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Departments of Clinical Pharmacology (S.Z., F.M., M.W.) and Internal Medicine II (S.Z., C.P., E.M., G.-H.S.), Medical University of Vienna, A-1090 Vienna, Austria
Address all correspondence and requests for reprints to: Dr. Sophie Ziegler, Department of Angiology, Allgemeines Krankenhaus Wien, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: sophie.ziegler{at}meduniwien.ac.at.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Subjects and Methods: Forty-nine subjects (15 women, 34 men) with PAD and fasting plasma total Hcy concentrations greater than 15 µmol/liter were randomized to receive either oral vit-B and folic acid therapy (n = 27) or placebo (n = 22) for 6 wk. Fasting venous blood samples were monitored for plasma total Hcy, vit-B12 and folate, ADMA, symmetric dimethylarginine, L-arginine, and high-sensitivity C-reactive protein.
Results: After 6 wk, plasma Hcy concentrations were decreased, and concentrations of vit-B12 and folate were elevated in patients with vitamin supplementation (all P < 0.05 vs. baseline) and unchanged in the placebo group. Dimethylarginine plasma concentrations were not affected by treatment. High-sensitivity C-reactive protein correlated with ADMA plasma concentrations (r = 0.29; P < 0.01).
Conclusion: The lack of vit-B and folic acid therapy on plasma concentrations of ADMA renders a role of extracellular methylarginines unlikely to be involved in the pathophysiology of hyperhomocyst(e)inemia and its complications.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In patients with peripheral artery disease (PAD), elevation of plasma homocyst(e)ine (Hcy) concentrations is a marker for progression of disease and an independent risk factor of mortality (4, 5, 6). In this group of patients, increased ADMA plasma levels have been linked to impaired vascular NO bioactivity and endothelial dysfunction (7). Furthermore, plasma ADMA is elevated in patients with hyperhomocyst(e)inemia (8). In cell culture experiments, increased ADMA concentrations were found after exposure to Hcy (9). Consistently, plasma concentrations of ADMA increased in animals and humans after an oral methionine load, which was paralleled by elevated Hcy and impaired endothelial-dependent vasodilation in humans (8, 10, 11, 12). Thus, it has been hypothesized that ADMA may contribute to hyperhomocyst(e)inemia-related vascular dysfunction.
Hcy plasma concentrations can be effectively lowered by oral vitamin B (vit-B) and folic acid treatment (13, 14, 15, 16). However, there is evidence that the improvement of endothelial dysfunction by vitamin supplementation in coronary artery disease is largely independent of Hcy (17) and mainly attributable to decreased production of oxygen-derived free radicals such as superoxide (18). In monkeys with hyperhomocyst(e)inemia, vitamin therapy could not reduce elevated plasma L-ARG to ADMA ratio and failed to restore endothelial dysfunction (11, 19). Furthermore, hyperhomocyst(e)inemia was not associated with increased levels of ADMA in patients with ischemic heart disease (20). Accordingly, there is a debate about the potential beneficial effects of vit-B and folic acid supplementation on impaired vascular function and its relationship with ADMA. The purpose of this study was to test whether a combined oral vit-B and folic acid supplementation reduces ADMA plasma concentrations in patients with hyperhomocyst(e)inemia and PAD in a double-blind, randomized, placebo-controlled study.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The study protocol was approved by the Ethics Committee of the University of Vienna and complied with the Declaration of Helsinki including current revisions and the Good Clinical Practice Guidelines of the European Union. Written informed consent was obtained from all patients before enrollment into the study.
Forty-nine subjects (15 women, 34 men) with PAD (mean age 69 yr; SD 7) and stable intermittent claudication and hyperhomocyst(e)inemia (fasting plasma total Hcy concentration > 15 µmol/liter) were recruited from the Division of Angiology, Department of Internal Medicine II, University Hospital of Vienna during a period of 6 months. Following a double-blind, parallel-group study design, patients were randomized to receive either a mixture of vit-B (50 mg vit-B1, 50 mg vit-B6, 0.05 mg vit-B12) and folic acid (5 mg) (Beneuran compositum tablets; Nycomed Austria GmbH, Linz, Austria) (n = 27) as an oral dose once daily or matching placebo (n = 22) over a 6-wk period. Compliance of patients was evaluated by pill count.
