- Email: [email protected]
Effect of rosuvastatin on plasma levels of asymmetric dimethylarginine in patients with hypercholesterolemia
Effect of Rosuvastatin on Plasma Levels of Asymmetric Dimethylarginine in Patients With Hypercholesterolemia Tse-Min Lu, MD, Yu-An Ding, MD, PhD, Hsin-Bang Leu, MD, Wei-Hsian Yin, Wayne Huey-Herng Sheu, MD, PhD, and Kai-Min Chu, MD, PhD
MD,
Elevated plasma levels of asymmetric dimethylarginine (ADMA) have been associated with attenuated endothelium-dependent vasodilation in hypercholesterolemic patients. However, whether lowering of plasma cholesterol concentration by hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) can reduce plasma ADMA levels is still not clear. This study was a multicenter, randomized, double-blind, placebo-controlled design including 46 patients with elevated low-density lipoprotein cholesterol levels. Patients were randomized into 2 groups: rosuvastatin 10 mg/day and placebo for 6 weeks. Plasma levels of ADMA, 8-isoprostane (as a marker of oxidative stress), homocysteine, and highsensitivity C-reactive protein were measured at baseline and 6 weeks later. Endothelial function assessed by flow-mediated vasodilation of the brachial artery was performed in 11 patients in the rosuvastatin group and
in 12 in the placebo group. Baseline characteristics of both groups were similar, and the plasma ADMA levels were significantly correlated with 8-isoprostane (r ⴝ 0.388, p ⴝ 0.008). After 6 weeks of treatment, plasma ADMA levels were significantly reduced in the rosuvastatin group (from 0.60 ⴞ 0.19 to 0.49 ⴞ 0.10 mol/L, p <0.001). Increases in flow-mediated vasodilation were positively correlated with reductions in plasma levels of ADMA (p ⴝ 0.017) and low-density lipoprotein cholesterol (p <0.001). Thus, our findings suggest that treatment with rosuvastatin in patients with hypercholesterolemia may lead to a significant reduction in plasma ADMA levels, which appear to be related to the improvement in endothelial function by rosuvastatin. 䊚2004 by Excerpta Medica, Inc. (Am J Cardiol 2004;94:157–161)
erangement of the -arginine–nitric oxide (NO) pathway by asymmetric dimethylarginine (ADMA), D which is characterized as a circulating endogenous com-
a novel risk factor of coronary artery atherosclerosis.9,10 However, whether elevated plasma concentrations of ADMA can be modified by drug treatment is still not clear. Because recent studies have suggested that native or oxidized low-density lipoprotein (LDL) cholesterol may increase the accumulation of ADMA,11–13 it is tempting to hypothesize that treating hypercholesterolemia with hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) may lower plasma ADMA levels, which may further contribute to the restoration of endothelial dysfunction. Hence, we designed a multicenter, randomized, double-blind, placebo-controlled study to investigate the influence of statin therapy on plasma levels of ADMA and endothelial function. We selected rosuvastatin, which is potent in lowering plasma cholesterol concentrations14 and has been reported to increase vascular endothelial NO production.15
L
petitive inhibitor of NO synthase, has been implicated as a possible contributing factor to endothelial dysfunction.1 Elevation of ADMA has been observed in plasma from patients with various risk factors of atherosclerosis,2 including hypercholesterolemia,3 hypertension,4 diabetes,5 and insulin resistance.6 Two recent studies have shown that plasma ADMA levels are both independent risk factors of cardiovascular mortality in patients with endstage renal disease and predictors of acute coronary events in middle-aged men.7,8 We also found that elevations in ADMA levels may be associated with later major adverse cardiovascular events after percutaneous coronary intervention, suggesting that ADMA may be From the Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, and School of Medicine, National YangMing University, Taiwan; the Division of Cardiology, Cheng-Hsin General Hospital, Taiwan; Department of Medical Education and Research, Taichung Veterans General Hospital, Taiwan; and Division of Cardiology, Department of Internal Medicine, Tri-Service General Hospital, Taiwan, Republic of China. This study was supported by Grants NSC91-2314-B075-081 and NSC92-2314-B075-114 from the National Science Council and Grants VGH92-035 and VGH92237 from the Veterans General Hospital Research Program, Taiwan, Republic of China, and in part by Astra-Zeneca, Taipei, Taiwan, Republic of China. Manuscript received December 16, 2003; revised manuscript received and accepted March 26, 2004. Address for reprints: Yu-An Ding, MD, PhD, Division of Cardiology, Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road, Taipei, Taiwan, Republic of China. E-mail: [email protected]. ©2004 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 94 July 15, 2004
METHODS
Study patients and protocol: This study was a multicenter, randomized, double-blind, placebo-controlled, parallel group study and enrolled patients with hypercholesterolemia with fasting plasma LDL cholesterol ⬎160 mg/dl and triglyceride levels ⬍350 mg/dl after an initial 6 weeks of diet control. Patients with renal dysfunction (serum creatinine ⬎2.0 mg/dl), hepatic or thyroid disease, chronic or acute inflammation, history of malignancy, uncompensated heart failure, uncontrolled hypertension, or diabetes mellitus were excluded. Patients with acute coronary syndromes occurring within 1 month were also excluded. During a 6-week dietary run-in 0002-9149/04/$–see front matter doi:10.1016/j.amjcard.2004.03.052
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TABLE 1 Clinical Characteristics of Study Subjects
Variables Age (yrs) Men/women Systemic hypertension Body mass index (kg/m2) Diabetes mellitus Established CAD Serum creatinine (mg/dl) Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglycerides (mg/dl) Medications Aspirin Nitrate Calcium antagonists ACE inhibitors/ARBs  blockers L-arginine (M) ADMA (M) SDMA (M) L-arginine/ADMA ratio Homocysteine (M) 8-isoprostane (pg/ml) hs-CRP (mg/L)
Rosuvastatin (10 mg/d) (n ⫽ 23)
Placebo (n ⫽ 23)
p Values
62.8 ⫾ 11.2 10/13 14 (60.9%) 25.3 ⫾ 2.7 3 (13.0%) 8 (34.8%) 1.1 ⫾ 0.2 259.9 ⫾ 35.3 44.2 ⫾ 11.7 177.5 ⫾ 30.6 190.7 ⫾ 88.3
59.8 ⫾ 11.8 17/6 14 (60.9%) 24.8 ⫾ 2.9 0 (0.0%) 3 (13.0%) 1.1 ⫾ 0.2 256.4 ⫾ 31.9 48.8 ⫾ 13.5 178.3 ⫾ 30.3 146.4 ⫾ 54.5
0.32 0.07 1.00 0.55 0.12 0.16 0.75 0.73 0.22 0.94 0.09
7 (30.4%) 3 (13.0%) 7 (30.4%) 9 (39.1%) 7 (30.4%) 81.9 ⫾ 22.3 0.60 ⫾ 0.19 0.59 ⫾ 0.24 151.1 ⫾ 61.3 12.3 ⫾ 6.2 52.2 ⫾ 20.0 2.0 ⫾ 2.2
5 (21.7%) 3 (13.0%) 11 (47.8%) 7 (30.4%) 4 (17.4%) 83.3 ⫾ 24.4 0.54 ⫾ 0.14 0.65 ⫾ 0.45 167.7 ⫾ 68.6 10.5 ⫾ 5.7 55.7 ⫾ 13.5 2.5 ⫾ 2.4
0.59 1.00 0.22 0.23 0.20 0.84 0.16 0.57 0.39 0.31 0.49 0.22
ACE ⫽ angiotensin-converting enzyme; ARB ⫽ angiotensin-II receptor blocker; CAD ⫽ coronary artery disease; HDL ⫽ high-density lipoprotein; SDMA ⫽ symmetric dimethylarginine.
