -18.2% and -22.0%, respectively (both P <0.05).

Expression of DDAH2 mRNA was augmented by 87.6% after 48 h of incubation with 10.0 mg/L tobacco smoke concentrate, whereas changes in expression did not reach statistical significance at lower concentrations (Fig. 2B). Expression of DDAH1 was not significantly altered (all P >0.05). None of the concentrations of CSC was associated with significant increases in LDH activity (as indicator of cytotoxicity; Fig. 2C).


The major finding of this prospective population-based study of 342 men (88 incident cases and 254 age-matched controls) without a history of CHD or diabetes mellitus at baseline is that increase of ADMA is an independent risk factor for future fatal and nonfatal coronary events in nonsmoking men but not in smoking men. With respect to ADMA and smoking, our findings point to a significant interaction of smoking and ADMA-associated cardiovascular risk. Furthermore, our cell culture experiments indicate that CSC may enhance degradation of ADMA by upregulation of DDAH2.


So far, the effect of smoking on ADMA has been discussed quite controversially (23). In a previous study in elderly men with a high cardiovascular risk profile, significantly lower plasma ADMA concentrations were no longer statistically significant after correction for baseline variables and morbidity (24). Another recent study by Schnabel et al. (9) found higher ADMA concentrations in smoking compared with nonsmoking men and women in a population of patients with preexisting CHD. The most plausible explanation for apparent discrepancies regarding the effects of smoking on ADMA reported by different investigators is that ADMA plasma concentrations are regulated by multiple factors and that in health and disease the relative contributions of these mechanisms to ADMA concentrations may be quite different (1, 25).

Most data available today suggest that degradation of ADMA by DDAH, rather than asymmetrical methylation of L-arginine residues and proteolysis, plays the key role in the regulation of ADMA concentrations (1, 25). Both isoforms of DDAH have been shown to be sensitive to oxidative stress (26), and ADMA concentrations tend to rise under conditions of oxidative stress (27). Hence, differences in systemic oxidative stress in patients with and without underlying cardiovascular disease may explain some of the apparent discrepancies.

A previous analysis of the MONICA Augsburg 1994/ 1995 cohort found a strong association between smoking and various markers of systemic inflammation in men (28). Unlike oxidative stress, inflammation is associated with lower ADMA concentrations (29). Although in our study there was no formal statistical interaction between ADMA and CRP, smoking-induced inflammation may have contributed to lower ADMA concentrations.

This study was the first to investigate a complete (lipid and water soluble) extract of cigarette smoke; previous studies reporting divergent results on ADMA and DDAH expression used only the water-soluble fraction of cigarette smoke (30) or nicotine at cytotoxic levels (31) or investigated ADMA concentrations in regenerated endothelial cells from carotid arteries obtained 6 weeks after endothelial removal in rabbits chronically exposed to nicotine (32).

A further explanation for divergent observations may be that CSCs from different cigarette brands were found to differ in their effect on the expression of a subset of >1000 genes (33).


This is the first prospective study investigating the association of ADMA and cardiovascular risk in a nested case-control design in a large cohort of primarily healthy participants. All previous studies on ADMA involved patients with preexisting CHD (6, 9) or organ failure (11, 12, 34) or mixed cohorts (7). In the whole cohort on which the present study was based (n = 3022), the incidences of fatal and nonfatal coronary events per 100 000 persons for current smokers, former smokers, and men who never smoked were 723, 578, and 399, respectively. With this in mind, it is striking that in the present study we observed little or no additive effect of smoking on risks in the middle and top ADMA tertiles (Fig. 1). Even more surprising is the observation that in men with low ADMA values, smoking was associated with the highest HR. Based on our in vitro data (which suggest that cigarette smoke may enhance metabolism of ADMA) a simple explanation for this apparent paradox may be that with respect to ADMA-associated risk, smoking may present a classical confounding factor. High cigarette consumption may lower ADMA concentrations while contributing itself to a higher incidence of cardiovascular events via multiple other mechanisms.


Upregulation of DDAH expression should not raise too much enthusiasm in smokers, though. Smoking clearly associated with a higher risk for cancer, and promotion of NO-mediated angiogenesis could have catastrophic effects when occurring in tumors. Indeed, recent studies suggest that DDAH activity and expression are increased in human tumors (35) and may enhance tumor growth (36). Thus, induction of DDAH expression may be an additional mechanism linking smoking and cancer.