Fasting venous blood samples were taken at baseline and after 6 wk of treatment for the measurement of plasma total Hcy, vit-B12, folate, L-ARG, ADMA, symmetric dimethylarginine (SDMA), C-reactive protein, creatinine, and lipid profile.
Concomitant medication comprised antithrombotic therapy and oral anticoagulation, antihypertensive therapy, and lipid-lowering therapy (Table 1
) and remained unchanged during the observation period. None of the participants was taking additional vitamin supplements during the study.
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Methods
Plasma Hcy concentrations were measured by a commercially available fluorescence polarization immunoassay (IMx analyzer; Abbott Laboratories, Abbott Park, IL). Plasma concentrations of vit-B12 and folate were quantified with a radioassay (Simul TRAC-SNB; ICN Pharmaceuticals Inc., Costa Mesa, CA). Plasma high-sensitivity C-reactive protein (hs-CRP) was measured using a high-sensitivity assay (N Latex CRP Mono; DADE Behring, Deerfield, IL) with a lower detection level of 0.03 mg/dl and a coefficient of variation of 4.6%. Plasma creatinine, total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride concentrations were determined spectrophometrically using standard laboratory methods. Low-density lipoprotein (LDL) cholesterol concentrations were either calculated or measured, depending on triglyceride values. If triglycerides were less than 400 mg/dl, LDL cholesterol was calculated using the Friedwald equation. In case of higher triglyceride values, LDL cholesterol was quantified using a commercially available, enzymatic color test kit (Olympus, Vienna, Austria) (21).
Determination of L-ARG, ADMA, and SDMA
For measurement of L-ARG, ADMA, and SDMA, plasma was subjected to cation exchange solid-phase extraction and analyzed by HPLC (22). The coefficients of variation for inter- and intrasample variations tested with a pooled plasma sample were less than 3% for all analytes. The detection limit for dimethylarginines was 0.04 µmol/liter (23).
Statistics
Patients were eligible for analysis of efficacy if compliance was 80% or more. Outcome parameters were tested for normal distribution and log transformed if not normally distributed. Between- and within-group differences were analyzed by Student’s unpaired and paired t test, respectively. The effect of vitamin supplementation on primary outcome parameters was assessed by a repeated-measure ANOVA model. Correlations between outcome parameters were calculated for pooled data sets and subgroups using Pearson's correlation and a multiple regression analysis was applied. P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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). There was no discontinuation or withdrawal of patients, and all subjects under study were eligible for analysis. Study drug treatment was tolerated without adverse drug reactions, and mean compliance to therapy was 92%. Plasma concentrations of total Hcy, vit-B12, folate, L-ARG, ADMA, SDMA, hs-CRP, and creatinine and lipid profile (with the exception of HDL cholesterol) were also comparable between groups (Table 2). Creatinine values were mildly to moderately elevated above the normal range (1.1 mg/dl) in seven subjects (four of the verum group and three of the placebo group). Treatment with vit-B and folic acid significantly decreased plasma Hcy concentrations [6.95 µmol/liter (confidence interval [CI] 4.43; 9.46)] and increased concentrations of vit-B12 [66.59 pmol/liter (CI 37.64; 95.54)] and folate [108.89 nmol/liter (CI 71.31; 146.47)], compared with baseline (Table 2). Vitamin supplementation had no effect on L-ARG, ADMA, or SDMA plasma concentrations. In subjects randomized to placebo, no change of outcome parameters over time was detectable. hs-CRP tended to decrease over time in both groups under study. According to a two-way ANOVA, vitamin supplementation resulted in significantly lower concentrations of plasma Hcy (P < 0.01, ANOVA) and higher plasma folate (P < 0.03, ANOVA) vs. placebo. No effect was detectable regarding L-ARG, ADMA, SDMA, hs-CRP, or vit-B12 plasma concentrations between groups in response to treatment.
