Influence of Oral Vitamin E Therapy on Micro-Inflammation and Cardiovascular Disease Markers in Chronic Hemodialysis Patients

Abstract

Background. The aim of this study was to evaluate the influence of oral vitamin E therapy on serum concentrations of several markers of micro-inflammation and cardiovascular disease in chronic hemodialysis (HD) patients. Methods. 29 HD patients were randomized into two groups: 15 patients were treated orally with 400mg of vitamin E daily for a period of five weeks, and 14 patients received no antioxidant supplementation. Before and after vitamin E therapy, serum concentrations of vitamin E (high-performance liquid chromatography), pregnancy-associated plasma protein-A (immunochemical – TRACE assay), C-reactive protein (nephelometry), intercellular adhesion molecule-1 (ELISA), and E-selectin (ELISA) were measured. HD patients were compared with 16 healthy controls. Results. Baseline serum concentrations of PAPP-A and CRP were significantly higher in HD patients than in healthy controls (PAPP-A: 26.23±11.94 vs. 11.41±1.94 mIU/L, p<0.001; CRP: 5.20±3.50 vs. 3.40±3.80 mg/L, p<0.05). After five weeks of oral vitamin E intake, serum PAPP-A, CRP, ICAM-1, and E-selectin concentrations remained unchanged in both groups of HD patients. Conclusion. Chronic micro-inflammation in HD patients is documented by the elevation of CRP and PAPP-A. A daily oral dose of 400 mg of vitamin E does not seem to be able to reduce enhanced oxidative stress and micro-inflammation in chronic HD patients.

Keywords:

• atherosclerosis
• CRP
• E-selectin
• ICAM-1
• hemodialysis
• oxidative stress
• PAPP-A
• vitamin E

INTRODUCTION

Chronic hemodialysis (HD) patients are at a significantly higher risk of atherosclerotic complications when compared to normal population.[1] Patients undergoing maintenance HD are chronically exposed to increased oxidative stress, which is interrelated with micro-inflammation and progressive atherosclerosis.[2]

Closely linked to atherosclerosis, pregnancy-associated plasma protein-A (PAPP-A) is a high molecular weight, zinc-binding metalloproteinase, which has recently been reported as a marker of acute coronary syndromes.[3] The authors' previous studies demonstrated a significant elevation of circulating PAPP-A in HD patients and a close correlation with markers of oxidative stress (advanced oxidation protein products, AOPP) and inflammation (C- reactive protein, CRP).[4],[5]

Elevated circulating levels of soluble cell adhesion molecules have been reported in HD patients,[6],[7] endothelial expression of adhesion cell molecules being one of the key events in the pathogenesis of atherosclerosis.[8] Vitamin E has been shown to inhibit the production of chemokines and inflammatory cytokines and reduce the expression of adhesion molecules in human aortic endothelial cells in vitro.[9]

Although antioxidant therapy seems to be a promising approach to reduce cardiovascular disease in HD patients, it is still unclear in what doses, when, and whether antioxidants should be routinely administered to chronic HD patients at all.

The aim of this study was to evaluate the influence of oral vitamin E therapy on serum concentrations of several markers of micro-inflammation and cardiovascular disease in chronic HD patients.