TABLE 2 Changes in Plasma Parameters After Treatment With Rosuvastatin or Placebo Rosuvastatin (10 mg/d) (n ⫽ 23)
Total cholesterol LDL cholesterol HDL cholesterol Triglycerides L-arginine ADMA SDMA L-arginine/ADMA ratio Homocysteine 8-isoprostane hs-CRP
Placebo (n ⫽ 23)
Change (%)
p Values
Change (%)
p Values
⫺30.2 ⫺43.4 ⫹14.0 ⫺19.4 ⫹1.0 ⫺22.4 ⫹16.3 ⫹23.2 ⫺17.4 ⫹7.7 ⫺40.6
⬍0.0001 ⬍0.0001 ⬍0.0001 0.003 0.89 ⬍0.001 0.33 0.009 0.08 0.21 0.06
⫹3.3 ⫹2.3 ⫹2.4 ⫹11.2 ⫹0.1 ⫺0.7 ⫺2.7 ⫹1.5 ⫺4.4 ⫹5.7 ⫺25.8
⬍0.08 0.40 0.53 0.28 0.99 0.79 0.76 0.79 0.55 0.34 0.07
Abbreviations as in Table 1.
period for determining eligibility for treatment, patients were encouraged to adhere to the American Heart Association step I diet. Eligible patients were randomized to either the rosuvastatin group (10 mg/day) or the placebo group and received treatment for 6 weeks. Fasting blood samples were collected before and 6 weeks after treatment in all patients for measurements of plasma ADMA, L-arginine, 8-isoprostane (8-epi-prostaglandin F2␣), homocysteine, high-sensitivity C-reactive protein (hsCRP), lipid profile, creatinine, and blood glucose. Symmetric dimethylarginine, the biologically inactive stereoisomer of ADMA, was also determined. Before blood sampling, all medications were withdrawn for 12 hours. Cigarette smoking and beverages containing alcohol or caffeine were also avoided for ⱖ12 hours. This 158 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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study was approved by local ethics committees and written informed consent was obtained from all patients before participating in this study. Laboratory analysis: Blood samples were centrifuged at 3,000 rpm for 10 minutes at 4°C immediately after collection. Plasma samples were then kept frozen at ⫺70°C until analysis. Plasma L-arginine and ADMA concentrations were determined by high-performance liquid chromatography using precolumn derivatization with o-phthaldialdehyde as previously described.9,10 The recovery rate for ADMA was ⬎90%, and the within-assay and betweenassay variation coefficients were not ⬎7% and 8%, respectively. Plasma homocysteine levels were determined by the isocratic high-performance liquid chromatography method. Determination of hs-CRP levels was performed using latex enhanced immunophelometric assays on a BN II analyzer (Dade Behring, Marburg, Germany). For determination of plasma 8-isoprostane, the samples were purified by using ethyl acetate containing 1% methanol on a C-18 solid-phase extraction cartridge, and then total plasma 8-isoprostane was determined by the enzyme-linked immunosorbent assay method using a commercialized kit (Cayman, Ann Arbor, Michigan). Fasting serum creatinine, total and high-density lipoprotein cholesterol, triglycerides, and blood sugar levels were determined by an autoanalyzer (model 747 to 100, Hitachi, Tokyo). LDL cholesterol levels were calculated according to the Friedewald formula. Measurement of flow-mediated vasodilation: Endothelial function as-
sessed as flow-mediated and nitroglycerin-mediated vasodilation of the brachial artery was performed in 11 patients in the rosuvastatin group and in 12 patients in the placebo group (patients enrolled in Taipei Veteran General Hospital). Studies were performed at baseline and 6 weeks later in the morning after a 12-hour fast. Each subject lay comfortably in bed in a quiet, dark, and air-conditioned room (22°C to 24°C) for ⬎20 minutes. Then, B-mode scans of the brachial artery were obtained in a longitudinal section 2 to 15 cm above the elbow by using a Hewlett-Packard Echo system (model 5500, Andover, Massachusetts) incorporated with a 7.5-MHz linear-arrayed vascular ultrasound transducer. Increased forearm flow was induced by inflating a blood pressure cuff around the widest part of the forearm to a systolic blood pressure of JULY 15, 2004
200 mm Hg or ⬎20 mm Hg above systolic blood pressure for exactly 5 minutes. Repeat brachial artery diameter scans were obtained immediately and 1 minute after release of occlusion. Brachial artery diameters scans at rest were repeated 15 minutes later. Sublingual nitroglycerin (400 g) was then administered, and final scans were recorded after 5 minutes. Images were recorded using the ultrasound digital storage and retrieval software system. Vessel diameters were measured with ultrasonic calipers by independent observers blinded to subject information. The diameter of the brachial artery was measured in electrocardiographic-gated and diastolic frames from 1 intima-media interface to the other in a 5-cm segment of vessel. Flow-mediated (endothelium-dependent) vasodilation was expressed as the relative percent increase in brachial artery diameter during hyperemia (flow-mediated vasodilation). Nitroglycerin-mediated (endothelium-independent) vasodilation was expressed in an analogous fashion. Statistical analysis: All parametric values are expressed as mean ⫾ SD. Parametric continuous data between treatment and placebo groups were compared by unpaired Student’s t test and nonparametric data by the Mann-Whitney test. Categorical data between the 2 groups were compared by the chi-square test or Fisher’s exact test. Baseline data and changes in all plasma variables in each group were tested by paired t test. Pearson’s correlation coefficients were calculated to examine possible correlations between continuous variables, and Spearman’s rank correlation coefficients were used for variables with non-normal distribution. A stepwise multiple regression analysis was performed to assess the relations between changes in plasma ADMA levels and other risk factors. We tested the following variables: age; gender; baseline data; changes in 8-isoprostane, homocysteine, and LDL cholesterol; and the use of angiotensin-converting enzyme inhibitors/angiotensin-II receptor blockers. A p value ⬍0.05 (2-tailed) was considered statistically significant. The SPSS 11.0 (SPSS Inc., Chicago, Illinois) software package was used for statistical analysis.