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Discussion |
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Hyperhomocyst(e)inemia has been associated with increased risk for cardiovascular mortality, although the exact pathomechanisms remain unclear (24, 25, 26). Hcy-induced generation of oxidant stress was proposed to impair vascular NO bioactivity, but a clear pathophysiological association of plasma Hcy and endothelial dysfunction is discussed controversially (14, 19, 27, 28, 29). In animal experiments and human trials, oral methionine load caused hyperhomocyst(e)inemia and endothelial dysfunction, which correlated with increased plasma concentrations of ADMA (11, 12).
Based on these findings, it was suggested that the detrimental effect of Hcy on endothelial function could be mediated by ADMA, whereby two different mechanisms were postulated. First, Hcy can inhibit dimethylarginine dimethylaminohydrolase activity (1), the enzyme involved in the degradation of ADMA. Second, elevated ADMA could result from increased availability of methionine (12), a substrate of methyltransferases, to form the NO inhibitor. However, oral methionine had no effect on ADMA plasma concentrations in different human experiments (30). Our present data show that lowering of elevated plasma Hcy is not accompanied by a reduction of plasma ADMA concentrations. This is consistent with animal experiments, in which vitamin supplementation also failed to affect plasma ADMA or restore endothelial dysfunction in monkeys with hyperhomocyst(e)inemia (11). Furthermore, our results corroborate findings from Jonasson et al. (20), who found out that substantial reduction of plasma total Hcy did not affect the level of plasma ADMA in an open-label study.
The present findings of decreased plasma Hcy concentrations by vitamin therapy are also in good agreement with other data (15, 16). Nevertheless, our results are in contrast to findings from Holven et al. (15), who reported decreased plasma ADMA after a 6-wk folic acid supplementation. However, none of the patients with hyperhomocyst(e)inemia included in the other study had PAD, and a different laboratory method was used. ADMA values showed a much greater variation between subjects than in our cohort. Further ADMA concentrations ranged above the values reported by most laboratories (7, 8, 20, 31). In addition, Holven et al. (15) performed an uncontrolled open study, whereas our investigation was done according to a double-blind, parallel-group design. It is rather unlikely that the discrepancy of findings is due to different compliance because an equipotent drug effect on Hcy concentrations was achieved. Thus, vitamin supplementation in the present investigation was appropriate to study clinically relevant changes in ADMA metabolism over time. It is, however, unclear whether the described lack of an effect of vitamin supplementation on ADMA is specific for PAD patients.
The association among renal function, ADMA, and Hcy is complex. In contrast to healthy subjects, patients with chronic renal disease accumulate ADMA and display 2- to 6-fold increased plasma concentrations, compared with controls (32). Because patients with mildly or moderately elevated creatinine values were equally distributed between groups in this study, a potentially confounding role of impaired renal function is unlikely.
Baseline HDL cholesterol was different between treatment groups. Previous studies demonstrated that ADMA concentrations are not associated with cholesterol levels (33). Whereas there is a debate whether statins may affect ADMA concentrations (34, 35), this potential confounding effect was excluded by avoiding changes in concomitant drug therapy during the study period.
In this population a positive correlation between the inflammatory marker hs-CRP and ADMA values was noted. It is tempting to speculate that ADMA may also be associated with cardiovascular events as described for hs-CRP in this group of patients with advanced atherosclerosis (36). Interestingly, hs-CRP slightly decreased over time, which was not seen for ADMA. Given the short plasma half-life of exogenously administered ADMA of about 24 min (37), it may be assumed that increased formation of dimethylarginines continues also in the absence of prolonged subclinical inflammatory conditions. This observation and its potential impact on the clinical course remain to be confirmed.
In conclusion, combined vit-B and folic acid therapy for 6 wk lowers plasma Hcy in patients with PAD independently of circulating ADMA concentrations. Therefore, extracellular methylarginines are unlikely involved in the pathophysiology of hyperhomocyst(e)inemia and its complications in PAD patients.
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Footnotes |
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Abbreviations: ADMA, Asymmetrical dimethylarginine; CI, confidence interval; Hcy, homocyst(e)ine; HDL, high-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; L-ARG, L-arginine; LDL, low-density lipoprotein; NO, nitric oxide; PAD, peripheral artery disease; SDMA, symmetric dimethylarginine; vit-B, vitamin B.
Received June 8, 2004.
Accepted December 22, 2004.
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