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Effects of vitamin E and C supplementation on oxidative stress and viral load in HIV-infected subjects

Allard, Johane P.1,2; Aghdassi, Elaheh1; Chau, Jenny1; Tam, Carolyn1; Kovacs, Colin M.1; Salit, Irving E.1; Walmsley, Sharon L.1

ARTICLES

Objectives: The HIV-infected population is known to be oxidatively stressed and deficient in antioxidant micronutrients. Since in vitro replication of HIV is increased with oxidative stress, this study assessed the effect of antioxidant vitamin supplementation on lipid peroxidation, a measure of oxidative stress, and viral load in humans.

Design: A randomized placebo-controlled, double-blind study.

Methods: Forty-nine HIV-positive patients were randomized to receive supplements of both DL-α-tocopherol acetate (800 IU daily) and vitamin C (1000 mg daily), or matched placebo, for 3 months. Plasma antioxidant micronutrient status, breath pentane output, plasma lipid peroxides, malondialdehyde and viral load were measured at baseline and at 3 months. New or recurrent infections for the 6-month period after study entry were also recorded.

Results: The vitamin group (n = 26) had an increase in plasma concentrations of α-tocopherol (P < 0.0005) and vitamin C (P < 0.005) and a reduction in lipid peroxidation measured by breath pentane (P < 0.025), plasma lipid peroxides (P < 0.01) and malondialdehyde (P < 0.0005) when compared with controls (n = 23). There was also a trend towards a reduction in viral load (mean ± SD changes over 3 months, −0.45 ± 0.39 versus +0.50 ± 0.40 log10 copies/ml; P = 0.1; 95% confidence interval, −0.21 to −2.14). The number of infections reported was nine in the vitamin group and seven in the placebo group.

Conclusion: Supplements of vitamin E and C reduce oxidative stress in HIV and produce a trend towards a reduction in viral load. This is worthy of larger clinical trials, especially in HIV-infected persons who cannot afford new combination therapies.

1Department of Medicine, University of Toronto, Toronto, Ontario, Canada.

2Requests for reprints to: Dr J.P. Allard, The Toronto Hospital, General Division, 200 Elizabeth Street, 9 EN-217A, Toronto, Ontario, Canada M5G 2C4.

Sponsorship: This study was supported by a grant from the Canadian Foundation for AIDS Research (CANFAR).

Date of receipt: 5 February 1998; revised: 13 May 1998; accepted: 19 May 1998.

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Introduction

HIV infection results in the progressive impairment of immune response leading to the development of AIDS. Amongst the mechanisms contributing to this progression, oxidative stress induced by the production of reactive oxygen species (ROS) during activation of polymorphonuclear leukocytes and macrophages [1] may play a critical role. In vitro experiments [2] have demonstrated that ROS can specifically activate the transcription factor nuclear factor (NF)-κB to induce the expression and replication of HIV [2,3]. Addition of antioxidant vitamins to the system blocked this activation and inhibited HIV replication [4–6].

Several studies have now documented an excessive production of ROS in the HIV-infected population, regardless of the extent of their immunosuppression, based on measurements of lipid peroxidation indices in plasma and expired breath [7–13]. This increase in lipid peroxidation may be moderated by a normal antioxidant defense system that scavenges the ROS. The latter depends on the integrity of an enzymatic system that requires adequate intake of trace minerals such as selenium, copper, zinc, and manganese, and on the presence of adequate levels of vitamin E, C, A and carotenoids in the cytoplasm and lipid membrane of the cells. Previous studies have shown that patients with HIV infection may have deficiencies in many of these components, including selenium [11,14], vitamin A [15], E [16], C [17] and carotenoids [11].

Of these antioxidants, α-tocopherol (vitamin E) is the most potent and most abundant lipophilic antioxidant in vivo [18] as well as an immunoenhancer [19]. Vitamin C is the major water-soluble antioxidant and acts as the first defense against ROS in whole blood [20] and plasma [21]. In addition, a cooperative interaction exists between the two vitamins, vitamin C being important in regenerating vitamin E during the antioxidant defense process [22].

Large sums of money are spent by the HIV-infected population on various vitamin and mineral supplements each year but no clinical trials have rigorously investigated the effect of supplementation of these antioxidant vitamins in the HIV-infected population. In this study, we hypothesized that supplementation of vitamin E (DL-α-tocopherol acetate) and vitamin C will result in a decrease in oxidative stress, which could have clinical implications on the course of HIV.