RESULTS
Patient characteristics: In all, 172 patients were enrolled in the run-in phase, of whom 122 were unable to fulfill the inclusion criteria. All of the 50 remaining eligible patients were randomized into the rosuvastatin or placebo groups. Two patients in each group were withdrawn from the study prematurely because of poor compliance, and finally data of 46 patients were analyzed. Baseline characteristics of the study patients are listed in Table 1 and showed no significant differences between both groups. In the total population, a significant correlation was observed between the plasma levels of 8-isoprostane and ADMA (r ⫽ 0.388, p ⫽ 0.008). In contrast, plasma ADMA levels did not correlate with plasma levels of homocysteine, hsCRP, creatinine, total cholesterol, high-density lipoprotein and LDL cholesterol, and triglycerides.
FIGURE 1. Changes in plasma ADMA levels in the rosuvastatin and placebo groups.
Reduction in plasma ADMA levels with rosuvastatin:
As expected, treatment with rosuvastatin for 6 weeks led to a significant reduction in plasma concentrations of total cholesterol, LDL cholesterol, triglycerides, and an increase in high-density lipoprotein cholesterol levels (Table 2). Compared with the placebo group, treatment with rosuvastatin significantly reduced plasma levels of ADMA (from 0.60 ⫾ 0.19 to 0.49 ⫾ 0.10 mol/L, p ⬍0.001; Table 2 and Figure 1). In contrast, the plasma levels of symmetric dimethylarginine remained unchanged after treatment. Moreover, treatment with rosuvastatin caused a borderline reduction in plasma hs-CRP levels and did not lower the plasma concentrations of 8-isoprotane and homocysteine (Table 2). All of these variables did not change significantly in the placebo group. In the total population, the reduction in plasma ADMA levels was positively correlated with baseline plasma concentrations of 8-isoprostane (r ⫽ 0.429, p ⫽ 0.003) and homocysteine (r ⫽ 0.338, p ⫽ 0.02). In addition, there were significantly positive correlations between changes in plasma ADMA levels and changes in LDL cholesterol (r ⫽ 0.459, p ⫽ 0.001; Figure 2) and homocysteine (r ⫽ 0.504, p ⬍0.001). In stepwise multiple regression analysis, changes in ADMA levels were significantly associated with baseline plasma 8-isoprostane levels (p ⫽ 0.008) and changes in plasma concentrations of LDL cholesterol (p ⬍0.001) and homocysteine (p ⫽ 0.02).
Improvement in endothelial function with rosuvastatin:
At baseline, no differences in percent flow-mediated dilation and percent nitroglycerin-mediated vasodilation were found between 2 groups, and the baseline percent flow-mediated dilation did not correlate with plasma levels of LDL cholesterol, ADMA, and other variables. After treatment for 6 weeks, the percent flow-mediated dilation in patients receiving rosuvastatin was significantly improved (p ⫽ 0.001; Figure 3), whereas the percent flow-mediated dilation in patients receiving placebo was unchanged. In contrast, percent nitroglycerin-mediated vasodilation was similar before and after treatment in both groups (Figure 3). Changes in percent flow-mediated dilation before
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ment with rosuvastatin in hypercholesterolemic patients with more increased oxidative stress may lead to a greater decrease in ADMA levels. Formation of 8-isoprostane, a marker of oxidative stress, has been reported to be increased in patients with hypercholesterolemia,19 and ADMA concentrations were reported to be elevated not only in patients with hypercholesterolemia but also in other conditions of increased oxidative stress, such as hypertension,4 hyperglycemia,6 and hyperhomocysteinemia.20 Lin et al21 recently reported that hyperglycemia-induced oxidative stress can impair the activity of dimethylarginine dimethylaminohydrolase, the enzyme responsible for the degradation of ADMA, resulting in an accumulation of ADMA in diabetic rats. On the other FIGURE 2. Correlations between reduction in plasma ADMA levels and plasma hand, in contrast to L-arginine as the LDL cholesterol in the rosuvastatin (black circles) and placebo (white circles) substrate for NO synthase, ADMA may groups. also uncouple the electron transfer between NO synthase and L-arginine, increase oxidative stress, and further imand after treatment were positively correlated with pair the availability of endothelium-derived NO.1 reductions in plasma ADMA levels (r ⫽ 0.493, p ⫽ Bo¨ ger et al13 recently demonstrated that ADMA may 0.017) and LDL cholesterol (r ⫽ 0.702, p ⬍0.001). be a proatherogenic molecule by increasing endothelial oxidative stress and potentiating monocyte adhesion; in hypercholesterolemic rabbits, this effect is DISCUSSION The results of this study showed that in patients reversed by supplementation of L-arginine.22 This evwith hypercholesterolemia, elevated plasma ADMA idence, together with our findings, suggest that elelevels were associated with increased oxidative stress. vated concentrations of ADMA may be associated In addition, to improve endothelium-dependent vaso- with increased oxidative stress that may decrease the dilation, treatment with rosuvastatin for 6 weeks also bioavailability of NO and further contribute to the led to a significant decrease in plasma ADMA levels, development of endothelial dysfunction. which was closely correlated with the reduction in Recent studies demonstrated that elevated concenplasma LDL cholesterol. Moreover, improvement in trations of ADMA may be involved in the pathogenendothelial function was significantly correlated with esis of hyperhomocysteinemia-mediated endothelial a reduction in plasma ADMA and LDL cholesterol dysfunction.23,24 In this study, after treatment with levels. These findings support the hypothesis that re- rosuvastatin, the changes in plasma ADMA levels ducing plasma concentrations of ADMA, probably were associated positively with changes in plasma through lowering LDL cholesterol levels, may contribute homocysteine levels. However, no significant correlato the restoration of endothelial dysfunction in hypercho- tion was found between baseline plasma ADMA and lesterolemic patients treated with rosuvastatin. homocysteine levels, and in accordance with previous Statins can increase vascular endothelial NO pro- studies, plasma homocysteine levels did not decrease duction, and their effects on up-regulation of endothe- significantly after treatment with statins.25 Because lial NO synthase and antioxidation have been sug- hyperhomocysteinemia (⬎15 mol/L) was noted in gested to be the possible underlying mechanisms.15–17 only 5 of our patients, the role of homocysteine in the Because ADMA has been associated with attenuation mechanism of reducing ADMA with statins needs to of endothelium-dependent vasodilation,3 and a direct be elucidated in hyperhomocysteinemic patients. There are several limitations to this study. First, infusion of ADMA into human brachial arteries causes a dose-dependent decrease in forearm blood there were no correlations between baseline plasma flow,18 taken together, our data suggest that reducing levels of LDL cholesterol and ADMA, which was ADMA levels may be another possible mechanism of inconsistent with previous studies3 but was similar to the vasoprotective effects of statins. those reported by Miyazaki et al2 and Pa¨ iva¨ et al.26 Our findings showed that in hypercholesterolemic This may be due to different ethnic and demographic patients, there was a positive correlation between characteristics (e.g., older age and more cardiovascubaseline plasma ADMA and 8-isoprostane levels. lar risk factors in addition to hypercholesterolemia in Moreover, the decrease in ADMA levels after treat- our patients). Second, only some of the patients in this ment with rosuvastatin correlated positively with the study underwent assessment of endothelial function baseline 8-isoprostane levels, suggesting that treat- by measuring flow-mediated vasodilation, which may 160 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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FIGURE 3. Percent FMD (A) and percent nitroglycerin-mediated vasodilation (%NMD) (B) in the rosuvastatin and placebo groups at baseline and after treatment for 6 weeks.
have led to a selection bias. These patients were enrolled in 1 center unselectively, and the baseline characteristics in these patients were not different from those of total population. Moreover, in these patients, the reduction in plasma ADMA levels and the positive correlation between reduction of ADMA and LDL cholesterol after rosuvastatin treatment remained significant. Finally, in contrast to our findings, several previous studies have shown that treatment of hypercholesterolemia with various statins, including simvastatin, atorvastatin,26 and pravastatin,27 failed to reduce plasma ADMA levels despite adequate reduction of plasma cholesterol concentrations. This is likely to be related to different characteristics in our population, but may also due to the specific effect of rosuvastatin. Nevertheless, our results need to be corroborated by further studies. Acknowledgment: We are indebted to Shu-Chuan Lin and Pui-Ching Lee for their excellent technical and statistical assistance.
