Vitamin C and E supplements improve the impaired antioxidant status and decrease plasma lipid peroxides in hemodialysis patients.

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Vitamin C and E supplements improve the impaired antioxidant status

and decrease plasma lipid peroxides in hemodialysis patients

夞

Jane C.-J. Chao

a,

*, PhD, Mi-Duo Yuan

a

, MS, Pei-Yuan Chen

b

, MD, Shiaw-Wen Chien

b

, MD

a_{Graduate Institute of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan}

b_{Department of Internal Medicine, Taipei Medical University, Taipei, Taiwan} Received 3 January 2002; received in revised form 19 April 2002; accepted 6 May 2002

Abstract

This study investigated the supplementation with vitamin C or/and E on the antioxidant system in hemodialysis patients. Thirty-eight hemodialysis patients (27 males and 11 females) with the average of 60 years old were divided into four groups: placebo (400 mg starch/time), vitamin C (400 mg/time)-, vitamin E (400 mg d,l-␣-tocopheryl acetate/time)-, and vitamin C (400 mg/time) ⫹ E (400 mg

d,l-␣-tocopheryl acetate/time)-supplemented groups for 6-week supplementation. The patients orally received three capsules of placebo or

antioxidant(s) three times a week after finishing hemodialysis. Thirty-six healthy subjects (22 males and 14 females) with the average of 58 years old were recruited as the control group. Hemodialysis patients significantly decreased plasma vitamin C by 32%, erythrocyte glutathione by 26%, and plasma total antioxidant status by 9%, but increased plasma lipid peroxide levels by 102% compared with the control group at the baseline. The levels of plasma vitamin C and total antioxidant status significantly decreased by 24% and 18%, respectively, from the post-dialysate compared with those from the pre-dialysate. At week 6, vitamin C ⫹ E-supplemented group significantly increased plasma vitamin C and E, erythrocyte glutathione, and plasma antioxidant status, and inhibited plasma lipid peroxides compared with placebo group. Additionally, vitamin C ⫹ E-supplemented group had higher plasma vitamin C, vitamin E, and total antioxidant status, and lower plasma lipid peroxides than placebo group even at least 2 weeks after the termination of the supplements. Therefore, antioxidant vitamin supplements could improve antioxidant status and decrease lipid peroxides of hemodialysis patients. © 2002 Elsevier Science Inc. All rights reserved.

Keywords: Vitamin C; Vitamin E; Hemodialysis; Antioxidant status; Lipid peroxidation

1. Introduction

A profound imbalance between oxidants and antioxi-dants as well as the abnormality of plasma lipid profile have been observed in chronic renal failure (CRF) patients [1– 6]. Previous studies reported that dialysis patients had an im-paired antioxidant system, including antioxidant status [7–10], antioxidant enzyme activities [11–17], and reduced antioxidant defense against lipid peroxidation [4,5,14 –18]. Due to hemobioincompatibality of the dialysis system, the formation of free oxygen radical species and trace amounts of endotoxins were induced during the inflammatory state in hemodialysis patients [6]. Both the abnormalities in the

antioxidant defense system and the increased oxidative stress may increase their susceptibility to lipid peroxidation in low density lipoprotein (LDL), which may lead to the subsequent development of atherosclerotic cardiovascular disease in hemodialysis patients [5–7].

The concentrations of plasma antioxidants changed in hemodialysis patients. Plasma retinol significantly elevated in hemodialysis patients [8,9], but absolute plasma concen-trations of ␣- or ␤-carotene did not differ between CRF patients with or without hemodialysis and the control [8]. Plasma␣-tocopherol concentration in hemodialysis patients did not differ from the control group [7–10]. Additionally, plasma␣-tocopherol concentration was not affected during a single session of hemodialysis [7–10], and no ␣-tocoph-erol was detected in the dialysate [10]. However, the hemo-dialysis process acutely lowered plasma antioxidant status, and plasma total ascorbate, thiol, and uric acid levels, which can be lost in the dialysate due to their water-soluble prop-erty [7–10]. During a 3-h hemodialysis session, the mean

夞 Supported by National Science Council of Taiwan, R.O.C. (grant

NSC 89 –2320-B-038 – 036 and NSC 89 –2320-B-038 – 050).

* Corresponding author. Tel.: ⫹886-2-2736-1661 #6550⬃6556 Ext.117; fax:⫹886-2-2737-3112.

E-mail address: [email protected] (J. C-J Chao).

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decrease in total vitamin C was 40% [10]. Additionally, the ascorbyl free radical/vitamin C ratio elevated after the he-modialysis session [7]. Some studies have demonstrated that vitamin supplements are required to meet the special-ized needs of renal failure or to improve the redox status in hemodialysis patients [5,6,19]. Vitamin E supplementation via oral administration or bound on dialyzer membrane improved the redox status by increasing antioxidant levels and lowering oxidative damage in hemodialysis patients [5,20 –23]. However, it is unclear for the effect of combined antioxidant vitamin supplements on the antioxidant system during and after supplementation in a manner of time course. Of potential antioxidants in the foods, vitamin C and E are the principal dietary antioxidants to protect from reactive oxygen species (ROS) damage. Due to the conflict-ing findconflict-ings for the effect of␤-carotene on ROS-associated diseases and elevated plasma retinol level in hemodialysis patients, ␤-carotene supplement is not considered in this study. Therefore, the proposes of the study was to investi-gate the effect of oral vitamin C or/and vitamin E supple-ments on the antioxidant defense system, and to follow up the effects after the termination of the supplements in he-modialysis patients. The antioxidant capacity was evaluated by blood antioxidant levels, total antioxidant status, and lipid peroxidation.