Of all risk markers evaluated (including CRP and oxidized LDL), we found a positive correlation of ADMA concentrations only with age and a negative correlation only with GFR. Conflicting results regarding the correlation or association of ADMA and other risk markers have been previously noted (1) and have been attributed at least in part to differences in concomitant diseases and risk factors. In contrast to most previous studies, we investigated a rather healthy, unselected, population-based study cohort. Negative findings in our population certainly do not preclude significant correlations in higher-risk populations with more extreme deviations from the normal range of BMI or blood pressure. In this respect, it is of interest to note that in healthy men an infusion of ADMA (leading to a more than 20-fold increase of plasma ADMA concentration) resulted in an increase of the mean arterial blood pressure by as little as 4.5 mm Hg (37).


The major strengths of the present study are its population-based design and the length of follow-up. We took all incident cases from 2 random samples of the general population and age-matched controls from the same source. Also, we analyzed only the hard endpoints fatal and nonfatal myocardial infarction and sudden cardiac death. Nonetheless, there are limitations of the present study that need to be considered. Whereas the sample size in this event-based nested case-control design was clearly adequate to detect or exclude clinically meaningful differences in outcome for the primary comparison, sample size for the subgroup analysis of smokers and nonsmokers was small. This resulted in relatively wide CIs, indicating limited power of the subgroup analyses, especially with regard to negative findings and absolute effect sizes. In conclusion, in an apparently healthy population with a low to moderate cardiovascular risk, we found no significant relationship between plasma ADMA concentration and long-term cardiovascular outcome for the cohort at large. This apparent lack of association could be attributed at least in part to a substantial interaction of smoking status, plasma ADMA concentration, and cardiovascular risk. When accounting for this interaction, our data actually provide first evidence that ADMA is an independent risk marker in healthy nonsmoking men.

R.H.B. and R.M. have filed patents related to NOS inhibitors.

Received October 18, 2006; accepted January 19, 2007. Previously published online at DOI: 10.1373/clinchem.2006.081893


(1.) Valiance P, Leiper J. Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohydrolase pathway. Arterioscler Thromb Vasc Biol 2004;24:1023-30.

(2.) Boger RH. When the endothelium cannot say 'NO' anymore. ADMA, an endogenous inhibitor of NO synthase, promotes cardiovascular disease [Editorial]. Eur Heart J 2003;24:1901-2.

(3.) Tsikas D, Rode I, Becker T, Nashan B, Klempnauer J, Frolich JC. Elevated plasma and urine levels of ADMA and 15(S)-8-isoPGF2alpha in end-stage liver disease. Hepatology 2003;38:1063-4.

(4.) Miyazaki H, Matsuoka H, Cooke JP, Usui M, Ueda S, Okuda S, et al. Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Circulation 1999;99:1141-6.

(5.) Kielstein JT, Boger RH, Bode-Boger SM, Frolich JC, Haller H, Ritz E, et al. Marked increase of asymmetric dimethylarginine in patients with incipient primary chronic renal disease. J Am Soc Nephrol 2002;13:170-6.

(6.) Stuhlinger MC, Abbasi F, Chu JW, Lamendola C, McLaughlin TL, Cooke JP, et al. Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor. JAMA 2002;287: 1420-6.

(7.) Valkonen VP, Paiva H, Salonen JT, Lakka TA, Lehtimaki T, Laakso J, et al. Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine. Lancet 2001;358:2127-8.

(8.) Lu TM, Ding YA, Lin SJ, Lee WS, Tai HC. Plasma levels of asymmetrical dimethylarginine and adverse cardiovascular events after percutaneous coronary intervention. Eur Heart J 2003;24: 1912-9.

(9.) Schnabel R, Blankenberg S, Lubos E, Lackner KJ, Rupprecht HJ, Espinola-Klein C, et al. Asymmetric dimethylarginine and the risk of cardiovascular events and death in patients with coronary artery disease: results from the AtheroGene Study. Circ Res 2005;97: e53-9.

(10.) Zoccali C, Benedetto FA, Maas R, Mallamaci F, Tripepi G, Malatino LS, et al. Asymmetric dimethylarginine, C-reactive protein, and carotid intima-media thickness in end-stage renal disease. J Am Soc Nephrol 2002;13:490-6.

(11.) Zoccali C, Bode-Boger S, Mallamaci F, Benedetto F, Tripepi G, Malatino L, et al. Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study. Lancet 2001;358:2113-7.