1. Leiper J, Vallance P. Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res 1999;43:542–548. 2. Miyazaki H, Matsuoka H, Cooke JP, Usui M, Ueda S, Okuda S, Imaizumi T.
Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Circulation 1999;99:1141–1146. 3. Bo¨ ger RH, Bode-Bo¨ ger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, Blaschke TF, Cooke JP. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 1998;98:1842–1847. 4. Surdacki A, Nowichi M, Sandmann J, Tsikas D, Bo¨ ger RH, Bode-Bo¨ ger SM, Kruszelnicka-Kwiatkowska O, Kokot F, Dubiel JS, Fro¨ lich JC. Reduced urinary excretion of nitric oxide metabolites and increased plasma levels of asymmetric dimethylarginine in men with essential hypertension. J Cardiovasc Pharmacol 1999;33:652–658. 5. Abbasi F, Asagmi T, Cooke JP, Lamendala C, McLaughlin T, Reaven GM, Stu¨ hlinger MC, Tsao PS. Plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus. Am J Cardiol 2001;88: 1201–1203. 6. Stu¨ hlinger MC, Abbasi F, Chu JW, Lamendola C, McLaughlin TL, Cooke JP, Reaven GM, Tsao PS. Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor. JAMA 2002;287:1420 –1426. 7. Zoccali C, Bode-Bo¨ ger SM, Mallamaci F, Benedetto FA, Tripepi G, Malatina LS, Cataliotti A, Bellanuova I, Ferno I, Fro¨ lich JC, Bo¨ ger RH. Plasma concentrations of asymmetrical dimethylarginine and mortality in patients with endstage renal disease: a prospective study. Lancet 2001;358:2113–2117. 8. Valkonen VP, Pa¨ lva¨ H, Salonen JT, Lakka TA, Lehtima¨ ki T, Laakso J, Laaksonen R. Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine. Lancet 2001;358:2127–2128. 9. Lu TM, Ding YA, Charng MJ, Lin SJ. Asymmetrical dimethylarginine: a novel risk factor for coronary artery disease. Clin Cardiol 2003;26:458 –464. 10. 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;24:1912–1919. 11. Ito A, Tsao PS, Adimoolam S, Kimoto M, Ogawa T, Cooke JP. Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. Circulation 1999;99:3092–3095. 12. Bo¨ ger RH, Syndow K, Borlak J, Tuhm T, Lenzen H, Schubert B, Tsikas D, Bode-Bo¨ ger SM. LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethioninedependent methyltransferases. Circ Res 2000;87:99 –105. 13. Bo¨ ger RH, Bode-Bo¨ ger SM, Tsao PS, Lin PS, Chan JR, Cooke JP. An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes. J Am Coll Cardiol 2000;36:2287–2295. 14. Olsson AG, Pears J, McKellar J, Mizan J, Raza A. Effect of rosuvastatin on low-density lipoprotein cholesterol in patients with hypercholesterolemia. Am J Cardiol 2001;88:504 –508. 15. Jones SP, Gibson MF, Rimmer DM, Gibson TM, Sharp BR, Lefer DJ. Direct vascular and cardioprotective effects of rosuvastatin, a new HMG-CoA reductase inhibitor. J Am Coll Cardiol 2002;40:1172–1178. 16. Laufs U, La Fata V, Plutzky J, Liao J. Upregulation of endothelial nitric oxide synthase by HMG-CoA reductase inhibitors. Circulation 1998;97:1129 –1135. 17. Shishehbor MH, Brennan ML, Aviles RJ, Fu X, Penn MS, Sprecher DL, Hazen SL. Statins promote potent systemic antioxidant effects through specific inflammatory pathways. Circulation 2003;108:426 –431. 18. Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992;339:572–575. 19. Reilly MP, Pratico` D, Delanty N, DiMinno G, Tremoli E, Rader D, Kapoor S, Rokach J, Lawson J, FitzGerald GA. Increased formation of distinct F2 isoprostanes in hypercholesterolemia. Circulation 1998;98:2822–2828. 20. Kanami PM, Sinkey CA, Browning RL, Allaman M, Knapp HR, Haynes WG. Role of oxidant stress in experimental dysfunction produced by experimental hyperhomocyst(e)inemia in humans. Circulation 1999;100:1161–1168. 21. Lin KY, Ito A, Asagami T, Tsao PS, Adimoolam S, Kimoto M, Tsuji H, Reaven GM, Cooke JP. Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase. Circulation 2002;106:987–992. 22. Bo¨ ger RH, Bode-Bo¨ ger SM, Mu¨ gge A, Kienke S, Brandes R, Dwenger A, Frohlich JC. Supplementation of hypercholesterolemic rabbits with L-arginine reduces the vascular release of superoxide anions and restores NO production. Atherosclerosis 1995;117:273–284. 23. Sydow K, Schwedhelm E, Arakawa N, Bode-Bo¨ ger SM, Tsikas D, Hornig B, Fro¨ lich JC, Bo¨ ger RH. ADMA and oxidative stress are responsible for endothelial dysfunction in hyperhomocysteinemia: effects of L-arginine and B vitamins. Cardiovasc Res 2003;57:244 –252. 24. Stu¨ hlinger MC, Oka RK, Graf EE, Schmo¨ lzer I, Upson BM, Kapoor O, Szuba A, Malinow R, Wascher TC, Pachinger O, Cooke JP. Endothelial dysfunction induced by hyperhomocyst(e)inemia: role of asymmetric dimethylarginine. Circulation 2003;108:933–938. 25. Giral P, Bruckert E, Jacob N, Chapman MJ, Foglietti MJ, Turpin G. Homocysteine and lipid-lowering agents: a comparison between atorvastatin and fenofibrate in patients with mixed hyperlipidemia. Atherosclerosis 2001;154:421–427. 26. Pa¨ iva¨ H, Laakso J, Lehtima¨ ki T, Isomustaja¨ rvi M, Ruokonen I, Laaksonen R. Effect of high-dose statin treatment on plasma concentrations of endogenous nitric oxide synthase inhibitors. J Cardiovasc Pharmacol 2003;41:219 –222. 27. Eid HM, Eritsland J, Larsen J, Arnesen H, Seljeflot I. Increased levels of asymmetrical dimethylarginine in populations at risk for atherosclerosis disease. Effects of pravastatin. Atherosclerosis 2003;166:279 –284.