2. Materials and methods

2.1. Subjects

Thirty-eight patients (27 males and 11 females) of the average 60 years old (27⬃85 years old) with chronic renal failure and receiving regular hemodialysis for at least 3 months (3⬃261 months) were recruited from the center of hemodialysis at Taipei Medical University Hospital. All patients regularly received 3–5 h hemodialysis carried on cellulose or poly methyl methacrylate membrane three times weekly. Thirty-six healthy subjects (22 males and 14 females) of the average 58 years old (28⬃81 years old) were recruited as the control group. The subjects were selected by interviewing their motivation, commitment to completing the experiment, dietary habits (the intake amount and frequency of alcohol, coffee, and tea), physical activity (the type and frequency of exercise), smoking habit (the amount, frequency, and duration), and medical history, as well as by laboratory examination of their blood pressure, plasma lipids, blood glucose, and general health conditions by the investigators and physicians. The exclusion criteria for hemodialysis patients included those with acute illness, severe diabetes (blood glucose⬎8.06 mmol/L ante cibum and ⬎11.11 mmol/L post cibum), severe hypertension (blood pressure⬎150/95 mmHg during hemodialysis), he-patic, respiratory, or chronic inflammatory diseases. The exclusion criteria for all subjects were obesity, malnutrition, drastic body weight changes (⬎ 2.5 kg in last 1 month),

hormone replacement therapy, heavy smoking, vegetarian diet, drug abuse, the habit of alcohol, coffee or tea drinking (⬎ 3 cups/day) in last 3 months, or dietary supplementation of high-dosage (⬎ 200% Recommended Dietary Allow-ances) vitamins, minerals, antioxidants, herbs, or fish oil in last 3 months. All qualified subjects were asked for attend-ing this study, and the informed consent was obtained prior to the beginning of the study. To prevent the potential risk for their illness, hemodialysis patients were allowed to take their regular medication without any antioxidant effect but decreasing the dosage to the minimum. If the medication has an antioxidant effect (such as probucol), it was replaced two weeks before the supplementation by the physicians. During the experimental period, the patients were followed up for their health conditions routinely by the physicians, and for their obedience and complaint irregularly by the investigators. If any serious illness or complaint occurs, the patient was permitted dropping out at any time. During the experimental period, five hemodialysis patients withdrew. One in placebo group was dead, one and two in vitamin C-and vitamin E-supplemented groups, respectively, were poor health conditions, and one in vitamin C⫹ E-supple-mented group transferred to other hospital. Other than those, the patients completed the experiment without any com-plaint. All subjects were allowed to maintain their physical activity and regular life style. The study was performed in accordance with the regulations of the ethics committee of Taipei Medical University Hospital.

2.2. Dietary intake

To evaluate the effect on the antioxidant system caused by antioxidant supplements rather than by dietary intake, the dietary intake was assessed in all subjects. The subjects were asked to record their daily intake once a week for at least three times. The investigators then interviewed the subjects for their dietary record one day after. Energy and nutrient intake was calculated by food composition analysis software developed by Institute of Biomedical Sciences, Academia Sinica.

2.3. Antioxidant supplements

It was designed as a double-blind control study. Accord-ing to age, sex ratio, the degree of the diseases, the medical treatment, and the duration of receiving hemodialysis ther-apy, the patients were divided into the following four groups: placebo (400 mg starch/time), vitamin C (400 mg L-ascorbate/time)-, vitamin E (400 mg d,l- ␣-tocopheryl acetate/time)-, and vitamin C (400 mg L-ascorbate/time)⫹ E (400 mg d,l- ␣-tocopheryl acetate/time)-supplemented groups for 6 weeks. To achieve the double-blind study, all supplements were filled into the same-looking hard capsules without any flavor, and given to hemodialysis patients by the nurses rather than by the investigators. Due to the limitation of the volume of the hard capsule, the amount of

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the supplement per time was evenly filled into three hard capsules produced by China Chemical & Pharmaceutical Co., Ltd. (GMP #99 –203791– 00, Taipei, Taiwan, R.O.C.). Considering the loss of the supplements during hemodialy-sis, the patients were orally given three capsules of the supplement after the end of hemodialysis under the super-vision of the nurse three times a week to ensure totally oral intake and to increase their compliance. After 6 weeks, the supplements were terminated and the antioxidant sta-tus of hemodialysis patients was followed up for another 4 weeks.

2.4. Biochemical analyses

Pre- and post-dialysis venous blood samples (6 mL) were drawn at week 0, 3, 6, 8, and 10. Blood samples were collected in EDTA-containing tubes for lipid peroxidation assay or heparinized tubes for other analyses, and centri-fuged at 2,500⫻ g at 4°C for 10 min to separate cells and plasma. The erythrocyte pellet was washed three times by 0.9% NaCl, and centrifuged for 5 min at 2,500 ⫻ g after each wash. The pellet was prepared freshly for glutathione analysis.

Vitamin C was analyzed by reverse phase HPLC [24]. The plasma (0.5 mL) was mixed with an equal volume of cold 10% (wt/vol) metaphosphoric acid (MPA), and frozen stored until analysis. Prior to apply to HPLC, samples were centrifuged at 10,000⫻ g, 4°C, for 10 min. The supernatant (50 ␮L) was run through C18 column (RP-18E, 4 ⫻ 250

mm, 5␮m, Merck Taiwan Ltd., Taipei, Taiwan, R.O.C.). The mobile phase of HPLC system (Jasco PU-980 Intelli-gent HPLC Pump, Jasco UV-975 IntelliIntelli-gent UV/VIS De-tector, Jasco AS-950 Intelligent Sampler, Jasco Cor., To-kyo, Japan) was 5 mM 1-pentane sulfuric acid sodium salt (pH 3.1), and the flow rate was 0.8 mL/min. Vitamin C was detected at UV 254 nm. Different concentrations of L-ascorbate in 10% MPA were used as vitamin C standards. The retention time for L-ascorbate was 4.5 min.

Vitamin E was analyzed by reverse phase HPLC as described previously [25]. The plasma (0.5 ml) was mixed with 2 mL of 1% pyrogallol in absolute alcohol, 0.1 mL of 12 M HCl, and 6 mL of n-hexane containing 0.125% bu-tylated hydroxytoluene (BHT) to extract vitamin E. The n-hexane layer containing vitamin E was evaporated under nitrogen, and the residue was solubilized in 0.1 mL meth-anol, and was run through C18 column (RP-18E, 4⫻ 250

mm, 5␮m). The mobile phase of HPLC system was 100% methanol, and the flow rate was 1 mL/min. Vitamin E was detected at UV 292 nm. Different concentrations of ␣-to-copherol in absolute alcohol containing 0.1% BHT were used as vitamin E standards. The retention time for ␣-to-copherol was 5.5 min. Vitamin C and E concentrations were analyzed by SISC-LAB (32) chromatographic analysis soft-ware (Scientific Information Service Cor., Taipei, Taiwan, R.O.C.).

