Comparative Effects of Silymarin and Vitamin E Supplementation on Oxidative Stress Markers, and Hemoglobin Levels Among Patients on Hemodialysis

Abstract

Background: The incidence of accelerated atherosclerosis among patients on hemodialysis is very high and oxidative stress (OS) is a potentially major contributor to their morbidity and mortality. Objective: To evaluate the effects of Silymarin and/or vitamin E on OS markers and hemoglobin levels in patients on hemodialysis. Methods: Eighty patients on hemodialysis were randomized into four groups: Group 1 received silymarin 140 mg 3 times daily; Group 2 received vitamin E 400 IU/day; Group 3 received silymarin 140 mg 3 times daily and vitamin E 400 IU/day; and Group 4 was the control. Samples were obtained at baseline and on day 21 for measurement of malondialdehyde (MDA), red blood cell (RBC) glutathione peroxidase (GPX), and hemoglobin. Results: Combination of silymarin and vitamin E led to a reduction in the MDA levels (7.84 ± 1.84 vs. 9.20 ± 2.74 nmol/mL; p = 0.008). There was a significant increase in RBC GPX levels in all treatment groups compared with controls after 3 weeks. This was more pronounced in the group receiving combination compared with the group receiving vitamin E or the control group (5.78 ± 3.51, 4.22 ± 1.63, and 3.16 ± 1.89 IU/grHb, respectively; p < 0.001). There was also a significant increase in mean hemoglobin of all treatment groups compared with the control. Conclusions: Oral supplementation with silymarin and vitamin E leads to reduction in MDA, increase in RBC GPX, and increase in hemoglobin levels in patients with end-stage renal disease. Studies with larger sample sizes and longer follow-up are required to investigate the effect of silymarin on cardiovascular outcomes and erythropoietin requirement.

• oxidative stress
• hemodialysis
• anemia
• vitamin E
• silymarin

INTRODUCTION

Atherosclerotic cardiovascular disease is the most common cause of morbidity and mortality in patients on maintenance dialysis. A proportion of these patients do not have traditional risk factors of atherosclerosis such as hypertension (HTN), hyperlipidemia, diabetes (DM), and smoking.^1–3 Recent clinical and experimental evidence suggests that oxidative stress (OS), chronic inflammation, malnutrition, and endothelial dysfunction are increased in end-stage renal disease (ESRD). OS may be implicated in the pathogenesis of atherosclerosis, malnutrition, anemia, and dialysis-induced amyloidosis.^4–8

There is ample evidence for increased OS in renal failure: chronic inflammation associated with uremia; presence of trace amounts of endotoxins in the dialysate; activation of oxidative metabolism in leukocytes by dialysis membrane; and depletion of antioxidants such as vitamin E, flavonoids, superoxide dismutase, glutathione, and glutathione peroxidase (GPX). Other evidences for increased OS in renal failure patients are increased levels of malondialdehyde (MDA) in serum, red blood cells (RBCs), platelets, and peripheral blood mononuclear cells; increased formation of advanced oxidation proteins and advanced glycosylation end products; and increased oxidation of lipoproteins. As a result, supplementation with antioxidants has been proposed as a reasonable strategy to prevent the deleterious effects of OS in this population. Vitamin E, an effective lipid-soluble antioxidant in biologic membranes, has been widely used in the defense against OS in patients with ESRD.^9–11

Silymarin, a polyphenolic flavonolignans from Silybum marianum plant, consists of four flavonolignans isomers – silybin, isosilybin, silydianin, and silychristin. It is orally absorbed and excreted mainly through bile as sulfates and conjugates. Although most of the evidence indicates a renal-protective impact of silymarin in animal models of acute kidney injury, the effect is inconclusive.^12–16 Silymarin has also been demonstrated to lead to enhanced recuperation of renal tissue damage among alloxan-induced diabetic rats.¹⁷

Silymarin has been used in alcoholic liver diseases, liver cirrhosis, Amanita mushroom poisoning, viral hepatitis, and toxic and drug-induced liver diseases and among diabetic patients. It has been claimed to promote protein synthesis, help regenerate liver tissue, control inflammation, enhance glucuronidation, and protect against glutathione depletion.^18,19 Clinical and experimental studies have shown silymarin to act by antioxidative, anti-lipid peroxidative, antifibrotic, anti-inflammatory, membrane stabilizing, and immunomodulatory mechanisms. In the kidney cells damaged in vitro by paracetamol, cisplatin, and vincristin, silybin can reduce or avoid nephrotoxic effects.^18–20