SUBJECTS AND METHODS

Patients

The present study was performed in a cohort of 29 HD patients and 16 age-matched healthy controls. HD patients were randomized into two groups: group A (15 patients: 4 males and 11 females, mean age 63 ± 6 years) and group B (14 patients: 6 males and 8 females, mean age 60 ± 8 years). In group A, the etiology of renal failure was tubulointerstitial nephritis in ten cases and polycystic kidney disease in five cases, the mean duration of chronic HD treatment in group A being 32 ± 20 months (range 12–84 months). In group B, the etiology of renal failure was tubulointerstitial nephritis in 11 cases and polycystic kidney disease in three cases, the mean duration of chronic HD treatment in group B being 32 ± 19 months (range 12–82 months). All patients included in this study were treated for hypertension; three patients in group A and two patients in group B had a history of previous cardiovascular disease. All patients were in a stable clinical status. Patients with clinical signs of acute inflammation and patients with diabetes, liver diseases, malignancies, and autoimmune diseases were excluded. All patients were hemodialyzed three times weekly, 4 hours per session, using low-flux, single-use polysulphone dialyzers (F6; Fresenius Medical Care, Bad Homburg, Germany) and bicarbonate-based dialysate. The criteria of adequate dialysis (Kt/V>1.2) were fulfilled in all patients. All patients received intravenous erythropoietin therapy (average dose 88 IU/kg of body weight weekly) and iron therapy (62.5 mg of sodium ferric gluconate once a week). All patients were treated with phosphate binders and platelet antiaggregants. Patients in group A and B were comparable by antihypertensive treatment and other medication. (They all received angiotensin-converting enzyme inhibitors and/or beta-blockers; three patients in group A and two patients in group B were treated with nitrates.) The group of healthy controls consisted of 5 males and 11 females (mean age 57 ± 7 years). Four weeks prior to entering the study, none of the subjects received any antioxidants, antibiotics, or immunosuppressive drugs, and they all gave their written informed consent to enter the study. The study was carried out according to the Declaration of Helsinki and was approved by the local institutional ethical committee.

Study Design and Blood Sampling

For a period of five weeks, patients in group A were treated with oral vitamin E (Vitamin E 400, Slovakofarma®; i.e., alpha-tocopherol 400mg = 888 IU of vitamin E daily). Group B patients did not receive any antioxidant supplementation. Blood samples were taken via the arterial-venous fistula prior to HD session before starting vitamin E supplementation and after five weeks of oral vitamin E therapy. Blood was centrifuged at 1450g (4°C) for ten minutes. Serum was stored at –20°C and processed within three months. In each sample, serum concentrations of vitamin E, pregnancy-associated plasma protein-A (PAPP-A), C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), and E-selectin were measured, all measurements being performed randomly in parallel.

Laboratory Methods

Serum vitamin E concentration was measured by high-performance liquid chromatography (HPLC) with a coefficient of variation of 4.2%. PAPP-A was measured immunochemically using TRACE (Time Resolved Amplified Cryptate Emission) technology, based on non-radiating energy transfer. Commercial kit KRYPTOR-PAPP-A (Brahms, Germany, and Cezanne, France) contains two different monoclonal antibodies: one conjugated with europium cryptate and one with fluorescent agent XL 665. The antigens (PAPP-A) present in serum samples are sandwiched between the two conjugates. The fluorescent signal measured during the formation of the antigen-antibody complex by the KRYPTOR analyzer (Cezanne, France) is proportional to the antigen concentration. The coefficient of variation for the PAPP-A measurement was 8.8%. The serum CRP concentration was assessed by nephelometry (routine laboratory method recommended by the International Federation of Clinical Chemistry and Laboratory Medicine [IFCC], with a coefficient of variation of 5%). Serum ICAM-1 and E-selectin concentrations were determined using standard enzyme-linked immunosorbent assay (ELISA) kits (BioSource International, Camarillo, California, USA). The coefficient of variation for both ELISA measurements was 8%.

Statistical Analysis

The results are expressed as mean ± standard deviation (SD). Due to high interindividual variability, CRP is expressed as a median and interquartile range. A random allocation of patients to the two groups was performed using computer software (Statistica 6.1,® Stat Soft Inc., Tulsa, Oklahoma, USA). A nonparametric Wilcoxon matched pairs test and Mann-Whitney test (Statistica 6.1,® Stat Soft Inc., Tulsa, Oklahoma, USA) were used for analysis of the data. The results were considered statistically significant at p <0.05.

