Impact of vitamin E on plasma asymmetric dimethylarginine (ADMA) in chronic kidney disease (CKD): a pilot study

Rajiv Saran1, James E. Novak1, Anjali Desai2, Emil Abdulhayoglu1, Jeffrey S. Warren2, Rami Bustami3, Garry J. Handelman4, Damian Barbato4, William Weitzel1, Louis G. D'Alecy5 and Sanjay Rajagopalan6

1Division of Nephrology, 2Department of Pathology, 5Department of Physiology and 6Department of Cardiovascular Medicine, University of Michigan, 3University Renal Research and Education Institute, Ann Arbor, MI and 4University of Massachusetts at Lowell, Boston, MA, USA

Correspondence and offprint requests to: Rajiv Saran, MD MS, Assistant Professor, Division of Nephrology, University of Michigan, Kidney Epidemiology and Cost Center, 315 West Huron, Suite 240, Ann Arbor, MI 48103-4262, USA. Email: rsaran{at}umich.edu



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase and a proposed cardiovascular risk factor, is elevated in chronic kidney disease (CKD). Pharmacological strategies that lower plasma concentration of ADMA may be expected to increase nitric oxide (NO·) bioavailability and potentially limit atherosclerosis. We hypothesized that the antioxidant {alpha}-tocopherol (vitamin E) reduces ADMA levels in CKD.

Methods. An open-label pilot interventional study using 800 IU of vitamin E was undertaken in eight stable out-patients with non-diabetic CKD (creatinine clearance <30 ml/min/1.73 m2) and six healthy controls, with the objective of measuring plasma ADMA levels at baseline and after 8 weeks of treatment. Plasma ADMA, symmetric dimethylarginine (SDMA) and {alpha}-tocopherol concentrations were determined at study entry and exit using high-performance liquid chromatography, while plasma total F2-isoprostanes, an index of oxidative stress, were measured using a commercially available enzyme-linked immunosorbent assay kit.

Results. ADMA and SDMA concentrations were significantly higher in the plasma of patients compared with that of controls (P <= 0.001). After treatment with vitamin E, ADMA decreased by 23% in six of eight patients (P <0.001). The remaining two patients showed either an increase or no change (overall, P = 0.16). There was no significant change in plasma F2-isoprostanes with vitamin E treatment for 8 weeks.

Conclusions. Antioxidant therapy with vitamin E has the potential to lower ADMA levels in CKD patients, implying increased NO· availability. This strategy merits further exploration in the setting of CKD prior to renal replacement.

Keywords: asymmetric dimethylarginine (ADMA); chronic kidney disease; isoprostane; nitric oxide; {alpha}-tocopherol; vitamin E



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Mortality rates continue to be unacceptably high in end-stage renal disease (ESRD). Atherosclerotic cardiovascular disease is the single greatest threat to survival in ESRD patients and accounts for half of all deaths [1]. The risk for cardiovascular events increases markedly with the onset of ESRD and, therefore, strategies aimed at limiting atherosclerosis and its complications are more likely to be successful if they are initiated prior to ESRD.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), has been shown to be an independent predictor of cardiovascular and overall mortality in ESRD [2]. Plasma ADMA levels are increased in both early and late renal failure [3,4]. This increase is thought to result from both decreased renal clearance and metabolism by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) [3,5]. Oxidant stress may increase the synthesis of ADMA by stimulating S-adenosylmethionine-dependent methyltransferases [6] and/or by decreasing the metabolism of ADMA by reducing the activity of DDAH [7]. Antioxidants have been shown to decrease ADMA levels in cultured human endothelial cells [6] and in vivo in rats exposed to native low-density lipoprotein (LDL) [8]. Chronic kidney disease (CKD) is associated with activation of multiple redox pathways as evidenced by elevated levels of advanced glycation end-products (AGEs), C-reactive protein (CRP) and F2-isoprostanes (F2-ISPs), presumably due to a constellation of abnormalities including elevation in LDL cholesterol, triglyceride-rich lipoproteins, homocysteine and AGEs, all of which have been associated with enhanced free radical production.