The concentrations of reduced glutathione were

mea-sured spectrophotometrically at 400 nm using a commercial kit (Calbiochem 354102, Calbiochem-Novabiochem Cor., La Jolla, CA) [26]. The erythrocyte pellet (50 ␮L) was resuspended in 200 ␮L of 6% MPA at 4°C followed by centrifugation at 3,000 ⫻ g for 15 min. The supernatant (100␮L) was diluted with 800 ␮L of assay buffer (200 mM potassium phosphate, pH 7.8/0.2 mM diethylene-triamine-pentaacetic acid/0.025% LUBROL). The diluted superna-tant was added 50␮l of the chromogenic reagent (12 mM solution in 0.2 M HCl) followed by mixed with 50␮L of 30% NaOH at room temperature. After 10-min reaction in the dark, glutathione levels were determined at 400 nm.

Plasma total antioxidant status was determined by eval-uation of the ability of the antioxidants in the samples to inhibit the oxidation of ABTS®(2,2 ⬘-azino-di-[3-ethylben-zthiazoline sulfonate]) to ABTS® Y⫹ by metmyoglobin (a peroxidase) using a commercial kit (Randox NX 2332, Randox Laboratories Ltd., Antrim, UK). The amount of ABTS® Y⫹produced can be monitored at 600 nm [27]. The standard (1.65 mmol/L 6-hydroxy-2,5,7,8-tetramethylchro-man-2-carboxylic acid) and plasma (20␮L) was mixed with 1 mL chromogen (5 ␮M metmyoglobin and 500 ␮M ABTS®in 66 mM phosphate buffer saline, pH 7.4), and the initial absorbance was read at 600 nm at 37°C. After the incubation with 250 ␮M H2O2for 3 min, the final

absor-bance was read at 600 nm at 37°C.

The concentration of lipid peroxides in the plasma was assessed colorimetrically at 586 nm using a commercial kit (Calbiochem 437634, Calbiochem-Novabiochem Cor., La Jolla, CA) [28]. The plasma (200␮l) was mixed with 650 ␮l of Reagent 1 (7.7 mM N-methyl-2-phenylindole in 75% acetonitrile and 25% methanol) and 150 ␮L of Reagent 2 (15.4 M methanesulfonic acid) at 45°C for 40 min. The levels of malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE), the end products derived from peroxida-tion of polyunsaturated fatty acids and related esters, were measured at 586 nm.

2.5. Statistical analysis

All data were analyzed by Statistical Analysis System (SAS) software (v6.12, SAS Institute Inc., Cary, NC). The differences of sex ratio and the numbers of hemodialysis patients with cardiovascular disease, diabetes mellitus, or erythropoietin therapy were analyzed by chi square. The mean differences between healthy subjects and hemodialy-sis patients were analyzed by Student’s t test, and those between the pre- and post-dialysis in hemodialysis patients were analyzed by paired t test. General linear model was used to analyze the differences among four supplemented groups at different time points. Fisher’s least significant difference test was used to make post-hoc comparisons if the treatment effect was demonstrated. Statistical signifi-cance is assigned at the 0.05 level.

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3. Results

3.1. Baseline data and dietary intake

Clinical data of the control and hemodialysis subjects at the baseline were shown in Table 1. Sex ratio (male:fe-male⫽ 61%:39% vs 71%:29%), age (58 ⫾ 19 vs 60 ⫾ 13 years), and body mass index (23.7 ⫾ 3.1 vs 22.4 ⫾ 2.7) were not different between the control and hemodialysis groups. The numbers of hemodialysis patients with cardio-vascular disease, diabetes mellitus, or recombinant human erythropoietin therapy, the averages for duration of hemo-dialysis therapy (59 months), plasma albumin (39 g/L), total cholesterol (4.00 mmol/L), and triglyceride levels (2.58 mmol/L) were not significantly different among four hemo-dialysis groups. The averages of plasma albumin and total cholesterol concentrations in hemodialysis patients were in the normal ranges. However, the average of plasma triglyc-eride concentration was higher than the normal range in all hemodialysis groups.

The dietary intake of the control and hemodialysis sub-jects were shown in Table 2. Due to no significant differ-ence among dietary assessments from different days and among hemodialysis groups, the daily average intake of the control and hemodialysis groups was used. The daily di-etary intake for energy, carbohydrate, protein, lipid, satu-rated fatty acids (SFA), unsatusatu-rated fatty acids (UFA), vi-tamin C, vivi-tamin E, and crude fiber did not significantly differ between the control and hemodialysis groups. The intake for carbohydrate, protein, and lipid in the control and hemodialysis groups was 48.8%, 18.6%, and 32.6%, as well as 53.6%, 14.5%, and 31.9% energy, respectively. The UFA/SFA ratio in the control and hemodialysis groups was 2.9 and 2.2, respectively. The average vitamin C intake in the control and hemodialysis groups was beyond Dietary Reference Intakes (DRI) [29]. Whereas the average vitamin E intake in both groups was less than DRI.

3.2. Plasma vitamin C and E concentrations

Plasma vitamin C concentrations in hemodiaytic patients significantly lowered by 32% (P⬍ 0.05) than those in the control group at the baseline (Table 3). Plasma vitamin C levels also significantly lowered by 24% (P⬍ 0.05) from the post-dialysis compared with those from the pre-dialysis of hemodialysis patients during 10 weeks. Vitamin C ⫹ E-supplemented group had lower plasma vitamin C concen-tration (P⬍ 0.05) than other hemodialysis groups at week 0. During the supplemented period, plasma vitamin C levels significantly increased by 33% and 63% (P ⬍ 0.05) in vitamin C-supplemented group, and by 35% and 72% (P⬍ 0.05) in vitamin C⫹ E-supplemented group at week 3 and 6 compared with the respective baselines. Both vitamin C-and vitamin C⫹ E-supplemented groups had higher plasma

Table 1

Clinical data of the control group and the four subgroups of hemodialysis patients at the baseline1 Control (n⫽ 36) Hemodialysis Placebo (n⫽ 8) Vitamin C (n⫽ 10) Vitamin E (n⫽ 10) Vitamin C⫹ E (n⫽ 10) Sex M22, F14 M6, F2 M7, F3 M7, F3 M7, F3 Cardiovascular disease, n — 7 8 8 6 Diabetes mellitus, n — 3 2 3 2 Erythopoietin therapy, n — 7 10 7 6 Age, year 58⫾ 19 66⫾ 12 57⫾ 14 62⫾ 8 58⫾ 17

Body mass index, kg/m2 _23.7_{⫾ 3.1} _23.6_{⫾ 3.2} _19.7_{⫾ 2.4} _22.9_{⫾ 2.7} _23.8_{⫾ 2.5}