Silymarin has also been used in the treatment of patients suffering from end-stage diabetic nephropathy (ESDN). The molecular basis of the anti-inflammatory effect of silybin/silymarin is yet unknown. This might be related to inhibition of the nuclear factor NF-κB, which regulates and coordinates the expression of various genes involved in the inflammatory process and in cytoprotection. NF-κB seems to be subject to redox regulation, thus suggesting an important role of antioxidants in its inactivation. The treatment with silybin or silymarin led to a restoration of the thiol status both in vitro and in vivo. These data provide the rationale for using flavonolignans in ESDN to normalize immunoregulatory defects through restoration of the cellular thiol status.^20,21 Considering the importance of this subject and lack of experience with silymarin among hemodialysis patients, we investigated the effect of this flavonoid alone, and in combination with vitamin E, on biomarkers of OS in this patient population.

SUBJECTS AND METHODS

This study is in conformity with the Declaration of Helsinki and was approved by the institutional ethics committee of Shiraz University of Medical Sciences. All the patients provided informed consent form. Subjects were recruited from among clinically stable hemodialysis patients (age range = 18–60 years) in Ebrahimi Hemodialysis Center in Sadra City (Shiraz, Iran). All patients received 4-hour hemodialysis treatments, 3 times per week. The membrane and the general dialysis prescription were similar for all patients. Exclusion criteria consisted of history of a cardiovascular event within the previous 12 months and intake of vitamin E, levothyroxin, and oral contraceptives. Patients with any active infection including hepatitis B or C and New York Heart Association class III and IV heart failure were also excluded. All patients were instructed to maintain a renal prudent diet and were asked not to change their diet or level of physical activity throughout the course of this study.

Eighty consenting patients were randomized into four equal groups: Group 1 received silymarin 140 mg 3 times daily, Group 2 received vitamin E 400 IU/day, Group 3 received both Silymarin 140 mg 3 times daily and vitamin E 400 IU/day, and Group 4 was the control group and received no study-related intervention. The duration of this study was 3 weeks. Each 140 mg coated tablet of silymarin (Livergol^®; Goldaru Herbal Products Pharmaceutical Company, Isfahan, Iran) contains the dried extracts of Silybum marianum equivalent to 140 mg silymarin. Vitamin E (Zahravi Pharmaceutical Company, Tabriz, Iran) was administered as 400 mg pearls. Fasting blood samples (10 mL) were collected in EDTA-containing tubes from the arterial line immediately before a midweek dialysis session, before heparin administration, immediately before supplementation (baseline), and on day 21 after the intervention period. Samples were immediately centrifuged and frozen at -70°C.

Analytical Procedures

MDA, an indirect index of lipid peroxidation, was assayed as thiobarbituric acid reactive substances (TBARS) using a colorimetric method.²² Briefly, 0.5 mL of diluted plasma (1:1, v/v) was mixed with 2 mL TBA reagent containing trichloroacetic acid [15% (w/v)], thiobarbituric acid [0.375% (w/v)], and hydrochloric acid (0.25 N) and the mixture placed in a boiling water bath for 15 min. The samples were cooled and centrifuged at 3000 g for 15 min at 4°C. The absorbance of the supernatant was measured at 532 nm. The TBARS concentration was calculated using 1,1,3,3,-tetraethoxy propane as a standard. Results are expressed as nmol/mL.

The GPX activity was measured in RBC hemolysate according to the method of Paglia and Valentine²³ by decrease in absorption at 340 nm due to oxidation of NADPH to the NADP⁺, when oxidized glutathione was reduced by glutathione reductase (GR). Oxidized glutathione had been formed earlier by reaction of its reduced form with t-butyl hydroperoxide (t-BuOOH) and GPX. To obtain erythrocyte hemolysate, 100 µL of packed erythrocytes was hemolyzed by adding 9 volumes of cold distilled water. The resulting suspension was centrifuged twice to eliminate all of the cell membranes. Each assay consisted of 0.25 mM GSH, 0.38 mM NaN₃, 0.23 mM EDTA, 0.175 mM NADPH, 0.1 units of GR, 0.05 mM t-BuOOH in 37.6 mM phosphate buffer (pH 7.2), and an appropriate amount of hemolysate in a final volume of 600 µL. The units of enzymatic activity were calculated using an extinction coefficient of 6.22/mM/cm for NADPH. One unit was equivalent to the oxidation of 1 µmol of NADPH per minute. GPX activity in RBC was expressed as IU/grHb. The hemoglobin concentration was determined using an autoanalyser SE-9000 (Sysmex KX-21N Instruments, Mundelein, IL, USA).