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MD,
Elevated plasma levels of asymmetric dimethylarginine (ADMA) have been associated with attenuated endothelium-dependent vasodilation in hypercholesterolemic patients. However, whether lowering of plasma cholesterol concentration by hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) can reduce plasma ADMA levels is still not clear. This study was a multicenter, randomized, double-blind, placebo-controlled design including 46 patients with elevated low-density lipoprotein cholesterol levels. Patients were randomized into 2 groups: rosuvastatin 10 mg/day and placebo for 6 weeks. Plasma levels of ADMA, 8-isoprostane (as a marker of oxidative stress), homocysteine, and highsensitivity C-reactive protein were measured at baseline and 6 weeks later. Endothelial function assessed by flow-mediated vasodilation of the brachial artery was performed in 11 patients in the rosuvastatin group and
in 12 in the placebo group. Baseline characteristics of both groups were similar, and the plasma ADMA levels were significantly correlated with 8-isoprostane (r ⴝ 0.388, p ⴝ 0.008). After 6 weeks of treatment, plasma ADMA levels were significantly reduced in the rosuvastatin group (from 0.60 ⴞ 0.19 to 0.49 ⴞ 0.10 mol/L, p <0.001). Increases in flow-mediated vasodilation were positively correlated with reductions in plasma levels of ADMA (p ⴝ 0.017) and low-density lipoprotein cholesterol (p <0.001). Thus, our findings suggest that treatment with rosuvastatin in patients with hypercholesterolemia may lead to a significant reduction in plasma ADMA levels, which appear to be related to the improvement in endothelial function by rosuvastatin. 䊚2004 by Excerpta Medica, Inc. (Am J Cardiol 2004;94:157–161)
erangement of the -arginine–nitric oxide (NO) pathway by asymmetric dimethylarginine (ADMA), D which is characterized as a circulating endogenous com-
a novel risk factor of coronary artery atherosclerosis.9,10 However, whether elevated plasma concentrations of ADMA can be modified by drug treatment is still not clear. Because recent studies have suggested that native or oxidized low-density lipoprotein (LDL) cholesterol may increase the accumulation of ADMA,11–13 it is tempting to hypothesize that treating hypercholesterolemia with hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) may lower plasma ADMA levels, which may further contribute to the restoration of endothelial dysfunction. Hence, we designed a multicenter, randomized, double-blind, placebo-controlled study to investigate the influence of statin therapy on plasma levels of ADMA and endothelial function. We selected rosuvastatin, which is potent in lowering plasma cholesterol concentrations14 and has been reported to increase vascular endothelial NO production.15
L
petitive inhibitor of NO synthase, has been implicated as a possible contributing factor to endothelial dysfunction.1 Elevation of ADMA has been observed in plasma from patients with various risk factors of atherosclerosis,2 including hypercholesterolemia,3 hypertension,4 diabetes,5 and insulin resistance.6 Two recent studies have shown that plasma ADMA levels are both independent risk factors of cardiovascular mortality in patients with endstage renal disease and predictors of acute coronary events in middle-aged men.7,8 We also found that elevations in ADMA levels may be associated with later major adverse cardiovascular events after percutaneous coronary intervention, suggesting that ADMA may be From the Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, and School of Medicine, National YangMing University, Taiwan; the Division of Cardiology, Cheng-Hsin General Hospital, Taiwan; Department of Medical Education and Research, Taichung Veterans General Hospital, Taiwan; and Division of Cardiology, Department of Internal Medicine, Tri-Service General Hospital, Taiwan, Republic of China. This study was supported by Grants NSC91-2314-B075-081 and NSC92-2314-B075-114 from the National Science Council and Grants VGH92-035 and VGH92237 from the Veterans General Hospital Research Program, Taiwan, Republic of China, and in part by Astra-Zeneca, Taipei, Taiwan, Republic of China. Manuscript received December 16, 2003; revised manuscript received and accepted March 26, 2004. Address for reprints: Yu-An Ding, MD, PhD, Division of Cardiology, Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road, Taipei, Taiwan, Republic of China. E-mail: [email protected]. ©2004 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 94 July 15, 2004
METHODS
Study patients and protocol: This study was a multicenter, randomized, double-blind, placebo-controlled, parallel group study and enrolled patients with hypercholesterolemia with fasting plasma LDL cholesterol ⬎160 mg/dl and triglyceride levels ⬍350 mg/dl after an initial 6 weeks of diet control. Patients with renal dysfunction (serum creatinine ⬎2.0 mg/dl), hepatic or thyroid disease, chronic or acute inflammation, history of malignancy, uncompensated heart failure, uncontrolled hypertension, or diabetes mellitus were excluded. Patients with acute coronary syndromes occurring within 1 month were also excluded. During a 6-week dietary run-in 0002-9149/04/$–see front matter doi:10.1016/j.amjcard.2004.03.052
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TABLE 1 Clinical Characteristics of Study Subjects
Variables Age (yrs) Men/women Systemic hypertension Body mass index (kg/m2) Diabetes mellitus Established CAD Serum creatinine (mg/dl) Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglycerides (mg/dl) Medications Aspirin Nitrate Calcium antagonists ACE inhibitors/ARBs  blockers L-arginine (M) ADMA (M) SDMA (M) L-arginine/ADMA ratio Homocysteine (M) 8-isoprostane (pg/ml) hs-CRP (mg/L)
Rosuvastatin (10 mg/d) (n ⫽ 23)
Placebo (n ⫽ 23)
p Values
62.8 ⫾ 11.2 10/13 14 (60.9%) 25.3 ⫾ 2.7 3 (13.0%) 8 (34.8%) 1.1 ⫾ 0.2 259.9 ⫾ 35.3 44.2 ⫾ 11.7 177.5 ⫾ 30.6 190.7 ⫾ 88.3
59.8 ⫾ 11.8 17/6 14 (60.9%) 24.8 ⫾ 2.9 0 (0.0%) 3 (13.0%) 1.1 ⫾ 0.2 256.4 ⫾ 31.9 48.8 ⫾ 13.5 178.3 ⫾ 30.3 146.4 ⫾ 54.5
0.32 0.07 1.00 0.55 0.12 0.16 0.75 0.73 0.22 0.94 0.09
7 (30.4%) 3 (13.0%) 7 (30.4%) 9 (39.1%) 7 (30.4%) 81.9 ⫾ 22.3 0.60 ⫾ 0.19 0.59 ⫾ 0.24 151.1 ⫾ 61.3 12.3 ⫾ 6.2 52.2 ⫾ 20.0 2.0 ⫾ 2.2
5 (21.7%) 3 (13.0%) 11 (47.8%) 7 (30.4%) 4 (17.4%) 83.3 ⫾ 24.4 0.54 ⫾ 0.14 0.65 ⫾ 0.45 167.7 ⫾ 68.6 10.5 ⫾ 5.7 55.7 ⫾ 13.5 2.5 ⫾ 2.4
0.59 1.00 0.22 0.23 0.20 0.84 0.16 0.57 0.39 0.31 0.49 0.22
ACE ⫽ angiotensin-converting enzyme; ARB ⫽ angiotensin-II receptor blocker; CAD ⫽ coronary artery disease; HDL ⫽ high-density lipoprotein; SDMA ⫽ symmetric dimethylarginine.
