primary aldosteronism ( pa ) has been traditionally considered a rare cause of hypertension ( 1 ) . however , recent studies have shown a higher prevalence , ranging between 5% - 20 % among hypertensive patients ( 2 ) . moreover , recent studies have demonstrated a higher frequency of cardiovascular complications in pa compared to patients with essential hypertension ( 3 , 4 ) . endothelial dysfunction could play a role in the deleterious cardiovascular effects of aldosterone , as it predicts long - term atherosclerotic disease progression and the risk of cardiovascular events ( 5 , 6 ) . asymmetric dimethylarginine ( adma ) is one of the most widely used and validated markers for evaluating the endothelial function . elevated plasma adma has been identified as a predictor of acute coronary events and an independent risk factor for all - cause and cardiovascular mortality ( 8 , 9 ) . several studies have reported elevated amda concentration in patients with different cardiovascular and metabolic disorders as essential hypertension ( 10 ) , diabetes mellitus ( 11 ) , hypercholesterolemia ( 12 ) , coronary artery disease ( 13 ) , but reports on adma levels in endocrine hypertension are scarce . increased plasma concentrations of adma could contribute to endothelial dysfunction , increased atherogenesis and elevated cardiovascular risk in patients with pa . the aim of this study was to compare the levels of adma among patients with pa , controls with essential hypertension ( eh ) and healthy participants . the study population consisted of 18 patients with pa , 18 controls with eh , and 18 healthy participants . all studies were performed after obtaining a written informed consent form according to a protocol approved by the ethics committee of sofia medical university . hypertensive patients were referred to the clinical center of endocrinology in sofia for diagnosis and treatment during june 2009 - june 2011 . in patients with eh other secondary forms of hypertension were ruled out , using medical history , physical examination , biochemical and hormonal investigations and imaging studies . the diagnosis of pa was established , using the following algorithm : medications that can interfere with aldosterone and renin measurements ( beta - blockers , diuretics , angiotensin ii receptor blockers , angiotensin - converting enzyme inhibitors ) were discontinued for at least 7 - 10 days , and spironolactone was stopped for at least 45 days prior to blood sampling . centrally acting medicaments ( moxonodine , rilmenidine ) , alpha - blockers , and calcium channel blockers were used to treat hypertension . blood samples for plasma renin activity ( pra ) and aldosterone were taken in the morning between 8 - 10 hours a.m. after the patient had been in a sitting position for 30 minutes . the arr was then calculated and a confirmatory test , captopril test , was performed in patients with high arr ( above 750 pmol / l per ng / ml / h ) ( 10 ) and aldosterone above 416 pmol / l . the diagnosis of pa was considered if the arr was > 970 after the oral administration of 50 mg of captopril , while the patient has been sitting for 90 minutes ( 14 ) . plasma renin activity ( ng / ml / h ) was determined by quantitative determination of angiotensin i , using a commercially available radioimmunoassay kit ( diasorin s.p.a . serum aldosterone was measured by radioimmunoassay ( immunotech , beckman coulter company , marseille , france ) and expressed in picomols per liter ( pmol / l ) . the analytical sensitivity of the method was 0.20 ng / ml , inter - assay cv 7.5% , respectively , and intra - assay cv 5.4% . the cross reactivity with heptapeptide , angiotensin ii , and hexapeptide was below 0.02% . we elaborated our own reference range for pra in a sitting position ( 0.3 - 3.5 ng / ml / h ) ( 15 ) . in cases in which pra was < 0.3 ng / ml / h or undetectable , serum aldosterone was measured by ria ( immunotech , beckman coulter ) . for intra - assay assessment , the analytical and functional sensitivity were 1.44 pg / ml , and 6.98 pg / ml , respectively . according to the manufacturer antibody used in this immunoassay was highly specific for aldosterone . extremely low cross reactivities were found against other naturally occurring steroids ( cortisone , corticosterone , dheas , etc  ) serum adma levels were determined , using competitive enzyme - linked immunosorbent assay ( elisa ) ( human adma elisa kit , promokine , heidelberg , germany ; thermo electron corporation : appliscan ) . intra - assay and inter - assay cv for adma were 4.1 % and 4.5 % , respectively . glucose was measured , using enzymatic reference method with hexokinase ( cobas integra , roche diagnostics ) . intra - assay and inter - assay cv for glucose were 1.7% and 2.6% at 5.3 serum lipid parameters were assessed , using an enzymatic colorimetric method ( cobas integra , roche diagnostics ) . intra - assay and inter - assay cv for total cholesterol were 1.6% and 1.6% , respectively . intra - assay and inter - assay cv for triglycerides were 1.6% and 3.7% , respectively . intra - assay and inter - assay cv for hdl- cholesterol were 2.5 and 2.7% , respectively . the inter - assay ( qc low and qc high ) was calculated , using mean analytic value of all plates according to the formula : cv ( % ) = standard deviation / mean value of analytic * 100 , which corresponds to ifcc protocol . according to generally accepted quality criteria cited above , cv% for inter- and intra - assay was acceptable , which ensured the reliability of the results . statistical analysis was performed using spss 17.0 for windows ( chicago , illinois , usa ) . gaussian - distributed data are shown as mean  sd . student s t - test for independent samples was used to compare the equality of means for the two groups , and anova was used when testing three groups . the study population consisted of 18 patients with pa , 18 controls with eh , and 18 healthy participants . all studies were performed after obtaining a written informed consent form according to a protocol approved by the ethics committee of sofia medical university . hypertensive patients were referred to the clinical center of endocrinology in sofia for diagnosis and treatment during june 2009 - june 2011 . in patients with eh other secondary forms of hypertension were ruled out , using medical history , physical examination , biochemical and hormonal investigations and imaging studies . the diagnosis of pa was established , using the following algorithm : medications that can interfere with aldosterone and renin measurements ( beta - blockers , diuretics , angiotensin ii receptor blockers , angiotensin - converting enzyme inhibitors ) were discontinued for at least 7 - 10 days , and spironolactone was stopped for at least 45 days prior to blood sampling . centrally acting medicaments ( moxonodine , rilmenidine ) , alpha - blockers , and calcium channel blockers were used to treat hypertension . blood samples for plasma renin activity ( pra ) and aldosterone were taken in the morning between 8 - 10 hours a.m. after the patient had been in a sitting position for 30 minutes . the arr was then calculated and a confirmatory test , captopril test , was performed in patients with high arr ( above 750 pmol / l per ng / ml / h ) ( 10 ) and aldosterone above 416 pmol / l . the diagnosis of pa was considered if the arr was > 970 after the oral administration of 50 mg of captopril , while the patient has been sitting for 90 minutes ( 14 ) . plasma renin activity ( ng / ml / h ) was determined by quantitative determination of angiotensin i , using a commercially available radioimmunoassay kit ( diasorin s.p.a . , saluggia ( vc ) , italy ) . serum aldosterone was measured by radioimmunoassay ( immunotech , beckman coulter company , marseille , france ) and expressed in picomols per liter ( pmol / l ) . the analytical sensitivity of the method was 0.20 ng / ml , inter - assay cv 7.5% , respectively , and intra - assay cv 5.4% . the cross reactivity with heptapeptide , angiotensin ii , and hexapeptide was below 0.02% . we elaborated our own reference range for pra in a sitting position ( 0.3 - 3.5 ng / ml / h ) ( 15 ) . in cases in which pra was < 0.3 ng / ml / h or undetectable , serum aldosterone was measured by ria ( immunotech , beckman coulter ) . for intra - assay assessment , for inter - assay assessment , the samples were assayed in duplicate in 10 different series . the analytical and functional sensitivity were 1.44 pg / ml , and 6.98 pg / ml , respectively . according to the manufacturer antibody used in this immunoassay was highly specific for aldosterone . extremely low cross reactivities were found against other naturally occurring steroids ( cortisone , corticosterone , dheas , etc  ) serum adma levels were determined , using competitive enzyme - linked immunosorbent assay ( elisa ) ( human adma elisa kit , promokine , heidelberg , germany ; thermo electron corporation : appliscan ) . intra - assay and inter - assay cv for adma were 4.1 % and 4.5 % , respectively . glucose was measured , using enzymatic reference method with hexokinase ( cobas integra , roche diagnostics ) . intra - assay and inter - assay cv for glucose were 1.7% and 2.6% at 5.3 serum lipid parameters were assessed , using an enzymatic colorimetric method ( cobas integra , roche diagnostics ) . intra - assay and inter - assay cv for total cholesterol were 1.6% and 1.6% , respectively . intra - assay and inter - assay cv for triglycerides were 1.6% and 3.7% , respectively . intra - assay and inter - assay cv for hdl- cholesterol were 2.5 and 2.7% , respectively . the inter - assay ( qc low and qc high ) was calculated , using mean analytic value of all plates according to the formula : cv ( % ) = standard deviation / mean value of analytic * 100 , which corresponds to ifcc protocol . according to generally accepted quality criteria cited above , cv% for inter- and intra - assay was acceptable , which ensured the reliability of the results . statistical analysis was performed using spss 17.0 for windows ( chicago , illinois , usa ) . student s t - test for independent samples was used to compare the equality of means for the two groups , and anova was used when testing three groups . the baseline demographic and metabolic characteristics of those patients with primary aldosteronism , essential hypertension and normotensive controls are demonstrated in table 1 . no significant differences were observed among the three groups in age , gender and fasting blood glucose . patients with pa had higher body mass index and triglycerides and lower high- density ( hdl ) lipoprotein cholesterol compared to normotensive controls ( p = 0.001 , 0.016 , 0.002 , respectively ) . patients with eh had higher levels of total cholesterol compared to patients with pa ( p = 0.024 ) . patients with eh presented with significantly higher total cholesterol , low- density lipoprotein ( ldl ) cholesterol and triglycerides and lower hdl cholesterol than the control group ( p = 0.025 , p = 0.012 , p = 0.012 and p = 0.011 , respectively ) . abbreviations : bmi , body mass index ; hdl , high - density lipoprotein ; ldl , low - density lipoprotein . adma levels were compared in patients with pa , eh and healthy controls ( figure 1 ) . concentrations of adma were significantly higher in patients with pa than in healthy controls ( 0.49  0.09 vs. 0.43  0.05 there was no difference in adma levels between cases with pa and eh ( 0.49  0.09 vs. 0.48  0.08 the difference between participants with eh and the normotensive control group did not reach statistical significance ( p = 0.06 ) . no significant correlations were found between adma and some metabolic parameters in patients with pa ( table 2 ) . abbreviations : adma , asymmetric dimethylarginine ; bmi , body mass index ; hdl , high- density lipoprotein ; ldl , low - density lipoprotein . endothelial dysfunction ( ed ) is characterized with a tendency to proinflammatory and prothrombotic state and reduced vasodilatation . it is now well established that ed is related to the development of atherosclerosis ( 16 ) , myocardial infarction ( 17 ) , congestive heart failure ( 18 ) , diabetes mellitus ( 19 ) , and peripheral arterial diseases ( 20 ) . one of the possible mechanisms leading to ed is decreased nitric oxide ( no ) bioavailability , which can be caused by decreased no synthesis . with regards to this mechanism , adma , which is an endogenous analogue of l - arginine , was firstly recognized in 1992 as a naturally occurring inhibitor of no synthase ( 21 ) . moreover , elevated plasma adma concentration has been identified as a predictor of acute coronary events , and an independent risk factor for all - cause and cardiovascular mortality ( 8 , 9 ) . although elevated concentrations of adma have been found in several cardiovascular and metabolic diseases ( 10 - 13 , 22 - 24 ) , to our knowledge , to date , there have been no reports on adma levels in cases with pa . recent studies have demonstrated a higher frequency of cardiovascular events in pa compared to patients with eh ( 3 , 4 ) . recent