(12.) Nijveldt RJ, Teerlink T, Van Der Hoven B, Siroen MP, Kuik DJ, Rauwerda JA, et al. Asymmetrical dimethylarginine (ADMA) in critically ill patients: high plasma ADMA concentration is an independent risk factor of ICU mortality. Clin Nutr 2003;22:2330.

(13.) WHO-MONICA Project Principal Investigators. The World Health Organization MONICA Project (Monitoring Trends and Determinants in Cardiovascular Disease): a major international collaboration. J Clin Epidemiol 1988;41:105-14.

(14.) WHO MONICA Project. WHO MONICA project: objectives and design. Int J Epidemiol 1989;18(Suppl 1):29-37.

(15.) Loewel H, Lewis M, Hoermann A, Keil U. Case finding, data quality aspects and comparability of myocardial infarction registers: results of a south German register study. J Clin Epidemiol 1991;44:249-60.

(16.) Hense HW, Filipiak B, D6ring A, Stieber J, Liese A, Keil U. Ten-year trends of cardiovascular risk factors in the MONICA Augsburg region in southern Germany: results from 1984/1985, 1989/ 1990, and 1994/1995 surveys. CVD Prevention 1998;1:31827.

(17.) Koenig W, Sund M, Doring A, Ernst E. Leisure-time physical activity but not work-related physical activity is associated with decreased plasma viscosity: results from a large population sample. Circulation 1997;95:335-41.

(18.) Edgell CJ, McDonald CC, Graham JB. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Natl Acad Sci U S A 1983;80:3734-7.

(19.) Hutchinson WL, Koenig W, Frohlich M, Sund M, Lowe GD, Pepys MB. Immunoradiometric assay of circulating C-reactive protein: age-related values in the adult general population. Clin Chem 2000;46:934-8.

(20.) Schwedhelm E, Tan-Andresen J, Maas R, Riederer U, Schulze F, Boger RH. Liquid chromatography-tandem mass spectrometry method for the analysis of asymmetric dimethylarginine in human plasma. Clin Chem 2005;51:1268-71.

(21.) Schulze F, Wesemann R, Schwedhelm E, Sydow K, Albsmeier J, Cooke JP, et al. Determination of asymmetric dimethylarginine (ADMA) using a novel ELISA assay. Clin Chem Lab Med 2004;42: 1377-83.

(22.) Levey AS, Greene T, Kusek JW, Beck GJ, Group MS. A simplified equation to predict glomerular filtration rate from serum creatinine [Abstract]. J Am Soc Nephrol 2000;11:A0828.

(23.) Kielstein JT, Peter C, Adams MC. Cigarettes and ADMA: the smoke hasn't cleared yet. Hypertension 2006;48:E20.

(24.) Eid HM, Arnesen H, Hjerkinn EM, Lyberg T, Seljeflot I. Relationship between obesity, smoking, and the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine. Metabolism 2004;53:1574-9.

(25.) Maas R. Pharmacotherapies and their influence on asymmetric dimethylarginine (ADMA). Vasc Med 2005;10(Suppl 1):S49-57.

(25.) Leiper JM, Santa Maria J, Chubb A, MacAllister RJ, Charles IG, Whitley GS, et al. Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases. Biochem J 1999; 343:209-14.

(26.) Murray-Rust J, Leiper J, McAlister M, Phelan J, Tilley S, Santa Maria J, et al. Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylamin ohydrolase. Nat Struct Biol 2001;8:679-83. [Erratum in Nat Struct Biol 2001;8:818.]

(27.) Sydow K, Munzel T. ADMA and oxidative stress. Atheroscler Suppl 2003;4:41-51.

(28.) Frohlich M, Sund M, Lowel H, Imhof A, Hoffmeister A, Koenig W. Independent association of various smoking characteristics with markers of systemic inflammation in men. Results from a representative sample of the general population (MONICA Augsburg Survey 1994/95). Eur Heart J 2003;24:1365-72.

(29.) Zoccali C, Maas R, Cutrupi S, Pizzini P, Finocchiaro P, Cambareri F, et al. Asymmetric dimethyl-arginine (ADMA) response to inflammation in acute infections. Nephrol Dial Transplant 2006 [Epub ahead of print] doi:10.1093/ndt/gf1719.