Duration of hemodialysis therapy, month — 61⫾ 55 61⫾ 77 57⫾ 67 57⫾ 78

Plasma albumin, g/L 35–552 ₃₇_{⫾ 5} ₃₇_{⫾ 3} ₃₉_{⫾ 3} ₃₇_{⫾ 4}

Plasma total cholesterol, mmol/L 3.36–5.692 _3.54_{⫾ 0.77} _4.21_{⫾ 0.83} _3.96_{⫾ 0.90} _4.20_{⫾ 0.89} Plasma triglycerides, mmol/L 0.34–1.952 _2.47_{⫾ 2.85} _2.13_{⫾ 1.08} _3.02_{⫾ 2.79} _2.69_{⫾ 1.80}

1_{Values are mean}_{⫾ SD (P ⬎ 0.05).} 2_{Normal range values in laboratory data.}

Table 2

The dietary intake of the control and hemodialysis subjects1 Control (n⫽ 36) Hemodialysis (n⫽ 38) Energy, kcal 2028⫾ 432 1935⫾ 647 kJ 8484⫾ 1805 8097⫾ 2707 Carbohydrate, g 247.9⫾ 98.7 259.5⫾ 75.8 % energy 48.8 53.6 Protein, g 94.4⫾ 31.7 70.3⫾ 23.5 % energy 18.6 14.5 Lipid, g 73.5⫾ 29.3 68.8⫾ 11.4 % energy 32.6 31.9

Saturated fatty acids, g 18.9⫾ 10.4 21.1⫾ 12.4

% energy 8.3 9.8

% lipid 25.7 30.6

Unsaturated fatty acids, g 53.5⫾ 20.2 46.7⫾ 17.5

% energy 23.7 21.7

% lipid 72.8 67.8

Vitamin C, mg 126.1⫾ 92.6 98.6⫾ 74.9

Vitamin E, mg␣-TE 9.75⫾ 4.54 8.04⫾ 3.71

Crude fiber, g 3.64⫾ 1.75 2.83⫾ 2.09

1_{Values are mean}_{⫾ SD (P ⬎ 0.05).}

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vitamin C levels (P ⬍ 0.05) than placebo and vitamin E groups. After the termination of the supplements, plasma vitamin C levels remained the same in vitamin C- and vitamin C ⫹ E-supplemented groups for another 2 and 4 weeks, respectively. At week 8, plasma vitamin C levels significantly increased (P⬍ 0.05) in vitamin C-, vitamin E-, and vitamin C ⫹ E-supplemented groups compared with placebo group. At week 10, plasma vitamin C levels in vitamin C- and vitamin C⫹ E-supplemented groups were higher (P⬍ 0.05) compared with placebo group, but only in vitamin C ⫹ E-supplemented group was not significantly different from those of the control group.

Plasma vitamin E levels were not different between the control and hemodialysis groups, between the post- and pre-dialysis, and among the hemodialysis groups at week 0 (Table 4). Plasma vitamin E concentrations significantly elevated by 35% and 70% (P⬍ 0.05) in vitamin E-supple-mented group, and by 36% and 73% (P⬍ 0.05) in vitamin C⫹ E-supplemented group at week 3 and 6 compared with the respective baselines. During the supplemented period, both vitamin E- and vitamin C ⫹ E-supplemented groups had higher plasma vitamin E levels (P⬍ 0.05) than placebo and vitamin C groups. At week 6, plasma vitamin E levels were higher (P ⬍ 0.05) in vitamin E- and vitamin C ⫹ E-supplemented groups than those in the control group. Placebo and vitamin C did not affect plasma vitamin E levels during 10 weeks. After the termination of the sup-plements, plasma vitamin E concentrations remained higher (P⬍ 0.05) in vitamin E- and vitamin C ⫹ E-supplemented groups compared with placebo and vitamin C-supplemented groups. At week 10, plasma E levels in vitamin

E-supple-mented group were still higher (P⬍ 0.05) than those in the control group.

3.3. Erythrocyte glutathione concentration

Hemodialysis patients significantly lowered erythrocyte glutathione levels by 26% (P ⬍ 0.05) compared with the control group (Table 5). Except for lower erythrocyte glu-tathione in placebo group at week 10 from the post-dialysis, erythrocyte glutathione concentrations in the pre- and post-dialysate did not significantly differ (Table 5). Erythrocyte glutathione levels increased with both the supplemented duration and antioxidant vitamin supplements (P⬍ 0.05). At week 3 and 6, all antioxidant supplemented groups had higher erythrocyte glutathione levels (P ⬍ 0.05) than the respective baselines and placebo group. After the termina-tion of the supplements, erythrocyte glutathione levels in vitamin C- and vitamin E-supplemented groups were not significantly different from those at week 6 and from those in the control group. Additionally, erythrocyte glutathione levels were not significantly different among the hemodial-ysis groups at week 8 and 10.

3.4. Plasma total antioxidant status

Except for placebo group, hemodialysis patients had sig-nificantly decreased plasma total antioxidant status by 9% (P⬍ 0.05) compared with the control group at the baseline (Table 6). Plasma total antioxidant status significantly im-paired by 18% (P⬍ 0.05) from the post-dialysis compared with that from the pre-dialysis throughout 10 weeks. At the

Table 3

Plasma vitamin C concentrations of the control and hemodialysis subjects before, during, and after antioxidant vitamin supplements1 Plasma vitamin C concentration,␮mol/L (%)2