TABLE 2 Changes in Plasma Parameters After Treatment With Rosuvastatin or Placebo Rosuvastatin (10 mg/d) (n ⫽ 23)
Total cholesterol LDL cholesterol HDL cholesterol Triglycerides L-arginine ADMA SDMA L-arginine/ADMA ratio Homocysteine 8-isoprostane hs-CRP
Placebo (n ⫽ 23)
Change (%)
p Values
Change (%)
p Values
⫺30.2 ⫺43.4 ⫹14.0 ⫺19.4 ⫹1.0 ⫺22.4 ⫹16.3 ⫹23.2 ⫺17.4 ⫹7.7 ⫺40.6
⬍0.0001 ⬍0.0001 ⬍0.0001 0.003 0.89 ⬍0.001 0.33 0.009 0.08 0.21 0.06
⫹3.3 ⫹2.3 ⫹2.4 ⫹11.2 ⫹0.1 ⫺0.7 ⫺2.7 ⫹1.5 ⫺4.4 ⫹5.7 ⫺25.8
⬍0.08 0.40 0.53 0.28 0.99 0.79 0.76 0.79 0.55 0.34 0.07
Abbreviations as in Table 1.
period for determining eligibility for treatment, patients were encouraged to adhere to the American Heart Association step I diet. Eligible patients were randomized to either the rosuvastatin group (10 mg/day) or the placebo group and received treatment for 6 weeks. Fasting blood samples were collected before and 6 weeks after treatment in all patients for measurements of plasma ADMA, L-arginine, 8-isoprostane (8-epi-prostaglandin F2␣), homocysteine, high-sensitivity C-reactive protein (hsCRP), lipid profile, creatinine, and blood glucose. Symmetric dimethylarginine, the biologically inactive stereoisomer of ADMA, was also determined. Before blood sampling, all medications were withdrawn for 12 hours. Cigarette smoking and beverages containing alcohol or caffeine were also avoided for ⱖ12 hours. This 158 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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study was approved by local ethics committees and written informed consent was obtained from all patients before participating in this study. Laboratory analysis: Blood samples were centrifuged at 3,000 rpm for 10 minutes at 4°C immediately after collection. Plasma samples were then kept frozen at ⫺70°C until analysis. Plasma L-arginine and ADMA concentrations were determined by high-performance liquid chromatography using precolumn derivatization with o-phthaldialdehyde as previously described.9,10 The recovery rate for ADMA was ⬎90%, and the within-assay and betweenassay variation coefficients were not ⬎7% and 8%, respectively. Plasma homocysteine levels were determined by the isocratic high-performance liquid chromatography method. Determination of hs-CRP levels was performed using latex enhanced immunophelometric assays on a BN II analyzer (Dade Behring, Marburg, Germany). For determination of plasma 8-isoprostane, the samples were purified by using ethyl acetate containing 1% methanol on a C-18 solid-phase extraction cartridge, and then total plasma 8-isoprostane was determined by the enzyme-linked immunosorbent assay method using a commercialized kit (Cayman, Ann Arbor, Michigan). Fasting serum creatinine, total and high-density lipoprotein cholesterol, triglycerides, and blood sugar levels were determined by an autoanalyzer (model 747 to 100, Hitachi, Tokyo). LDL cholesterol levels were calculated according to the Friedewald formula. Measurement of flow-mediated vasodilation: Endothelial function as-
sessed as flow-mediated and nitroglycerin-mediated vasodilation of the brachial artery was performed in 11 patients in the rosuvastatin group and in 12 patients in the placebo group (patients enrolled in Taipei Veteran General Hospital). Studies were performed at baseline and 6 weeks later in the morning after a 12-hour fast. Each subject lay comfortably in bed in a quiet, dark, and air-conditioned room (22°C to 24°C) for ⬎20 minutes. Then, B-mode scans of the brachial artery were obtained in a longitudinal section 2 to 15 cm above the elbow by using a Hewlett-Packard Echo system (model 5500, Andover, Massachusetts) incorporated with a 7.5-MHz linear-arrayed vascular ultrasound transducer. Increased forearm flow was induced by inflating a blood pressure cuff around the widest part of the forearm to a systolic blood pressure of JULY 15, 2004
200 mm Hg or ⬎20 mm Hg above systolic blood pressure for exactly 5 minutes. Repeat brachial artery diameter scans were obtained immediately and 1 minute after release of occlusion. Brachial artery diameters scans at rest were repeated 15 minutes later. Sublingual nitroglycerin (400 g) was then administered, and final scans were recorded after 5 minutes. Images were recorded using the ultrasound digital storage and retrieval software system. Vessel diameters were measured with ultrasonic calipers by independent observers blinded to subject information. The diameter of the brachial artery was measured in electrocardiographic-gated and diastolic frames from 1 intima-media interface to the other in a 5-cm segment of vessel. Flow-mediated (endothelium-dependent) vasodilation was expressed as the relative percent increase in brachial artery diameter during hyperemia (flow-mediated vasodilation). Nitroglycerin-mediated (endothelium-independent) vasodilation was expressed in an analogous fashion. Statistical analysis: All parametric values are expressed as mean ⫾ SD. Parametric continuous data between treatment and placebo groups were compared by unpaired Student’s t test and nonparametric data by the Mann-Whitney test. Categorical data between the 2 groups were compared by the chi-square test or Fisher’s exact test. Baseline data and changes in all plasma variables in each group were tested by paired t test. Pearson’s correlation coefficients were calculated to examine possible correlations between continuous variables, and Spearman’s rank correlation coefficients were used for variables with non-normal distribution. A stepwise multiple regression analysis was performed to assess the relations between changes in plasma ADMA levels and other risk factors. We tested the following variables: age; gender; baseline data; changes in 8-isoprostane, homocysteine, and LDL cholesterol; and the use of angiotensin-converting enzyme inhibitors/angiotensin-II receptor blockers. A p value ⬍0.05 (2-tailed) was considered statistically significant. The SPSS 11.0 (SPSS Inc., Chicago, Illinois) software package was used for statistical analysis.
RESULTS
Patient characteristics: In all, 172 patients were enrolled in the run-in phase, of whom 122 were unable to fulfill the inclusion criteria. All of the 50 remaining eligible patients were randomized into the rosuvastatin or placebo groups. Two patients in each group were withdrawn from the study prematurely because of poor compliance, and finally data of 46 patients were analyzed. Baseline characteristics of the study patients are listed in Table 1 and showed no significant differences between both groups. In the total population, a significant correlation was observed between the plasma levels of 8-isoprostane and ADMA (r ⫽ 0.388, p ⫽ 0.008). In contrast, plasma ADMA levels did not correlate with plasma levels of homocysteine, hsCRP, creatinine, total cholesterol, high-density lipoprotein and LDL cholesterol, and triglycerides.