Statistical Analysis

All data were analyzed using SPSS statistical software (version 15.1) and degree of significance was set at 0.05. The analysis of variance (ANOVA) and chi-square test were used for statistical comparisons of demographic variables among groups. For comparing the values of variables after 21 days of supplementations, adjusted for baseline value, analysis of covariance (ANCOVA) test was used.

RESULTS

The mean age was 46.2 ± 13.6. Only eight (10%) patients were smokers. Fifty-eight patients (72.5%) were male. Approximately 31% were diabetic and 67.5% were hypertensive (n = 25 and 54, respectively). Table 1 describes patient characteristics in each group. Mean age was not significantly different among the four groups (p = 0.225). Similarly, there were no statistically significant differences among the groups regarding sex, HTN, and DM. All 80 participants successfully completed this study. Patients confirmed that no changes were made to their diet or level of physical activity during the study period. In addition, none of the subjects reported any adverse effects with Silymarin and vitamin E supplementation. Table 2 shows plasma hemoglobin and MDA, as well as RBC GPX levels, at baseline and after 21 days of silymarin and/or vitamin E supplementation.

Table 1. Group characteristics

	Control	Silymarin	Vitamin E	Silymarin + vitamin E	p-Value
Age (mean ± SD)	44 ± 17	50 ± 10.3	43 ± 13.4	48.5 ± 13	0.225
Sex (M/F)	16/4	18/2	11/9	13/7	0.065
HTN (±)	11/9	14/6	14/6	15/5	0.562
DM (±)	4/16	7/13	9/11	5/15	0.330

Note: HTN, hypertension; DM, diabetes.

Table 2. Oxidative stress markers and hemoglobin changes in four groups of study patients

Study period			p-Value	Estimated means (post 3 weeks)	Pairwise significant results (LSD)
	Baseline	3 weeks
Hb (mg/dL)
Control	11.01 ± 2.58	10.92 ± 1.68	0.002	11.05	2, 3, 4 vs. 1
Silymarin	10.93 ± 1.53	12.55 ± 1.79		12.72
Vitamin E	11.29 ± 2.46	12.61 ± 2.18		12.60
Combined	11.88 ± 1.63	12.68 ± 1.90		12.36
GPX (IU/grHb)
Control	5.62 ± 2.85	3.16 ± 1.89	0.001	2.70	2, 3, 4 vs. 1 and 4 vs. 3
Silymarin	3.52 ± 1.54	4.28 ± 1.94		4.51
Vitamin E	3.93 ± 2.18	4.22 ± 1.63		4.43
Combined	3.98 ± 2.03	5.78 ± 3.51		5.89
MDA (nmol/mL)
Control	7.82 ± 1.56	9.20 ± 2.74	0.049	9.64	4 vs. 1
Silymarin	9.67 ± 3.08	9.40 ± 2.54		9.12
Vitamin E	9.08 ± 2.83	8.42 ± 2.63		8.38
Combined	9.27 ± 2.62	7.84 ± 1.84		7.71

Notes: GPX, glutathione peroxidase; MDA, mlondialdehyde; Hb, hemoglobin; LSD, least significant difference.

*Based on ANCOVA: post 3 weeks measurement of each variable as “dependent” and “baseline” as “covariate” (significance level = 0.05).

The mean values of RBC GPX in all three treatment groups were significantly higher than in the control group after 3 weeks of treatment, and adjusted with baseline values using ANCOVA and least significant difference (LSD) tests. This effect was more pronounced when the supplements were used in combination, leading to a significantly higher value compared with the group receiving vitamin E alone (Table 2). Regarding plasma MDA level, a significantly decreased level was observed after 3 weeks only in the group receiving the combination compared with the control. Although not significant, MDA levels also were lower after 3 weeks in the groups receiving vitamin E or silymarin alone.