(30.) Zhang WZ, Venardos K, Chin-Dusting J, Kaye DM. Adverse effects of cigarette smoke on NO bioavailability: role of arginine metabolism and oxidative stress. Hypertension 2006;48:278-85.

(31.) Jiang DJ, Jia SJ, Yan J, Zhou Z, Yuan Q, Li YJ. Involvement of DDAH/ADMA/NOS pathway in nicotine-induced endothelial dysfunction. Biochem Biophys Res Commun 2006;349:683-93.

(32.) Hamasaki H, Sato J, Masuda H, Tamaoki S, Isotani E, Obayashi S, et al. Effect of nicotine on the intimal hyperplasia after endothelial removal of the rabbit carotid artery. Gen Pharmacol 1997;28: 653-9.

(33.) Jorgensen ED, Dozmorov I, Frank MB, Centola M, Albino AP. Global gene expression analysis of human bronchial epithelial cells treated with tobacco condensates. Cell Cycle 2004;3:1154-68.

(34.) Mallamaci F, Tripepi G, Cutrupi S, Malatino LS, Zoccali C. Prognostic value of combined use of biomarkers of inflammation, endothelial dysfunction, and myocardiopathy in patients with ESRD. Kidney Int 2005;67:2330-7.

(35.) Kostourou V, Robinson SP, Whitley GS, Griffiths JR. Effects of overexpression of dimethylarginine dimethylaminohydrolase on tumor angiogenesis assessed by susceptibility magnetic resonance imaging. Cancer Res 2003;63:4960-6.

(36.) Kostourou V, Robinson SP, Cartwright JE, Whitley GS. Dimethylarginine dimethylaminohydrolase I enhances tumour growth and angiogenesis. Br J Cancer 2002;87:673-80.

(37.) Kielstein JT, Impraim B, Simmel S, Bode-Boger SM, Tsikas D, Frolich JC, et al. Cardiovascular effects of systemic nitric oxide synthase inhibition with asymmetrical dimethylarginine in humans. Circulation 2004;109:172-7.

[4] Nonstandard abbreviations: ADMA, asymmetric dimethylarginine; NOS, nitric oxide synthase; CHD, coronary heart disease; MONICA, Monitoring of Trends and Determinants in Cardiovascular Disease; KORA, Kooperative Gesundheitsforschung in der Region Augsburg; BMI, body mass index; CSC, cigarette smoke condensate; DDAH, dimethylarginine dimethylaminohydrolase; TC, total serum cholesterol; HDL-C, HDL cholesterol; CRP, C-reactive protein; LDH, lactate dehydrogenase; GFR, glomerular filtration rate; MDRD, Modification of Diet in Renal Disease; HR, hazard ratio; CI, confidence interval.


[1] Institute of Experimental and Clinical Pharmacology and Toxicology, University Hospital Hamburg-Eppendorf, Hamburg, Germany.

[2] GSF, Research Center for Environment and Health, Institute of Epiderniology, Neuherberg, Germany.

[3] Department of Internal Medicine II-Cardiology, University of Ulm Medical Center, Ulm, Germany.

* Address correspondence to this author at: Department of Internal Medicine II-Cardiology, University of Ulm Medical Center, Robert-Koch Strasse 8, D-89081 Ulm, Germany. Fax 49-731-500-45021; e-mail
Table 1. Demographic and clinical characteristics of men with
(cases) and without (controls) incident coronary event. (a)

Characteristics Cases Controls

n 88 254
Age, years 61.1 (8.6) 61.2 (8.4)
Education, 12 years (b) 76.1 75.2
Body mass index, kg/[m.sup.2] 28.6 (4.0) 27.7 (3.7)
Actual hypertension (b) 76.1 56.7
Systolic blood pressure, mmHg 146.0 (22.3) 138.8 (18.2)
Diastolic blood pressure, mmHg 82.6 (13.2) 81.9 (10.7)
Physical activity (b) 31.8 29.5
Smoking status (b)
 Never smoker 21.6 31.9
 Former smoker 42.0 46.9
 Current smoker 36.4 21.3
Alcohol intake (b)
 0 g/day 18.2 16.9
 0.1-39.9 g/day 48.9 53.5
 [greater than or 33.0 29.5
 equal to]40 g/day
TC, mmol/L 6.6 (1.1) 6.3 (1.1)
HDL-C, mmol/L 1.3 (0.4) 1.3 (0.4)
TC/HDL-C 5.5 (1.6) 5.0 (1.8)
Oxidized LDL, U/L 109.8 (32.1) 93.2 (28.0)
CRP, mg/L (c) 2.8 (3.2) 1.7 (3.0)
Creatinine, [micro]mol/L 97.2 (26.5) 97.2 (17.7)
GFR, mL/min (d) 75.5 (18.7) 77.4 (17.3)
ADMA, [micro]mol/L 0.80 (0.22) 0.79 (0.21)