Week 0 Week 3 Week 6 Week 8 Week 10

Control 17.0⫾ 3.1 (n ⫽ 36) Hemodialysis 11.6⫾ 3.13_(n_{⫽ 38)} Placebo (n⫽ 8) (n⫽ 8) (n⫽ 8) (n⫽ 7) (n⫽ 7) Pre-dialysis 13.5⫾ 3.1b# _12.7_{⫾ 3.3}#₍₉₄₎a _12.0_{⫾ 3.0}#₍₈₉₎a _10.5_{⫾ 3.1}#₍₇₈₎a _11.7_{⫾ 3.3}#₍₈₇₎a Post-dialysis 10.3⫾ 3.2¶ _9.5_{⫾ 2.8}¶ _9.2_{⫾ 2.5}¶ _8.0_{⫾ 2.9} _8.8_{⫾ 2.5}¶ Vitamin C (n⫽ 10) (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) Pre-dialysis 10.7⫾ 3.1ab# _14.2_{⫾ 2.5*}#₍₁₃₃₎b _17.4_{⫾ 4.0* (163)}b _15.9_{⫾ 2.8 (149)}c _12.2_{⫾ 2.6}†#₍₁₁₄₎b Post-dialysis 8.1⫾ 2.4¶ _10.7_{⫾ 3.2}¶ _13.1_{⫾ 3.6}¶ _12.1_{⫾ 2.8}¶ _9.3_{⫾ 2.4}¶ Vitamin E (n⫽ 10) (n⫽ 9) (n⫽ 8) (n⫽ 8) (n⫽ 8) Pre-dialysis 12.5⫾ 3.2b# _13.2_{⫾ 3.6}#₍₁₀₆₎a _13.5_{⫾ 3.9 (108)}a _13.4_{⫾ 4.1 (107)}b _13.2_{⫾ 3.7}#₍₁₀₆₎ab Post-dialysis 9.2⫾ 3.2¶ _9.9_{⫾ 3.3} _10.2_{⫾ 3.2}¶ _10.2_{⫾ 3.9} _10.1_{⫾ 3.0}¶ Vitamin C⫹ E (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) (n⫽ 9) Pre-dialysis 9.6⫾ 2.9a# _13.0_{⫾ 3.0*}#₍₁₃₅₎b _16.5_{⫾ 3.2* (172)}b _15.9_{⫾ 4.0 (166)}c _13.9_{⫾ 3.2 (145)}c Post-dialysis 7.1⫾ 2.5 9.7⫾ 2.4¶ _12.5_{⫾ 3.5}¶ _12.1_{⫾ 3.1}¶ _10.6_{⫾ 3.0}¶

1_{Values are mean}_{⫾ SD.}

2_{Values are the percentage of the respective group at week 0 from the pre-dialysis in hemodialysis patients.} 3_{Values from the pre-dialysis of all hemodialysis patients at week 0 vs the control group, P}_{⬍ 0.05.}

Values of hemodialysis patients in each column not sharing the same letter significantly differ (P⬍ 0.05) by Fisher’s least significant difference test. * Week 3, 6 (during the supplemented period) vs week 0 (baseline), P⬍ 0.05.

†_{Week 8, 10 (the terminated period without supplementation) vs week 6 (the end of the supplemented period), P}_{⬍ 0.05.} #_{The hemodialysis group vs the control group, P}_{⬍ 0.05.}

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baseline, plasma total antioxidant status was below the normal range (1.30⬃1.77 mmol/L) from the pre-dialysis in eleven (29%) patients, and from the post-dialysis in all patients (100%). Total antioxidant status significantly en-hanced with the duration of antioxidant vitamin supple-ments (P⬍ 0.05). Vitamin C-, vitamin E-, and vitamin C ⫹ E-supplemented groups significantly elevated plasma total

antioxidant status by 9%, 26%, and 24% (P⬍ 0.05) at week 3, and by 21%, 33%, and 33% (P ⬍ 0.05) at week 6 compared with the respective baselines. Vitamin E- and vitamin C ⫹ E-supplemented groups had greater plasma total antioxidant status (P⬍ 0.05) than the control, placebo, and vitamin C-supplemented groups at week 3. At week 6, all antioxidant vitamin supplements significantly elevated

Table 4

Plasma vitamin E concentrations of the control and hemodialysis subjects before, during, and after antioxidant vitamin supplements1 Plasma vitamin E concentration,␮mol/L (%)2

Control 12.9⫾ 3.4 (n ⫽ 36) Hemodialysis 11.0⫾ 3.6 (n ⫽ 38) Placebo (n⫽ 8) (n⫽ 8) (n⫽ 8) (n⫽ 7) (n⫽ 7) Pre-dialysis 12.0⫾ 3.0a _11.7_{⫾ 3.1 (98)}a _12.4_{⫾ 3.3 (103)}a _11.8_{⫾ 2.9 (98)}a _12.7_{⫾ 2.8 (106)}a Post-dialysis 11.4⫾ 3.6 10.7⫾ 3.1 11.8⫾ 3.2 10.9⫾ 3.4 11.5⫾ 3.0 Vitamin C (n⫽ 10) (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) Pre-dialysis 11.0⫾ 3.8a _12.0_{⫾ 2.6 (109)}a _12.3_{⫾ 3.2 (112)}a _10.9_{⫾ 3.8 (99)}a _11.1_{⫾ 2.9 (101)}a Post-dialysis 11.2⫾ 3.8 11.1⫾ 3.2 11.7⫾ 3.0 10.2⫾ 3.8 9.8⫾ 3.4 Vitamin E (n⫽ 10) (n⫽ 9) (n⫽ 8) (n⫽ 8) (n⫽ 8) Pre-dialysis 11.3⫾ 4.1a _15.3_{⫾ 3.9* (135)}b _19.2_{⫾ 4.1*}#₍₁₇₀₎b _17.8_{⫾ 3.3}#₍₁₅₈₎b _16.5_{⫾ 4.6}#₍₁₄₆₎b Post-dialysis 9.9⫾ 3.5 15.4⫾ 3.4 18.6⫾ 3.4 16.7⫾ 3.5 15.3⫾ 3.5 Vitamin C⫹ E (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) (n⫽ 9) Pre-dialysis 9.8⫾ 3.3a _13.3_{⫾ 3.2* (136)}b _17.0_{⫾ 4.1*}#₍₁₇₃₎b _16.8_{⫾ 3.7}#₍₁₇₁₎b _15.9_{⫾ 3.6 (162)}b Post-dialysis 10.2⫾ 4.0 12.9⫾ 2.8 17.6⫾ 3.9 16.6⫾ 4.1 14.7⫾ 3.8

2_{Values are the percentage of the respective group at week 0 from the pre-dialysis in hemodialysis patients.}

#_{The hemodialysis group vs the control group, P}_{⬍ 0.05.}

Table 5

Erythrocyte glutathione concentrations of the control and hemodialysis subjects before, during, and after antioxidant vitamin supplements1 Erythrocyte glutathione concentration,␮mol/L (%)2