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Subjects and methods

This study is a randomized double-blind placebo-controlled trial. Stable HIV-positive subjects from amongst those of participating physicians were approached for the study between April 1995 and August 1996. Patients could be on any combination of antiviral therapy providing that any therapy was started 4 weeks prior to study entry and remained stable for the duration of the study. This trial was conducted before the widespread use of combination therapies and protease inhibitors. Subjects were eligible if they had no active opportunistic infection. Exclusion criteria were as follows: smoking, initiation of antioxidant vitamin therapy prior to the study, hyperlipidemia, diabetes, kidney/liver dysfunction, intractable diarrhea (at least six liquid stools daily), vomiting or evidence of gastrointestinal bleeding. Patients underwent an initial screening, which included history (medical, smoking, diet, alcohol and supplemental vitamin intake), anthropometric data (weight, height), and review of biochemical results (complete blood count, glucose, creatinine, urea, liver enzymes). Of 62 potentially eligible patients who were screened, nine patients did not want to participate and four were not recommended by their physicians because of poor drug compliance. Forty-nine subjects were enrolled and 40 completed the study. Amongst the nine who did not complete the study, three were randomized to the vitamin group (two had epigastric discomfort, one was unable to keep the 3-month follow-up appointment) and six were randomized to the placebo group (all were unable to keep their 3-month follow-up appointment). All nine patients had baseline and follow-up measurements (month 1 or 2 of the study) performed for all the parameters and were thus included in the statistical analysis on an intention-to-treat basis.

Patients enrolled were placed on a controlled diet that provided a polyunsaturated to saturated fatty acid ratio of 0.3 : 1 for 2 weeks prior to randomization and continued during the study period. They also received dietary counselling to maximize and standardize their intake of food rich in vitamins. Patients were randomly assigned (using a random number table) to receive daily either 800 IU DL-α-tocopherol acetate (vitamin E, two capsules) and ascorbic acid (vitamin C) 1000 mg daily (two tablets), or matched placebo (two capsules or two tablets), distributed by an independent nurse. The placebo for vitamin E contained soybean oil and the placebo for vitamin C contained dextrose. Both supplements and placebo were coded, had the same taste, appeared identical and were prepared specifically for this study (Hoffman-LaRoche, Nutley, New Jersey, USA). The code was not broken until all patients completed the therapy and all the analysis were performed. Therefore, subjects, observers and laboratory personnel were unaware of the nature of the supplement given. Compliance with the supplement or placebo intake was verified by counting leftover medication and by measuring plasma vitamin concentrations. Subjects were advised to continue their normal activity and to report any unusual symptoms. Patients were required to record their food intake for 3 days (2 week days and 1 weekend day) at the beginning and the end of the study, including all food and beverages consumed, their portion size and method of preparation. Patients were assessed at baseline and at 3 months. At each study visit, anthropometry, review of food records and laboratory tests were also performed.

Diagnosis of HIV-associated opportunistic and other infections were reported in the chart by physicians during the 6 months after randomization. Data were collected by chart review at the end of the study. A 6-month period was chosen because of a possible carryover effect from the vitamins. The sample size chosen was inadequate to demonstrate differences in the number of infections between groups. The recording was performed for the purpose of future clinical trials.

For vitamin levels, lipid peroxides, malondialdehyde (MDA) and viral load, blood was collected in EDTA, and for zinc and selenium, in trace-element-free tubes. The samples were put in ice and centrifuged promptly at 2400 r.p.m. at 4°C for 10 min. The plasma was removed and frozen at −70°C until analysis. Plasma for vitamin C assays was stabilized immediately with 100 g/l metaphosphoric acid (HPO3; 2.0 ml plasma plus 2.0 ml HPO3). The food records were analyzed using a nutrient database (The Minnesota Nutrition Data System, Version 2.7, Minneapolis, Minnesota, USA).

Written informed consent was obtained from all participants. The study protocol was approved by the Toronto Hospital Committee for Research on Human Subjects.

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Laboratory analyses

Breath alkane output

Breath analysis was performed as described previously [23]. Briefly, subjects were first required to breathe hydrocarbon-free air for 4 min to wash contaminating hydrocarbons from their lungs. Subsequently, expired air was collected for 2 min and analyzed by gas chromatography (Shimadzu 6-AM GC, Shimadzu Seisgkusho, Kyoto, Japan). Pentane was analyzed on a Porasil D column (Chromatographic Specialties, Inc., Brockville, Ontario, Canada) using a calibration curve derived from known concentrations of the gases. The concentration of breath pentane was expressed in pmol/kg/min.

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Lipid peroxide determination

Plasma lipid peroxides were determined using the Kamiya Biochemicals LPO kit (Thousand Oaks, California, USA). In this procedure, hemoglobin catalyses the reaction of hydroperoxides with a methylene blue derivative forming an equimolar concentration of methylene blue. Lipid peroxides are quantified by calorimetry at 675 nm and methylene blue formation is measured. For plasma high performance liquid chromatography (HPLC)-separated MDA determination, the method described by Draper [24] was used.