RESULTS

There was no significant difference between baseline vitamin E and E-selectin serum concentrations in HD patients and in healthy controls. Serum vitamin E levels were within normal ranges in both groups, showing that HD patients were replete with vitamin E at baseline. Serum ICAM-1 was higher in HD patients than in healthy controls, but this increase did not reach statistical significance (p=0.06). Baseline serum concentrations of PAPP-A and CRP were significantly higher in HD patients than in healthy controls (see Table 1).

Table 1 Laboratory parameters before vitamin E supplementation

Parameter	Hemodialysis patients	Healthy controls
Vitamin E (mg/L)	11.42 ± 3.23	11.41 ± 1.94
ICAM-1 (ng/mL)	434.8 ± 121.6	376.14 ± 79.0
E-selectin (ng/mL)	54.23 ± 32.12	43.64 ± 15.52
PAPP-A (mIU/L)	26.23 ± 11.94	11.41 ± 1.94
CRP (mg/L)	4.35 (1.69 – 5.69) ^†	1.55 (1.00 – 3.26)

^†p <0.05, versus healthy controls.

*p <0.001, versus healthy controls.

After five weeks of oral vitamin E intake, serum vitamin E increased significantly in the group of HD patients treated with vitamin E and remained unchanged in the untreated group. Serum PAPP-A, CRP, ICAM-1, and E-selectin concentrations remained unchanged in both groups of HD patients (see Table 2).

Table 2 Effect of vitamin E supplementation on laboratory parameters in hemodialysis patients

	Day 0		Day 35
Parameter	Group A (supplementation)	Group B(no supplementation)	Group A(supplementation)	Group B(no supplementation)
Vitamin E (mg/L)	11.03 ± 3.31	11.95 ± 3.07	20.71 ± 8.25	11.50 ± 2.46
ICAM-1 (ng/mL)	397.8 ± 94.0	474.4 ± 138.0	396.4 ± 95.9	459.0 ± 121.4
E-selectin (ng/mL)	45.69 ± 26.67	63.40 ± 35.79	43.43 ± 25.38	64.24 ± 33.73
PAPP-A (mIU/L)	26.29 ± 12.80	26.18 ± 11.43	28.15 ± 14.72	26.68 ± 10.29
CRP (mg/L)	4.28 (1.13 – 6.92)	4.89 (1.69 – 7.84)	3.56 (2.51 – 7.04)	6.02 (2.81 – 10.20)

*p < 0.001 versus day 0.

DISCUSSION

In this study, higher baseline serum concentrations of PAPP-A and CRP were observed in HD patients when compared to healthy controls. After five weeks of oral vitamin E supplementation (400mg of vitamin E daily), there were no significant changes in any of the studied parameters of micro-inflammation and cardiovascular disease.

In end-stage renal disease (ESRD) patients, mortality rate is significantly higher than in the normal population, cardiovascular disease (CVD) being the major cause of death in uremia.[1] Apart from traditional CVD risk factors, non-traditional, uremia-specific risk factors come into play in HD patients. HD patients are exposed to enhanced oxidative stress and chronic micro-inflammation that are closely interrelated and contribute to accelerated atherosclerosis.[10] Safe therapeutic interventions aimed at reducing oxidative stress and micro-inflammation appear to be a reasonable approach to improving patient outcome. Vitamin E supplementation seems to be a promising strategy in reducing enhanced oxidative stress in HD patients. Several studies have shown a potential benefit of vitamin E in ESRD patients. Vitamin E has been shown to inhibit the production of chemokines and inflammatory cytokines and reduce the expression of adhesion molecules in human aortic endothelial cells in vitro.[9] Another in vitro study shows the inhibition of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin induced by oxidized low-density lipoproteins (LDL) on human umbilical vein endothelial cells.[8] The beneficial effect of vitamin E-coated dialyzers has also been shown.[11] Yukawa and colleagues[12] found that oral supplementation for two weeks with 600 IU/day vitamin E improved LDL clearance from circulation and reduced malondialdehyde content of LDL in HD patients. A significant benefit from vitamin E supplementation was shown in the SPACE trial,[13] which tested the effect of vitamin E (800 IU/day) on a combined cardiovascular endpoint in 196 HD patients with pre-existing CVD. However, other clinical trials, such as the Heart Protection Study[14] and HOPE Study,[15] showed no benefit from vitamin E supplementation, and Miller's meta-analysis even showed an increase in all-cause mortality in high-dosage vitamin E clinical studies.[16]