FIGURE 1. Changes in plasma ADMA levels in the rosuvastatin and placebo groups.
Reduction in plasma ADMA levels with rosuvastatin:
As expected, treatment with rosuvastatin for 6 weeks led to a significant reduction in plasma concentrations of total cholesterol, LDL cholesterol, triglycerides, and an increase in high-density lipoprotein cholesterol levels (Table 2). Compared with the placebo group, treatment with rosuvastatin significantly reduced plasma levels of ADMA (from 0.60 ⫾ 0.19 to 0.49 ⫾ 0.10 mol/L, p ⬍0.001; Table 2 and Figure 1). In contrast, the plasma levels of symmetric dimethylarginine remained unchanged after treatment. Moreover, treatment with rosuvastatin caused a borderline reduction in plasma hs-CRP levels and did not lower the plasma concentrations of 8-isoprotane and homocysteine (Table 2). All of these variables did not change significantly in the placebo group. In the total population, the reduction in plasma ADMA levels was positively correlated with baseline plasma concentrations of 8-isoprostane (r ⫽ 0.429, p ⫽ 0.003) and homocysteine (r ⫽ 0.338, p ⫽ 0.02). In addition, there were significantly positive correlations between changes in plasma ADMA levels and changes in LDL cholesterol (r ⫽ 0.459, p ⫽ 0.001; Figure 2) and homocysteine (r ⫽ 0.504, p ⬍0.001). In stepwise multiple regression analysis, changes in ADMA levels were significantly associated with baseline plasma 8-isoprostane levels (p ⫽ 0.008) and changes in plasma concentrations of LDL cholesterol (p ⬍0.001) and homocysteine (p ⫽ 0.02).
Improvement in endothelial function with rosuvastatin:
At baseline, no differences in percent flow-mediated dilation and percent nitroglycerin-mediated vasodilation were found between 2 groups, and the baseline percent flow-mediated dilation did not correlate with plasma levels of LDL cholesterol, ADMA, and other variables. After treatment for 6 weeks, the percent flow-mediated dilation in patients receiving rosuvastatin was significantly improved (p ⫽ 0.001; Figure 3), whereas the percent flow-mediated dilation in patients receiving placebo was unchanged. In contrast, percent nitroglycerin-mediated vasodilation was similar before and after treatment in both groups (Figure 3). Changes in percent flow-mediated dilation before
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ment with rosuvastatin in hypercholesterolemic patients with more increased oxidative stress may lead to a greater decrease in ADMA levels. Formation of 8-isoprostane, a marker of oxidative stress, has been reported to be increased in patients with hypercholesterolemia,19 and ADMA concentrations were reported to be elevated not only in patients with hypercholesterolemia but also in other conditions of increased oxidative stress, such as hypertension,4 hyperglycemia,6 and hyperhomocysteinemia.20 Lin et al21 recently reported that hyperglycemia-induced oxidative stress can impair the activity of dimethylarginine dimethylaminohydrolase, the enzyme responsible for the degradation of ADMA, resulting in an accumulation of ADMA in diabetic rats. On the other FIGURE 2. Correlations between reduction in plasma ADMA levels and plasma hand, in contrast to L-arginine as the LDL cholesterol in the rosuvastatin (black circles) and placebo (white circles) substrate for NO synthase, ADMA may groups. also uncouple the electron transfer between NO synthase and L-arginine, increase oxidative stress, and further imand after treatment were positively correlated with pair the availability of endothelium-derived NO.1 reductions in plasma ADMA levels (r ⫽ 0.493, p ⫽ Bo¨ ger et al13 recently demonstrated that ADMA may 0.017) and LDL cholesterol (r ⫽ 0.702, p ⬍0.001). be a proatherogenic molecule by increasing endothelial oxidative stress and potentiating monocyte adhesion; in hypercholesterolemic rabbits, this effect is DISCUSSION The results of this study showed that in patients reversed by supplementation of L-arginine.22 This evwith hypercholesterolemia, elevated plasma ADMA idence, together with our findings, suggest that elelevels were associated with increased oxidative stress. vated concentrations of ADMA may be associated In addition, to improve endothelium-dependent vaso- with increased oxidative stress that may decrease the dilation, treatment with rosuvastatin for 6 weeks also bioavailability of NO and further contribute to the led to a significant decrease in plasma ADMA levels, development of endothelial dysfunction. which was closely correlated with the reduction in Recent studies demonstrated that elevated concenplasma LDL cholesterol. Moreover, improvement in trations of ADMA may be involved in the pathogenendothelial function was significantly correlated with esis of hyperhomocysteinemia-mediated endothelial a reduction in plasma ADMA and LDL cholesterol dysfunction.23,24 In this study, after treatment with levels. These findings support the hypothesis that re- rosuvastatin, the changes in plasma ADMA levels ducing plasma concentrations of ADMA, probably were associated positively with changes in plasma through lowering LDL cholesterol levels, may contribute homocysteine levels. However, no significant correlato the restoration of endothelial dysfunction in hypercho- tion was found between baseline plasma ADMA and lesterolemic patients treated with rosuvastatin. homocysteine levels, and in accordance with previous Statins can increase vascular endothelial NO pro- studies, plasma homocysteine levels did not decrease duction, and their effects on up-regulation of endothe- significantly after treatment with statins.25 Because lial NO synthase and antioxidation have been sug- hyperhomocysteinemia (⬎15 mol/L) was noted in gested to be the possible underlying mechanisms.15–17 only 5 of our patients, the role of homocysteine in the Because ADMA has been associated with attenuation mechanism of reducing ADMA with statins needs to of endothelium-dependent vasodilation,3 and a direct be elucidated in hyperhomocysteinemic patients. There are several limitations to this study. First, infusion of ADMA into human brachial arteries causes a dose-dependent decrease in forearm blood there were no correlations between baseline plasma flow,18 taken together, our data suggest that reducing levels of LDL cholesterol and ADMA, which was ADMA levels may be another possible mechanism of inconsistent with previous studies3 but was similar to the vasoprotective effects of statins. those reported by Miyazaki et al2 and Pa¨ iva¨ et al.26 Our findings showed that in hypercholesterolemic This may be due to different ethnic and demographic patients, there was a positive correlation between characteristics (e.g., older age and more cardiovascubaseline plasma ADMA and 8-isoprostane levels. lar risk factors in addition to hypercholesterolemia in Moreover, the decrease in ADMA levels after treat- our patients). Second, only some of the patients in this ment with rosuvastatin correlated positively with the study underwent assessment of endothelial function baseline 8-isoprostane levels, suggesting that treat- by measuring flow-mediated vasodilation, which may 160 THE AMERICAN JOURNAL OF CARDIOLOGY姞
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FIGURE 3. Percent FMD (A) and percent nitroglycerin-mediated vasodilation (%NMD) (B) in the rosuvastatin and placebo groups at baseline and after treatment for 6 weeks.
have led to a selection bias. These patients were enrolled in 1 center unselectively, and the baseline characteristics in these patients were not different from those of total population. Moreover, in these patients, the reduction in plasma ADMA levels and the positive correlation between reduction of ADMA and LDL cholesterol after rosuvastatin treatment remained significant. Finally, in contrast to our findings, several previous studies have shown that treatment of hypercholesterolemia with various statins, including simvastatin, atorvastatin,26 and pravastatin,27 failed to reduce plasma ADMA levels despite adequate reduction of plasma cholesterol concentrations. This is likely to be related to different characteristics in our population, but may also due to the specific effect of rosuvastatin. Nevertheless, our results need to be corroborated by further studies. Acknowledgment: We are indebted to Shu-Chuan Lin and Pui-Ching Lee for their excellent technical and statistical assistance.