Control 309⫾ 40 (n ⫽ 36) Hemodialysis 228⫾ 443_(n_{⫽ 38)} Placebo (n⫽ 8) (n⫽ 8) (n⫽ 8) (n⫽ 7) (n⫽ 7) Pre-dialysis 236⫾ 39a# ₂₄₂_{⫾ 33}#₍₁₀₃₎a ₂₄₉_{⫾ 43}#₍₁₀₆₎a ₂₄₂_{⫾ 21}#₍₁₀₃₎a ₂₄₉_{⫾ 20}#₍₁₀₆₎a Post-dialysis 205⫾ 36 212⫾ 32 213⫾ 38 218⫾ 28 213⫾ 23¶ Vitamin C (n⫽ 10) (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) Pre-dialysis 231⫾ 54a# ₂₈₉_{⫾ 58* (125)}b ₃₀₄_{⫾ 41* (131)}b ₂₆₀_{⫾ 57 (113)}a ₂₅₆_{⫾ 57 (111)}a Post-dialysis 210⫾ 44 265⫾ 45 279⫾ 37 231⫾ 52 233⫾ 54 Vitamin E (n⫽ 10) (n⫽ 9) (n⫽ 8) (n⫽ 8) (n⫽ 8) Pre-dialysis 234⫾ 36a# ₃₀₀_{⫾ 50* (128)}b ₃₁₀_{⫾ 57* (132)}b ₂₇₀_{⫾ 46 (115)}a ₂₆₂_{⫾ 48 (112)}a Post-dialysis 199⫾ 38 262⫾ 48 271⫾ 50 234⫾ 42 230⫾ 45 Vitamin C⫹ E (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) (n⫽ 9) Pre-dialysis 210⫾ 48a# ₂₇₃_{⫾ 39* (130)}b ₂₉₄_{⫾ 56* (140)}b ₂₄₁_{⫾ 37}†#₍₁₁₅₎a ₂₃₈_{⫾ 38}†#₍₁₁₃₎a Post-dialysis 184⫾ 44 244⫾ 39 267⫾ 51 217⫾ 33 214⫾ 33

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plasma total antioxidant status (P⬍ 0.05) compared with the control and placebo groups. However, plasma total an-tioxidant status was not different among anan-tioxidant vitamin supplemented groups. After the termination of the supple-ments, vitamin E- and vitamin C⫹ E-supplemented groups still had greater plasma total antioxidant status (P⬍ 0.05) than placebo group at week 8. At week 10, only vitamin C

⫹ E-supplemented group had greater plasma total

antioxi-dant status (P ⬍ 0.05) than the control group. However, plasma total antioxidant status was not significantly differ-ent among the hemodialysis groups.

3.5. Plasma lipid peroxidation

Hemodialysis patients had significantly increased plasma lipid peroxidation by 102% (P⬍ 0.05) compared with the control group at the baseline (Table 7). However, plasma lipid peroxide levels were not significantly different be-tween the post- and pre-dialysis during 10 weeks (Table 7). At week 3, vitamin E-supplemented group significantly lowered plasma lipid peroxides (P⬍ 0.05) compared with placebo and vitamin C-supplemented groups. Plasma lipid peroxide levels significantly decreased by 29%, 31%, and 47% (P⬍ 0.05) in vitamin C-, vitamin E-, and vitamin C ⫹ E-supplemented groups at week 6 compared with those in placebo group and those in the respective baselines. Only in vitamin E- and vitamin C⫹ E-supplemented groups plasma lipid peroxide levels were not significantly different from those of the control group from week 6 to week 10. Plasma lipid peroxide levels were still lower in antioxidant vitamin

supplemented groups than placebo group at week 8. At week 10, placebo and vitamin C-supplemented groups sig-nificantly increased plasma lipid peroxide levels by 54% and 124% (P ⬍ 0.05) compared with the control group. Additionally, vitamin E- and vitamin C⫹ E-supplemented groups had lower plasma lipid peroxides (P ⬍ 0.05) than placebo and vitamin C-supplemented groups.

4. Discussion

Hemodialysis patients in the study had normal plasma albumin and total cholesterol levels, suggesting that the patients were not malnutrition. It is also supported by the results of dietary intake, energy and macronutrients intake in hemodialysis patients was not different from the control group. Although protein, vitamin C, and crude fiber intake tended to be slightly lower in hemodialysis patients, there was not significantly different from the control group. En-ergy, macronutrients, crude fiber, vitamin C and vitamin E intake was very similar between hemodialysis and con-trol groups. Therefore, the difference observed in this study was totally due to the supplementation rather than dietary intake.

Oral dosage of vitamins each time in this study was 4-and 5-fold of DRI for vitamin C in males (90 mg) 4-and females (75 mg), and 18-fold of DRI for vitamin E (15 mg

␣-tocopherol), respectively [29]. Because a certain part of

oxalate in the urine derived from metabolized vitamin C, high intake of vitamin C was susceptible to cause secondary

Table 6

Plasma total antioxidant status of the control and hemodialysis subjects before, during, and after antioxidant vitamin supplements1 Plasma total antioxidant status, mmol/L (%)2

Control 1.46⫾ 0.09 (n ⫽ 36) Hemodialysis 1.33⫾ 0.143_(n_{⫽ 38)} Placebo (n⫽ 8) (n⫽ 8) (n⫽ 8) (n⫽ 7) (n⫽ 7) Pre-dialysis 1.40⫾ 0.21a _1.36_{⫾ 0.16 (97)}a _1.42_{⫾ 0.18 (101)}a _1.28_{⫾ 0.16}#₍₉₁₎a _1.36_{⫾ 0.17 (97)}a Post-dialysis 1.10⫾ 0.24¶ _1.04_{⫾ 0.06}¶ _1.14_{⫾ 0.10}¶ _1.09_{⫾ 0.24} _1.14_{⫾ 0.21}¶ Vitamin C (n⫽ 10) (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) Pre-dialysis 1.34⫾ 0.09a# _1.46_{⫾ 0.18* (109)}a _1.62_{⫾ 0.19*}#₍₁₂₁₎b _1.39_{⫾ 0.23 (104)}ab _1.33_{⫾ 0.27}†₍₉₉₎a Post-dialysis 1.08⫾ 0.10¶ _1.12_{⫾ 0.17}¶ _1.33_{⫾ 0.15}¶ _1.10_{⫾ 0.17}¶ ₁₀₆_{⫾ 0.25}¶ Vitamin E (n⫽ 10) (n⫽ 9) (n⫽ 8) (n⫽ 8) (n⫽ 8) Pre-dialysis 1.26⫾ 0.16a# _1.59_{⫾ 0.14*}#₍₁₂₆₎b _1.68_{⫾ 0.15*}#₍₁₃₃₎b _1.47_{⫾ 0.13 (117)}bc _1.37_{⫾ 0.12 (109)}a Post-dialysis 1.17⫾ 0.07¶ _1.28_{⫾ 0.12}¶ _1.47_{⫾ 0.18}¶ _1.22_{⫾ 0.16}¶ _1.24_{⫾ 0.13} vitamin C⫹ E (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) (n⫽ 9) Pre-dialysis 1.35⫾ 0.11a# _1.67_{⫾ 0.30*}#₍₁₂₄₎b _1.80_{⫾ 0.25*}#₍₁₃₃₎b _1.67_{⫾ 0.26}#₍₁₂₄₎c _1.52_{⫾ 0.14}†#₍₁₁₃₎a Post-dialysis 1.08⫾ 0.14¶ _1.32_{⫾ 0.09}¶ _1.48_{⫾ 0.34} _1.40_{⫾ 0.22}¶ _1.23_{⫾ 0.27}¶