studies have demonstrated that aldosterone impairs endothelial function by suppression of the synthesis of no ( 25 - 27 ) . experimental studies reported that the cytokine stimulated no synthesis in vascular smooth muscle cells is inhibited by aldosterone in a dose dependent manner ( 25 ) . recently , nishizaka et al . ( 26 ) reported a significant association between aldosterone and impaired endothelial function in human subjects , measured by flow - mediated arterial vasodilatation . in view of these findings , we hypothesized that increased adma levels in pa could contribute to the impaired endothelial cell - dependent vasodilator responses . our results confirmed the possible role of adma as a cardiovascular risk marker in pa since patients with pa had higher adma levels than healthy controls . on the other hand , numerous reports demonstrate that adma concentration is increased in patients with eh compared to healthy individuals ( 10 ) . the latter findings raise the following question : is high adma concentration in pa related to the effect of aldosterone or to hypertension per se ? the comparable levels of adma in pa and eh suggest the responsible factor for adma dysregulation is hypertension itself rather than the specific aldosterone action . indeed , several hypotheses for the elevation of adma in eh have been formulated , although the mechanism remains unclear . ( 28 ) have shown that adma release is enhanced by shear stress via activation of the nf - kappab pathway . in addition , it is possible that the oxidative stress , a well- known feature of eh , induces elevated adma by suppressing the activity of its metabolizing enzyme - dimethylaminohydrolase ( 29 ) . another possibility for the elevated adma concentration in pa and eh in our study was the increased ldl cholesterol and triglyceride levels , since it has been shown that ldl cholesterol decreases the activity of dimethylaminohydrolase . on the other hand , we did not find any statistically significant correlation between adma and metabolic lipid parameters , which confirms the possibility , that hypertension , itself , rather than the deteriorated lipid profile , results in higher adma levels ( table 2 ) . similar results were found in another study that we published recently , comparing adma in patients with pheochromocytoma and the control group of the present study ( 30 ) . endothelial function was impaired in pheochromocytoma patients , as shown by the elevated circulating levels of adma . the lack of association of these markers with glucose , cateholamines and lipid abnormalities as well as their comparable levels in patients with eh suggests that endothelial dysfunction may be related to hypertension itself . this was the first report to investigate the adma levels in pa and provide insight into the possible mechanisms of cardiovascular damage in pa , which is the main strength of this study . small sample size was the main study limitation , which was due to the restricted number of patients with pa . adma is an endogenous analogue of l - arginine and a naturally occurring inhibitor of no synthase . this was the first study to show elevated adma levels in patients with pa compared to normotensive controls . our data raise the possibility for a role of adma dysregulation in the cardiovascular damage in pa . on the other hand , adma levels were similar in patients with pa and eh , which suggests that endothelial dysfunction is more likely related to hypertension per se , than to the specific etiology of elevated blood pressure . this was the first report to investigate the adma levels in pa and provide insight into the possible mechanisms of cardiovascular damage in pa , which is the main strength of this study . small sample size was the main study limitation , which was due to the restricted number of patients with pa . adma is an endogenous analogue of l - arginine and a naturally occurring inhibitor of no synthase . this was the first study to show elevated adma levels in patients with pa compared to normotensive controls . our data raise the possibility for a role of adma dysregulation in the cardiovascular damage in pa . on the other hand , adma levels were similar in patients with pa and eh , which suggests that endothelial dysfunction is more likely related to hypertension per se , than to the specific etiology of elevated blood pressure .