As shown in Table 2, there is also a statistically significant increase in mean hemoglobin at 3 weeks in all three treatment groups.

DISCUSSION

In this study, we showed that silymarin alone or in combination with vitamin E can reduce MDA levels and increase RBC GPX. The effect on RBC GPX was statistically significant in the groups treated with silymarin, vitamin E, and combination of both. To our knowledge, this is the first study to evaluate the antioxidative and anti-lipid peroxidative effects of silymarin alone or in combination with vitamin E in hemodialysis patients. One of the main results of our study was a decrease in the end product of lipid peroxidation and MDA and an increase in the RBC GPX, an antioxidant enzyme, after supplementation with vitamin E for 3 weeks. The effects of vitamin E on oxidative markers have been evaluated by many studies with variable dose and duration of therapy among hemodialysis patients. Vitamin E supplementation caused an increase in GPX and SOD activities, a decrease in TBARS concentrations, and a decrease in MDA in this population.^24–32 Coating of the dialyzer membrane with vitamin E has been used to prevent neutrophil activation.^33,34 Our study confirms the results of other studies regarding antioxidative and antiperoxidative effect of vitamin E in hemodialysis patients and extend these observations to show that administration of vitamin E at a dose of 400 mg/day for 3 weeks causes a reduction in MDA. This effect was further increased by dietary supplementation with silymarin, which suggests that increasing the flavonoid content of the diet may counteract the increase in OS burden in hemodialysis patients. Compared with previous studies, this beneficial effect on OS markers occurred with relatively lower dose and shorter duration of therapy. This can be attributed to potentiating and synergistic effect of combination supplements, implying that silymarin as a potent antioxidant intensified this beneficial effect. Galli et al. showed that vitamin E, 800 mg/day for only 3 weeks, led to a slight and nonsignificant decrease in TBARS levels in HD patients.³⁴ The treatment with complex of silymarin and silibinin by Dietzman et al. led to a restoration of cellular deficiency of thiol in patients with ESDN on hemodialysis. This was followed by downregulation of TNF-α.²⁰ In contrast to our study, only patients with type 2 DM were included and plasma MDA, RBC GPX, and hemoglobin values were not evaluated.

The efficacy of silymarin in type II DM was also evaluated in a randomized, double-blind study. A significant decrease in HbA1c, FBS, total cholesterol, LDL, and triglyceride levels was shown in Silymarin-treated patients compared with that in control group. In addition, silymarin and its major constituent (silibinin) led to reversal of deficiency of cellular thiol in patients with ESDN and an improvement in functional key elements of the T-cell system and downregulation of TNF-α.³⁵

Lipid peroxidation of erythrocyte membranes by toxic oxygen-free radicals plays a major role in anemia of patients on hemodialysis and administration of antioxidants contributes to an improvement of anemia independent of erythropoietin need.^36–41 The study by Cristol et al. in a group of hemodialysis patients revealed a significant increase in RBC and a reduction in erythropoietin dose with vitamin E supplementation while maintaining stable hemoglobin concentrations.²⁷ Usberti et al. reported that a combination of a vitamin E-bonded dialysis membrane and glutathione infusion achieved better control of anemia than a vitamin E-bonded membrane alone, with a significant reduction of erythropoietin requirement.^42,43 In this study, we demonstrated a statistically significant increase in the mean hemoglobin levels of all three treatment groups compared with that in the control. Previously mentioned studies as well as our study suggest that the OS could be one of the resistance factors to erythropoietin response among patients on hemodialysis and that Silymarin and/or vitamin E supplementation could have a sparing effect on erythropoietin dosage requirement. The two main limitations of this study (the small sample size and the short duration of follow-up) preclude us from drawing definite conclusions about the effect of the interventions on erythropoietin requirement.

CONCLUSIONS

The results of this study demonstrate that oral supplementation with silymarin alone or in combination with vitamin E leads to a decrease in plasma MDA levels, an increase in the level of RBC GPX, and an increase in hemoglobin levels in patients with ESRD. Studies with larger sample sizes and a longer duration of follow-up are required to investigate the effect of silymarin supplementation on a clinically meaningful reduction in cardiovascular morbidity and mortality, erythropoietin requirement, and subsequent reduction in healthcare costs.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.