In this study, baseline serum concentrations of CRP and PAPP-A were significantly higher in HD patients than in healthy controls. This is in accordance with our previous results[4],[5] and suggests chronic inflammation and increased cardiovascular risk in HD patients. ICAM-1 serum concentration was higher in HD patients than in healthy controls, but this elevation (unlike that in other studies[6],[7]) did not reach statistical significance (p = 0.06). This could be due to the relatively small numbers of subjects included in our study and high interindividual variability of ICAM-1 serum concentrations. The finding of unchanged serum E-selectin levels in HD patients in comparison to healthy controls is in accordance with previous studies[6],[7] and could just be due to the transitory expression of this protein on the surface of activated endothelial cells, quickly returning to basal levels and not excluding the possibility of E-selectin being involved in the process of atherogenesis.[6],[17]

Although the dose and administration route of vitamin E was the same as in the SPACE study, the present authors were unable to show evidence of any significant changes in serum concentrations in any of the studied parameters after five weeks of vitamin E supplementation. This finding is in accordance with the results of our previous study,[18] where short term oral vitamin E supplementation failed to show any effect on serum concentrations of advanced oxidation protein products (AOPP) as a marker of oxidative stress.

The finding of unchanged serum CRP concentration after alpha-tocopherol supplementation is in accordance with the results of Himmelfarb et al., who failed to show any effect on serum CRP after supplementation of 300 mg alpha-tocopherol daily to HD patients for a period of two weeks.[19] In the current study, however, due to high interindividual variability of CRP levels, a relatively small number of enrolled patients, and a need for the less powerful nonparametric statistical testing, subtle but significant differences between pre- and post-treatment CRP levels could have been overlooked. The potential beneficial effect of vitamin E supplementation on serum CRP could have also been masked by the frequent fluctuation of CRP levels in HD patients due to infections (e.g., silent infections in clotted HD access grafts[20]), although none of the patients showed any signs of acute inflammation in the course of the study.

The failure to show a beneficial effect on any of the studied parameters could be due to the relatively short course of vitamin E supplementation, despite the fact that other studies[12] had previously shown a positive effect of vitamin E in short term. Another explanation is that antioxidants in vivo can only operate effectively if all of the components of the antioxidant system are in balanced concentrations. For example, the carotenoid molecule repairs the vitamin E radical and is in turn repaired by ascorbic acid.[21] Thus, it is possible that the potential beneficial effects of vitamin E are masked by the overall dysbalance of the antioxidant system.[22] Moreover, α-tocopherol may act as an initiator of an auto-oxidation chain,[23] and its pro-oxidative effects were shown in co-antioxidant-depleted HD patients.[24] It is also possible that vitamin E supplementation does not affect the parameters examined in this study and that other mechanisms play a more important role in the possible protective effect of vitamin E. The possibility also exists that vitamin E supplementation is not helpful at all, as the SPACE trial is the only one relatively large randomized trial to show a benefit of vitamin E and may be just a chance finding.[15]

In summary, chronic micro-inflammation and increased cardiovascular risk in HD patients is documented by the significant elevation of CRP and PAPP-A as compared to healthy controls. A daily oral dose of 400mg of vitamin E is sufficient to increase serum vitamin E concentration significantly, but it does not seem to be able to reduce the enhanced oxidative stress and micro-inflammation in chronic HD patients, characterized by novel marker PAPP-A.