Characteristics P value

Age, years 0.898
Education, 12 years (b) 0.860
Body mass index, kg/[m.sup.2] 0.058
Actual hypertension (b) 0.001
Systolic blood pressure, mmHg 0.007
Diastolic blood pressure, mmHg 0.671
Physical activity (b) 0.686
Smoking status (b) 0.013
 Never smoker
 Former smoker
 Current smoker
Alcohol intake (b) 0.746
 0 g/day
 0.1-39.9 g/day
 [greater than or
 equal to]40 g/day
TC, mmol/L 0.014
HDL-C, mmol/L 0.262
TC/HDL-C 0.023
Oxidized LDL, U/L <0.001
CRP, mg/L (c) 0.001
Creatinine, [micro]mol/L 0.198
GFR, mL/min (d) 0.368
ADMA, [micro]mol/L 0.721

(a) Data are arithmetic mean (SD), P value from t-test, unless
noted otherwise.

(b) %, P value from [chi square] test.

(c) Geometric mean (geometric SD), P value from t-test after
log transformation (n 316, 235 controls, 81 cases).

(d) Calculated according to the abbreviated MDRD formula.

Table 2. Correlation and association between ADMA and cardiovascular
risk factors in cases and controls. (a)


Risk factor Cases P value

Age 0.414 <0.001
BMI -0.051 0.640
Systolic blood pressure -0.065 0.546
Diastolic blood pressure -0.112 0.297
TC -0.041 0.702
HDL-C -0.021 0.844
TC/HDL-C -0.070 0.517
Creatinine 0.198 0.002
GFR (b) -0.326 0.002
Oxidized LDL 0.054 0.395
Log CRP (c) 0.056 0.621
Actual hypertension (d) 0.606
No 0.78 (0.22)
Yes 0.81 (0.23)
Physical activity (d) 0.816
No 0.81 (0.24)
Yes 0.79 (0.19)
Smoker (d) 0.001
Never 0.88 (0.24)
Former 0.85 (0.22)
Current 0.69 (0.17)

Risk factor Controls P value

Age 6:48 <0.001
BMI -0.005 0.933
Systolic blood pressure 0.040 0.523
Diastolic blood pressure -0.079 0.209
TC -0.058 0.362
HDL-C -0.093 0.142
TC/HDL-C 0.038 0.551
Creatinine 0.229 0.032
GFR (b) -0.250 <0.001
Oxidized LDL -0.078 0.486
Log CRP (c) 0.047 0.473
Actual hypertension (d) 0.944
No 0.79 (0.21)
Yes 0.79 (0.22)
Physical activity (d) 0.848
No 0.79 (0.19)
Yes 0.80 (0.27)
Smoker (d) 0.165
Never 0.80 (0.20)
Former 0.81 (0.23)
Current 0.74 (0.20)

(a) Data are Pearson correlation coefficient r unless noted

(b) Calculated according to the abbreviated MDRD formula.

(c) 81 cases, 235 controls.

(d) Mean (SD).

Table 3. Relative risk for the incidence of a coronary event
estimated by HR with 95% CIs.

Tertile of ADMA n, Cases/ Low ADMA,
 controls 0.36-0.70

Model Reference

All participants 88/254

Unadjusted 1
Adjusted for age and survey 1
Multivariable adjustment (a) 1

Current smokers 32/54

Unadjusted 1
Adjusted for age and survey 1
Multivariable adjustment (b) 1

Former smokers 37/119

Unadjusted 1
Adjusted for age and survey 1
Multivariable adjustment (b) 1

Never smokers 19/81

Unadjusted 1
Adjusted for age and survey 1
Multivariable adjustment (b) 1

Never smokers and former smokers 56/200

Unadjusted 1
Adjusted for age and survey 1
Multivariable adjustment (b) 1

Tertile of ADMA Intermediate ADMA, P,
 0.71-0.85 intermediate
 [micro]mol/L vs low ADMA