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hyperoxalemia and oxalosis in hemodialysis patients. Costello et al. [30] reported that increased plasma vitamin C status was not correlated with plasma oxalate level. How-ever, Ono [31] found that there was a strong correlation between plasma vitamin C and oxalate levels (r⫽ 0.755, P⬍ 0.01). Vitamin C at a dose of 100 mg/day for 1 month had no significant effect on oxalate metabolism, including plasma concentration, metabolic pool size, tissue accumu-lation rate, production rate, and dialysis clearance in hemo-dialysis patients [32]. However, plasma oxalate levels changed from 50.4⫾ 8.2␮mol/L to 34.1 ⫾ 1.4, 33.3 ⫾ 3.7, and 25.7 ⫾ 3.9␮mol/L, respectively, in hemodialysis pa-tients with daily oral vitamin C intake from 500 mg for more than 6 months to 100, 50, and 0 mg for 4 weeks [31]. Similarly, oral administration of vitamin C at a dose of 500 mg/day for 3 weeks, plasma oxalate rose form 30.3⫾ 3.5 to 48.4 ⫾ 6.1␮mol/L in hemodialysis patients [33]. Oxalate levels in pre- and post-dialysate significantly increased fol-lowing an increase of vitamin C dosage from 100 to 500 mg/day [34]. It is suggested that hemodialysis patients who have a defect in vitamin C or oxalate metabolism may restrict daily vitamin C intake below 500 mg. Whereas vitamin C bioavailability in healthy males was 80%, 72%, and 63%, respectively, for oral administration of pure vita-min C at 100, 200, and 500 mg as a single dose [35,36]. In our study, oral intake vitamin C at 400 mg/time three times a week should not be risk for oxalosis or calcium oxalate kidney stones in hemodialysis patients, although we did not measure plasma oxalate.

Our study showed that plasma vitamin C levels signifi-cantly decreased by 24% during hemodialysis. Similar to a

previous finding [10], the mean decrease in plasma total vitamin C was 40% during a 3-h hemodialysis session. Additionally, plasma vitamin C levels significantly de-creased by 32% in hemodialysis patients compared with the control group at the baseline. Although dietary intake of vitamin C in hemodialysis patients was similar to the con-trol group, lower plasma vitamin C levels probably resulted from the loss during hemodialysis because of its water-soluble property, poor absorption of vitamin C in the gas-trointestinal tract, or/and the utilization for the defense of free radicals in hemodialysis patients. After 6-week supple-mentation, vitamin C- and vitamin C ⫹ E-supplemented groups had significantly greater plasma vitamin C levels than the baselines and placebo group. Additionally, plasma vitamin C concentrations were not different among vitamin C-, vitamin C⫹ E-supplemented, and the control groups at week 6, indicating vitamin C supplement alone or with vitamin E could elevate plasma vitamin C levels to the normal concentration in hemodialysis patients. After the termination of the supplements, plasma vitamin C levels maintained at the normal levels at least for another 2 weeks in vitamin C- and vitamin C ⫹ E-supplemented groups, which was supported by the half-life data of plasma vitamin C in previous studies [37,38]. Hornig [37] demonstrated that the overall half-life of plasma vitamin C was 10 –20 days for daily supplements of 30 –180 mg, and dependent upon plasma vitamin C levels. Blanchard [38] also showed that the half-life of vitamin C was inversely related to entry vitamin C levels in the plasma below 85␮mol/L, whereas above this concentration half-life was approximately con-stant and averaged 14.2 days [38]. In contrast with plasma

Table 7

Plasma lipid peroxide concentrations of the control and hemodialysis subjects before, during, and after antioxidant vitamin supplements1 Plasma malondialdehyde⫹ 4-hydroxy-2(E)-nonenal concentration,␮mol/L (%)2

Control 23.6⫾ 9.8 (n ⫽ 36) Hemodialysis 47.7⫾ 21.43_(n_{⫽ 38)} Placebo (n⫽ 8) (n⫽ 8) (n⫽ 8) (n⫽ 7) (n⫽ 7) Pre-dialysis 37.7⫾ 19.8a# _40.0_{⫾ 19.7}#₍₁₀₆₎b _42.4_{⫾ 22.2}#₍₁₁₂₎c _44.0_{⫾ 26.8}#₍₁₁₇₎c _36.3_{⫾ 24.6}#₍₉₆₎b Post-dialysis 38.4⫾ 20.2 42.6⫾ 20.2 41.2⫾ 23.7 44.4⫾ 23.6 38.0⫾ 24.5 Vitamin C (n⫽ 10) (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) Pre-dialysis 56.3⫾ 20.0a# _52.9_{⫾ 16.4}#₍₉₄₎b _40.0_{⫾ 11.1*}#₍₇₁₎b _45.3_{⫾ 18.9}#₍₈₀₎b _52.9_{⫾ 19.8}†#₍₉₄₎b Post-dialysis 56.1⫾ 19.7 54.3⫾ 19.0 41.6⫾ 16.8 43.2⫾ 20.1 59.5⫾ 20.2 Vitamin E (n⫽ 10) (n⫽ 9) (n⫽ 8) (n⫽ 8) (n⫽ 8) Pre-dialysis 43.5⫾ 17.3a# _36.0_{⫾ 15.4}#₍₈₃₎a _30.2_{⫾ 12.4* (69)}ab _22.7_{⫾ 17.1 (52)}a _25.5_{⫾ 19.1 (59)}a Post-dialysis 44.7⫾ 16.6 36.0⫾ 13.2 28.1⫾ 11.7 24.2⫾ 19.4 24.1⫾ 16.7 Vitamin C⫹ E (n⫽ 10) (n⫽ 10) (n⫽ 9) (n⫽ 9) (n⫽ 9) Pre-dialysis 53.1⫾ 28.4a# _42.7_{⫾ 20.9}#₍₈₀₎a _28.0_{⫾ 16.1* (53)}a _25.3_{⫾ 19.4 (48)}a _32.3_{⫾ 21.6 (61)}a Post-dialysis 52.4⫾ 26.0 40.2⫾ 21.7 29.4⫾ 15.2 25.7⫾ 22.8 33.2⫾ 20.1