{alpha}-Tocopherol (hereafter referred to as vitamin E) is a highly effective fat-soluble antioxidant and has been shown to reduce lipid peroxidation and improve NO· availability in the vasculature by inhibiting free radical generation. Despite the negative results with this antioxidant from large-scale clinical trials in patients with atherosclerosis, smaller studies indicate a possible benefit of vitamin E under conditions of enhanced oxidative stress, as is likely in patients with CKD [9,10]. It was postulated that administration of vitamin E in a CKD population would reduce both oxidative stress and circulating levels of ADMA.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
The subjects were recruited from the out-patient nephrology clinic of the University of Michigan Medical Center (patients) or from the staff at the University of Michigan (controls). Informed consent was obtained from each subject, and the local Institutional Review Board approved the study. Inclusion criteria were subjects older than 18 years of age with non-diabetic CKD and an estimated creatinine clearance of < 30 ml/min/1.73 m2. Creatinine clearance was estimated using the Cockcroft–Gault formula. Exclusion criteria were chronic liver disease, active inflammatory disease, current smoking, recent (<2 months) hospitalization, over-the-counter or prescribed antioxidant ingestion, warfarin therapy and pregnancy. Medications were recorded for all subjects. Fasting blood and urine samples were collected from all subjects. The blood samples (15 ml) were centrifuged within 15 min of collection and 1 ml aliquots of plasma were frozen at -80°C. Plasma samples to be used for F2-ISP determinations were snap-frozen in liquid nitrogen after the addition of butylated hydroxytoluene (BHT, 0.005%) and indomethacin (10 µM). All subjects were given an 8-week supply of 800 IU/day of natural vitamin E (Mason Vitamins, FL). Four weeks into the study, subjects received a phone call from a study coordinator reminding them to take their medication regularly and inquiring about any side effects. Subjects presented during the final week of the study to submit a second set of fasting blood and urine samples, which were collected and processed identically to those obtained at the beginning of the study.

Quantification of dimethylated arginine derivatives
ADMA and symmetric dimethylarginine (SDMA) in human plasma samples were quantified by reverse-phase liquid chromatography (Waters, Milford, MA). A two-pump gradient system (Waters HPLC Model 510 Solvent Delivery System) delivered 85–82% (at 27 min) to 0% of 10 mM sodium acetate trihydrate (pH 4.76) with balance methanol at 1 ml/min for 43 min. Following pre-column fluorescent derivatization (Waters AccQ·Fluor), analyate separation was performed on a 4.6 mm x 250 mm, 3.5 µm column (Waters Xterra MS C18) with an identical 3.9 mm x 20 mm guard column, both controlled at 36°C (Eppendorf Scientific Model CH-500). Standards, blanks and samples (10 µl) were injected automatically (Waters Model 712 Intelligent Sample Processor) and fluorescent peak height and area evaluated at an excitation of 250 nm and an emission of 395 nm (Waters Model 474 Scanning Fluorescence Detector) to 0.1 µM original concentrations. The average method detection limit (MDL) of three sets of 10 replicates in plasma or standards was SDMA = 0.07, ADMA = 0.13 (µM). The average intra-assay coefficient of variations (CVs) (two assays of n = 4) for ADMA and SDMA (1.5 µM standard) were 2.4 and 3.3%, respectively. Intra-assay CVs (n = 8) for ADMA and SDMA in unspiked pooled plasma (mean of 1.1 µM) were 2.2 and 3.2%, respectively. The inter-assay CV (n = 4) for 1.5 µM was 2.6% for ADMA and 0.3% for SDMA.

Quantification of F2-isoprostanes
F2-ISPs, specifically 8-iso-prostaglandin F2{alpha}, was measured in human plasma samples using an 8-isoprostane kit purchased from Cayman Chemical Company (Ann Arbor, MI). This method has been used for quantitation of plasma F2-ISPs in a number of animal and human clinical studies [11]. Samples were purified by solid phase extraction following the manufacturer’s instructions and were analysed in triplicate. The inter- and intra-assay variation in our laboratory was <7%.

Quantification of {alpha}-tocopherol levels
Serum {alpha}-tocopherol (vitamin E) was assayed using high-performance liquid chromatography (HPLC) on a C-18 column with a flourescence detector following the method of Handelman et al. [12]. This method has a precision of ±2%.

Biochemical measurements
Routine complete blood counts, chemistries, CRP, lipid profile and homocysteine levels were determined by the hospital laboratory, as was the urine albumin:creatinine ratio.