All participants

Unadjusted 1.09 (0.65-1.83) 0.742
Adjusted for age and survey 1.02 (0.60-1.73) 0.953
Multivariable adjustment (a) 0.97 (0.56-1.67) 0.901

Current smokers

Unadjusted 0.76 (0.33-1.73) 0.506
Adjusted for age and survey 0.75 (0.32-1.78) 0.515
Multivariable adjustment (b) 0.39 (0.15-1.04) 0.060

Former smokers

Unadjusted 1.12 (0.46-2.69) 0.808
Adjusted for age and survey 0.98 (0.40-2.39) 0.957
Multivariable adjustment (b) 1.01 (0.40-2.57) 0.986

Never smokers

Unadjusted 5.05 (1.09-23.44) 0.039
Adjusted for age and survey 4.95 (1.04-23.57) 0.045
Multivariable adjustment (b) 3.96 (0.79-19.93) 0.096

Never smokers and former smokers

Unadjusted 1.83 (0.88-3.82) 0.109
Adjusted for age and survey 1.66 (0.79-3.52) 0.184
Multivariable adjustment (b) 1.77 (0.83-3.78) 0.138

Tertile of ADMA High ADMA, P, high vs
 0.86-2.30 low ADMA


All participants

Unadjusted 1.27 (0.77-2.11) 0.356
Adjusted for age and survey 1.20 (0.70-2.04) 0.505
Multivariable adjustment (a) 1.35 (0.78-2.33) 0.282

Current smokers

Unadjusted 0.72 (0.27-1.92) 0.505
Adjusted for age and survey 0.71 (0.25-2.00) 0.517
Multivariable adjustment (b) 0.48 (0.16-1.46) 0.198

Former smokers

Unadjusted 1.74 (0.78-3.91) 0.179
Adjusted for age and survey 1.51 (0.66-3.48) 0.332
Multivariable adjustment (b) 1.55 (0.66-3.65) 0.315

Never smokers

Unadjusted 4.43 (0.94-20.86) 0.060
Adjusted for age and survey 4.38 (0.89-21.61) 0.070
Multivariable adjustment (b) 7.06 (1.30-38.40) 0.024

Never smokers and former smokers

Unadjusted 2.28 (1.12-4.64) 0.023
Adjusted for age and survey 2.07 (0.99-4.30) 0.053
Multivariable adjustment (b) 2.40 (1.14-5.08) 0.021

Tertile of ADMA P for


All participants

Unadjusted 0.354
Adjusted for age and survey 0.488
Multivariable adjustment (a) 0.262

Current smokers

Unadjusted 0.439
Adjusted for age and survey 0.468
Multivariable adjustment (b) 0.150

Former smokers

Unadjusted 0.148
Adjusted for age and survey 0.261
Multivariable adjustment (b) 0.263

Never smokers

Unadjusted 0.086
Adjusted for age and survey 0.111
Multivariable adjustment (b) 0.019

Never smokers and former smokers

Unadjusted 0.025
Adjusted for age and survey 0.058
Multivariable adjustment (b) 0.022

(a) HRs for Cox regression models adjusting for age, survey,
education level, alcohol consumption, obesity, physical
activity, hypertension, TC/HDL-C ratio, GFR, and smoking status.

(b) HRs for Cox regression models adjusted for age, survey,
education level, alcohol consumption, obesity, physical
activity, hypertension, TC/HDL-C ratio, and GFR.

Fig. 1. HRs for a 1st coronary event in
men categorized in tertiles of plasma
ADMA concentration and smoking status.
Cox regression model adjusted for age,
survey, education level, alcohol consumption,
obesity, physical activity, hypertension,
and TC/HDL-C ratio. [section], reference (low
ADMA tertile and nonsmokers).

 Hazard ration (HR) for coronary events

 Non-Smokers Current Smokers

High ADMA (T3) 2.37 2.36
medium ADMA (T2) 1.63 2.25
Low ADMA (T1) 1.00 [section] 4.49

Note: Table made from bar graph.
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Article Details
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Title Annotation:Lipids, Lipoproteins and Cardiovascular Risk Factors
Author:Maas, Renke; Schulze, Friedrich; Baumert, Jens; Lowel, Hannelore; Hamraz, Khatera; Schwedhelm, Edzar
Publication:Clinical Chemistry
Article Type:Survey
Date:Apr 1, 2007
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