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vitamin C levels, plasma vitamin E concentrations were not different between the pre- and post-dialysis, as well as the hemodialysis and the control groups. Previous studies also found that plasma ␣-tocopherol concentrations unchanged during hemodialysis [7–10]. However, Pastor et al. [39] showed erythrocyte vitamin E levels were lower in hemo-dialysis patients than in the control group. Plasma vitamin E levels significantly increased in vitamin E- and vitamin C⫹ E-supplemented groups at week 6. Similar to our results, both plasma and RBC vitamin E concentrations signifi-cantly increased in hemodialysis patients receiving daily oral supplement of vitamin E at 600 mg for 30 days [20]. Additionally, serum vitamin E significantly increased in hemodialysis patients with oral vitamin E at a dose of 300 [21] or 800 mg/day [5] for 8 or 3 weeks, respectively. Peuchant et al. [18] also found that increased erythrocyte vitamin E level was observed in CRF patients on the re-stricted protein diet (0.3 g/day) supplemented with iron and multivitamins, including 7.5 mg vitamin A, 1000 IU vita-min D3, 5 mg␣-tocopheryl acetate, 75 mg vitamin C, and

vitamin B complex (B1,B2,B6,and B12) daily for 6 months

compared with pre-diet results. Our data indicated that the increased effect of plasma vitamin E lasted for another 4 weeks during the termination of the supplements probably due to its water-insoluble property.

Erythrocyte glutathione concentrations significantly de-creased in hemodialysis patients compared with the control group, indicating hemodialysis patients may have impaired glutathione-related antioxidant enzyme system. The activi-ties of plasma and erythrocyte glutathione peroxidase (GSH-Px) significantly decreased in hemodialysis patients compared with the healthy subjects [13]. Erythrocyte or polymorphonuclear leukocyte GSH-Px activities were also lower in the patients after hemodialysis than the control group [11–13,17]. Plasma GSH-Px activity significantly el-evated during hemodialysis [13]. All antioxidant vitamin supplemented groups significantly increased erythrocyte glutathione levels to the normal level at week 6, suggesting that antioxidant vitamin supplementation may reserve the consumption of glutathione for the protection from free radical damage. After the termination of the supplements, erythrocyte glutathione concentrations were not signifi-cantly different among the hemodialysis groups. Both vita-min C- and vitavita-min E-supplemented groups had similar erythrocyte glutathione levels as the control group from week 3 to week 10.

Plasma total antioxidant status significantly decreased by 9% in hemodialysis patients compared with the control group at the baseline, and impaired by 18% during hemo-dialysis. It is indicated that plasma water-soluble antioxi-dants may be lost during hemodialysis, which results in impaired plasma antioxidant status in hemodialysis patients. After 6-week supplementation, all antioxidant vitamin sup-plements significantly enhanced plasma total antioxidant status, which could be accompanied by the increases in plasma vitamin C, vitamin E, and erythrocyte glutathione

levels. Although plasma total antioxidant status was lower at the baseline in hemodialysis patients, vitamin C ⫹ E supplementation could improve plasma total antioxidant status even greater than the control group throughout the supplementation and termination periods.

In consistent with the results of total antioxidant status, the data showed that plasma lipid peroxide levels signifi-cantly increased by 102% in hemodialysis patients com-pared with the control group. Similar to our results, previous studies indicated that lipid peroxidation products in plasma [4,5,10,16] and RBC [17,18] increased in hemodialysis pa-tients compared with the healthy subjects. It is suggested that oxidative damage occurs during hemodialysis. At week 6, all antioxidant vitamin supplemented groups significantly decreased plasma lipid peroxide levels compared with the respective baselines and placebo group. A previous study demonstrated that significantly reduced erythrocyte lipid peroxidation was found in CRF patients on the restricted protein diet (0.3 g/day) supplemented with iron and vitamin E-containing multivitamins [18]. Supplementation with vi-tamin E at 300 mg/day for 1 month significantly inhibited both plasma and erythrocyte lipid peroxidation in hemodi-alysis patients [22]. Additionally, daily oral administration of vitamin E at 600 mg for 2 weeks resulted in a significant decrease of LDL lipid peroxidation in hemodialysis patients [23]. However, Galli et al. [5] found that oral supplemen-tation with 800 mg/day vitamin E for 3 weeks did not significantly affect plasma lipid peroxide-thiobarbituric acid reactants in hemodialysis patients. After the termination of the supplements, vitamin E- and vitamin C ⫹ E-supple-mented groups still had similar plasma lipid peroxide levels as the control group. However, plasma lipid peroxide levels were not different between vitamin E- and vitamin C ⫹ E-supplemented groups. Whereas placebo and vitamin C-supplemented groups had higher plasma lipid peroxide lev-els than the control group throughout 10 weeks. The data suggest that the inhibition of plasma lipid peroxidation in hemodialysis patients may be primarily contributed by the antioxidant action of vitamin E.

In conclusion, vitamin C (400 mg L-ascorbate/time)⫹ E (400 mg d,l-␣-tocopheryl acetate/time) supplements three times a week for 6 weeks not only effectively corrected the decreased plasma vitamin C, erythrocyte glutathione and plasma total antioxidant status at least to the normal levels, but also inhibited the increased plasma lipid peroxidation to the normal level in hemodialysis patients. Additionally, vitamin C⫹ E supplements had the overall longer lasting period for maintaining antioxidant status after the termina-tion of the supplements. For clinical applicatermina-tion, it is feasi-ble and inexpensive that oral administration of combined vitamin C (400 mg) and E (400 mg) after finishing hemo-dialysis session is helpful to prevent the development of cardiovascular disease by improving antioxidant status and decreasing oxidative damage in hemodialysis patients.

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Acknowledgments

We thank the nurses for assisting oral administration of the supplements in the center of hemodialysis at Taipei Medical University Hospital.

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