Statistical analysis
All analyses were done using SAS version 8.2 (SAS Institute Inc., Cary, NC). A generalized linear model (GLM) with repeated measures was used to test both the baseline differences between patients and controls in vitamin E, ADMA, SDMA and F2-ISP levels, as well as the change in these levels en each group after vitamin E treatment. The GLM allowed for adjustments for factors (such as LDL) that might have an impact on the biomarker levels, whereas other statistical methods such as a t-test would have been a simple comparison of means without the ability to account for confounders. Values were expressed as mean ± SD. A P-value of <0.05 was considered statistically significant.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Nine CKD patients and seven healthy controls were recruited initially. Eight patients and six controls completed 8 weeks of vitamin E therapy. One patient with adult polycystic kidney disease elected to discontinue the medication after 4 weeks due to the development of flank pain of unclear origin, although he had experienced similar flank pain in the past. One control subject was lost to follow-up. Hence, complete data are available for eight patients (three females) and six controls (one female).

Patient demographics are shown in Table 1. The mean age of controls and patients was 46 ± 10 and 55 ± 18 years, respectively. Plasma creatinine for controls was <= 1.0 mg/dl, while that for patients was 4.0 ± 2.3 mg/dl. The mean estimated creatinine clearance for patients was 19.5 ± 7.4 ml/min/1.73 m2. Table 1 also lists the medications being received by the patients.


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Table 1. Subject demographics

 
Vitamin E, ADMA, SDMA, F2-ISP, LDL, albumin, CRP and homocysteine levels, obtained at the beginning and at the end of vitamin E supplementation, are displayed in Table 2. After supplementation with vitamin E for 2 months, the mean vitamin E levels increased from 21.06 to 40.75 µM in controls (P = 0.04) and from 24.86 ± 10.53 to 33.87 ± 9.21 µM in the patients (P = 0.006). The baseline levels among controls and patients were not statistically different (P = 0.47). When adjusted for mean LDL levels, there was no significant difference in the concentration of vitamin E in controls and patients (P = 0.37). At baseline, the concentration of ADMA in controls and patients was 0.55 ± 0.20 and 1.85 ± 0.60 µM, respectively (P < 0.001). In control subjects, ADMA did not change with vitamin E supplementation, but in six of eight CKD patients, ADMA levels decreased significantly (Figure 1; 2.02 ± 0.54 to 1.56 ± 0.44 µM, P < 0.001). Since the remaining two patients showed either no change or an increase in ADMA concentration, the trend for the entire CKD group was not significant (from 1.85 ± 0.60 to 1.60 ± 0.57 µM, P = 0.16). SDMA concentrations at baseline were 0.40 ± 0.07 and 1.18 ± 0.38 µM in controls and patients, respectively (P < 0.001). These values did not change significantly in either group following vitamin E supplementation, even in the six patients where ADMA levels had shown a significant decline post-treatment (P = 0.30). Plasma F2-ISP concentrations were not significantly different between patients and controls either before or after vitamin E treatment, and vitamin E supplementation did not affect F2-ISP levels in either group (Table 2). There were no significant changes in CRP, homocysteine, serum albumin, LDL (Table 2) or urinary albumin:creatinine ratio, complete blood count and other routine chemistries (data not shown) after treatment with vitamin E.


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Table 2. Plasma vitamin E, ADMA, SDMA, F2-ISP, LDL, ALB, CRP and HCY concentrations at baseline and after 8 weeks of vitamin E treatment

 


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Fig. 1. Change in plasma ADMA concentration before and after 8 weeks of vitamin E treatment in controls (open circles, dotted lines) and patients (closed squares, solid lines for six of eight with a decrease; open squares, broken lines for the remaining two).

 


   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The main findings of this pilot study are that administration of vitamin E results in reductions in ADMA levels without alterations in other circulating markers of oxidant stress in patients with non-diabetic CKD. To the best of our knowledge, this is the first report to suggest an effect of an antioxidant on ADMA levels in this patient population.

Our baseline data are consistent with previously reported ADMA concentrations in non-diabetic renal failure [3,13]. We observed a 23% decrease in ADMA after 8 weeks of vitamin E supplementation in six of the eight patients. In one patient, a 53-year-old African-American woman with adult polycystic kidney disease, there was no change in ADMA concentration and, in another, a 75-year-old man with diffuse atherosclerosis and renovascular disease, ADMA levels actually increased during the 8-week study. Hence, the downward trend in ADMA in CKD patients failed to achieve statistical significance (P = 0.16). The small sample size in this pilot study may have contributed to a type II error. Alternatively, the patient in whom levels were higher after the course of treatment may represent an example of a heterogeneous response to be expected in a larger study. Non-compliance with the medication certainly did not play a role in this elderly gentleman who lived alone and expired 3 months after the initiation of haemodialysis. A clear explanation of why he did not respond is not immediately obvious. The authors speculate that he was declining in vascular and renal health so rapidly that antioxidant therapy was without effect on decreasing NO availability. Mechanisms or predictors of non-response need to be investigated further in a larger study. The P-value becomes highly significant (<0.005) if this patient alone were to be excluded from analysis.