1. Leiper J, Vallance P. Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res 1999;43:542–548. 2. Miyazaki H, Matsuoka H, Cooke JP, Usui M, Ueda S, Okuda S, Imaizumi T.
Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Circulation 1999;99:1141–1146. 3. Bo¨ ger RH, Bode-Bo¨ ger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, Blaschke TF, Cooke JP. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 1998;98:1842–1847. 4. Surdacki A, Nowichi M, Sandmann J, Tsikas D, Bo¨ ger RH, Bode-Bo¨ ger SM, Kruszelnicka-Kwiatkowska O, Kokot F, Dubiel JS, Fro¨ lich JC. Reduced urinary excretion of nitric oxide metabolites and increased plasma levels of asymmetric dimethylarginine in men with essential hypertension. J Cardiovasc Pharmacol 1999;33:652–658. 5. Abbasi F, Asagmi T, Cooke JP, Lamendala C, McLaughlin T, Reaven GM, Stu¨ hlinger MC, Tsao PS. Plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus. Am J Cardiol 2001;88: 1201–1203. 6. Stu¨ hlinger MC, Abbasi F, Chu JW, Lamendola C, McLaughlin TL, Cooke JP, Reaven GM, Tsao PS. Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor. JAMA 2002;287:1420 –1426. 7. Zoccali C, Bode-Bo¨ ger SM, Mallamaci F, Benedetto FA, Tripepi G, Malatina LS, Cataliotti A, Bellanuova I, Ferno I, Fro¨ lich JC, Bo¨ ger RH. Plasma concentrations of asymmetrical dimethylarginine and mortality in patients with endstage renal disease: a prospective study. Lancet 2001;358:2113–2117. 8. Valkonen VP, Pa¨ lva¨ H, Salonen JT, Lakka TA, Lehtima¨ ki T, Laakso J, Laaksonen R. Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine. Lancet 2001;358:2127–2128. 9. Lu TM, Ding YA, Charng MJ, Lin SJ. Asymmetrical dimethylarginine: a novel risk factor for coronary artery disease. Clin Cardiol 2003;26:458 –464. 10. 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;24:1912–1919. 11. Ito A, Tsao PS, Adimoolam S, Kimoto M, Ogawa T, Cooke JP. Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. Circulation 1999;99:3092–3095. 12. Bo¨ ger RH, Syndow K, Borlak J, Tuhm T, Lenzen H, Schubert B, Tsikas D, Bode-Bo¨ ger SM. LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethioninedependent methyltransferases. Circ Res 2000;87:99 –105. 13. Bo¨ ger RH, Bode-Bo¨ ger SM, Tsao PS, Lin PS, Chan JR, Cooke JP. An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes. J Am Coll Cardiol 2000;36:2287–2295. 14. Olsson AG, Pears J, McKellar J, Mizan J, Raza A. Effect of rosuvastatin on low-density lipoprotein cholesterol in patients with hypercholesterolemia. Am J Cardiol 2001;88:504 –508. 15. Jones SP, Gibson MF, Rimmer DM, Gibson TM, Sharp BR, Lefer DJ. Direct vascular and cardioprotective effects of rosuvastatin, a new HMG-CoA reductase inhibitor. J Am Coll Cardiol 2002;40:1172–1178. 16. Laufs U, La Fata V, Plutzky J, Liao J. Upregulation of endothelial nitric oxide synthase by HMG-CoA reductase inhibitors. Circulation 1998;97:1129 –1135. 17. Shishehbor MH, Brennan ML, Aviles RJ, Fu X, Penn MS, Sprecher DL, Hazen SL. Statins promote potent systemic antioxidant effects through specific inflammatory pathways. Circulation 2003;108:426 –431. 18. Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992;339:572–575. 19. Reilly MP, Pratico` D, Delanty N, DiMinno G, Tremoli E, Rader D, Kapoor S, Rokach J, Lawson J, FitzGerald GA. Increased formation of distinct F2 isoprostanes in hypercholesterolemia. Circulation 1998;98:2822–2828. 20. Kanami PM, Sinkey CA, Browning RL, Allaman M, Knapp HR, Haynes WG. Role of oxidant stress in experimental dysfunction produced by experimental hyperhomocyst(e)inemia in humans. Circulation 1999;100:1161–1168. 21. Lin KY, Ito A, Asagami T, Tsao PS, Adimoolam S, Kimoto M, Tsuji H, Reaven GM, Cooke JP. Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase. Circulation 2002;106:987–992. 22. Bo¨ ger RH, Bode-Bo¨ ger SM, Mu¨ gge A, Kienke S, Brandes R, Dwenger A, Frohlich JC. Supplementation of hypercholesterolemic rabbits with L-arginine reduces the vascular release of superoxide anions and restores NO production. Atherosclerosis 1995;117:273–284. 23. Sydow K, Schwedhelm E, Arakawa N, Bode-Bo¨ ger SM, Tsikas D, Hornig B, Fro¨ lich JC, Bo¨ ger RH. ADMA and oxidative stress are responsible for endothelial dysfunction in hyperhomocysteinemia: effects of L-arginine and B vitamins. Cardiovasc Res 2003;57:244 –252. 24. Stu¨ hlinger MC, Oka RK, Graf EE, Schmo¨ lzer I, Upson BM, Kapoor O, Szuba A, Malinow R, Wascher TC, Pachinger O, Cooke JP. Endothelial dysfunction induced by hyperhomocyst(e)inemia: role of asymmetric dimethylarginine. Circulation 2003;108:933–938. 25. Giral P, Bruckert E, Jacob N, Chapman MJ, Foglietti MJ, Turpin G. Homocysteine and lipid-lowering agents: a comparison between atorvastatin and fenofibrate in patients with mixed hyperlipidemia. Atherosclerosis 2001;154:421–427. 26. Pa¨ iva¨ H, Laakso J, Lehtima¨ ki T, Isomustaja¨ rvi M, Ruokonen I, Laaksonen R. Effect of high-dose statin treatment on plasma concentrations of endogenous nitric oxide synthase inhibitors. J Cardiovasc Pharmacol 2003;41:219 –222. 27. Eid HM, Eritsland J, Larsen J, Arnesen H, Seljeflot I. Increased levels of asymmetrical dimethylarginine in populations at risk for atherosclerosis disease. Effects of pravastatin. Atherosclerosis 2003;166:279 –284.
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