A number of well-designed clinical trials have shown no benefit of as much as 800 IU of vitamin E on cardiovascular end points [14,15]. One potential problem with these trials, however, is that the inclusion of subjects without biochemical evidence of elevated oxidant stress could dilute the population that may benefit from antioxidant therapy, even assuming the functional importance of oxidant stress in the disease under evaluation. It is well known that CKD is a state of oxidant stress, and antioxidant strategies have been explored in the setting of haemodialysis [16]. While there is no consensus among nephrologists regarding antioxidant treatment in CKD, several clinical trials suggest that such therapy confers cardiovascular benefit. The SPACE trial, a randomized, placebo-controlled, multi-centre study from Israel, demonstrated a significant reduction in composite cardiovascular disease end points and myocardial infarction in haemodialysis patients with 800 IU/day of vitamin E during a median follow-up of 519 days [9]. Mune et al. used vitamin E-coated cellulose membrane dialysers in haemodialysis patients for 2 years and found a significant reduction in oxidized LDL and aortic calcification (a surrogate for atherosclerosis progression) [10]. Islam et al. demonstrated a decrease in the oxidative susceptibility of LDL in haemodialysis and peritoneal dialysis patients with 800 IU/day of vitamin E for 12 weeks [17].

The benefit of antioxidant therapy in renal failure may result from the interruption of several pathophysiological mechanisms. Vitamin E protects glomerular basement membrane integrity, prevents neutrophil chemotaxis and inhibits platelet aggregation [18]. It also inhibits 5-lipoxygenase in peripheral blood monocytes of haemodialysis patients, resulting in decreased lipid peroxidation and leukotriene B4 content [19]. Another form of vitamin E, {gamma}-tocopherol, is essential for detoxifying peroxynitrite radicals [18]. Our results would seem to suggest a role for vitamin E in reducing levels of ADMA that may be an important surrogate marker of risk in this patient population. Interestingly, in our patient population, baseline levels of F2-ISPs were not elevated beyond control subjects. This may have reflected the effect of concomitant therapies such as angiotensin-converting enzyme inhibitors/angiotensin receptor blockers and statin use, both of which effectively reduce oxidative stress indices including F2-ISPs. It is possible that we did not observe a reduction in F2-ISPs since there was no baseline elevation over controls. The differential effect of vitamin E on ADMA levels alone but not on F2-ISP levels may potentially imply a direct effect of vitamin E in modifying the activity of DDAH, an enzyme thought to be pivotally important in the regulation of ADMA levels, and may imply addititive effects of vitamin E above and beyond therapies that modulate oxidant stress. Alternatively, it is possible that vitamin E may function via modulation of upstream effectors such as oxLDL and tumour necrosis factor-{alpha} levels which are well known to regulate DDAH activity. The fact that we did not see decreases in F2-ISPs is similar to a study by Meagher et al. [19] where urinary isoprostanes were unchanged in a group of healthy adults treated with different dose regimens of vitamin E despite a 5-fold increase in serum vitamin E levels. In contrast, hypercholesterolaemic patients treated with 3200 IU of vitamin E/day and other antioxidant vitamins showed a 54% decline in urinary F2-ISP metabolite concentration [20]. In contrast to ADMA, SDMA, though elevated in CKD patients as has been previously reported [3,13], was not lowered in response to vitamin E. Similarly, we did not observe any changes in homocysteine or CRP concentrations, suggesting that an antioxidant approach alone may be insufficient to counter increases in these entities, or that a longer duration of therapy may be warranted to see the effect. Decreases in ADMA could potentially translate into improvements in NO availability with resultant ‘atheroprotective’ effects. It is unknown whether a reduction in ADMA concentration would translate into long-term clinical benefit, via reduction in major cardiovascular events.

In summary, this pilot study confirms that ADMA levels are elevated in renal insufficiency and that these levels may be reduced with high-dose vitamin E supplementation. Based on these preliminary results, we are encouraged to explore further the relationship between ADMA and renal failure, and also to consider ADMA measurement as an intermediate end point to study vasoprotective pharmacotherapy in CKD prior to the initiation of renal replacement. A larger clinical trial with vitamin E in CKD is currently in progress.



   Acknowledgments
 
We are grateful to the patients and staff who volunteered for the study. This study was funded by the Renal Research Institute, New York, NY.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 12. 6.02
Accepted in revised form: 13. 6.03