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Vitamin and trace element determination

Retinol and other carotenoids including β-carotene were analyzed by HPLC according to the method of Sapuntzakis et al. [25]. In this method, a reverse-phase C18 column was used with an isocratic solvent system (methanol–acetonitrile–tetrahydrofuran, 50 : 45 : 5 vol/vol/vol) following a hexane extraction using 200 μl serum sample. α- and γ-tocopherol were analyzed using an isocratic reverse-phase HPLC and fluorescence spectrophotometry at 294 nm according to the method of Nata et al. [26].

Samples were analyzed for vitamin C by spectrophotometry method [27]. In this method, total biologically active vitamin C concentrations were determined spectrophotometrically at 521 nm using 2,4-dinitrophenyl–hydrazine as chromogen.

Plasma zinc was analyzed using atomic absorption spectrophotometer (Varian Techtron Model 1200, Varian Associates Canada Ltd, Malton, Ontario, Canada) by the method described by Wolman et al. [28]. Plasma selenium was measured using atomic absorption spectrophotometry at 196 nm [29]. In this method, nickel salt was added as a matrix modifier to prevent volatilization of selenium during ashing.

Plasma HIV viral load was assessed using a standardized PCR kit for RNA with all reagents provided by Roche Molecular Systems (Mississauga, Ontario, Canada) [30].

Data were expressed as means (SEM). Statistical significance was defined as P < 0.05. The primary objective was to compare the change in measurements between 0 and 3 months, between the placebo and vitamin-supplemented groups, using unpaired t-test for continuous variables. Linear regression was performed to analyze the association between viral load at baseline versus the change after 3 months. Analysis were performed on an intention-to-treat basis. It was estimated that 20 patients in each group would be required to detect a difference of 20% in breath alkane output, with 90% power using a 5% significance level (two-sided). SAS software was used for the analysis [31].

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Results

The baseline characteristics of the patients are outlined in Table 1. There were no significant differences between the groups at baseline with respect to demography, CD4 cell counts, dietary intake and number of patients on various reverse transcriptase inhibitors. In the supplement group, four patients were on no anti-retrovirals, two were on monotherapy, and 20 were on a combination therapy. In the placebo group, four were on no antiretrovirals, one was on monotherapy, and 17 were on combination therapy. None of the patients received protease inhibitors during the study period. Twenty-three patients (22 men, one woman) were randomized to the placebo group and 26 (25 men, one woman) to the supplemented group. Compliance was excellent on the basis of the number of pills returned and was similar between groups (vitamin E, 93.5 ± 2.2%; vitamin C, 93.6 ± 2.2%; placebo E, 95.8 ± 2.2%; placebo C, 96.0 ± 2.2%).

Table 1

Table 1

During the study, there was no significant change in body mass index or dietary intake in either group. There was a significant increase in plasma vitamin E and C concentrations in the supplemented group compared with the placebo group (Table 2) as expected. Plasma retinol, carotenoids, zinc and selenium remained unchanged during the study, suggesting that other supplementation was not occurring.

Table 2

Table 2

Lipid peroxidation measured by breath pentane output, plasma MDA and lipid peroxides was not significantly different between the two groups at baseline, although there was a trend for the placebo group to have a higher breath pentane output and a lower MDA value (Table 3). There was a significant decrease in these measurements in the vitamin-supplemented group at 3 months when compared with the placebo group.

Table 3

Table 3

Plasma HIV viral load (Table 3) was similar at baseline between the two groups. The change after 3 months of supplementation was observed to be more favorable in the vitamin-supplemented group with a mean decrease of 0.45 ± 0.39 log10 copies/ml compared with a mean increase of 0.50 ± 0.40 log10 copies/ml for the placebo group (P = 0.1; 95% confidence interval, −0.21 to −2.14). Linear regression analysis used to evaluate the association between viral load at baseline and the change after 3 months was significant (P = 0.026) indicating that the change after 3 months depended on the initial score.

There was no relationship between baseline plasma vitamin E or C concentrations and viral load and the response did not differ between those who had lower concentrations of antioxidant vitamins compared with those who had higher concentrations. There was no statistically significant difference between the groups with regard to new AIDS-defining opportunistic infections, HIV-associated or other infections (Table 4).

Table 4

Table 4

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Discussion

This study is the first randomized controlled trial to demonstrate that, in an HIV-positive population, daily supplementation of 800 IU vitamin E and 1000 mg vitamin C significantly decreases oxidative stress and produces a trend towards a reduction in HIV viral load.

HIV-infected populations have been shown to be oxidatively stressed and to have significantly lower plasma antioxidant micronutrient concentrations such as vitamin C, vitamin E, β-carotene and selenium than seronegative controls [11]. In the present study, the plasma concentrations of these antioxidant vitamins were also low at baseline. Others have also reported reduced plasma selenium [32,33], vitamin E [16] and vitamin C concentrations [17]. It was therefore appropriate to test the effect of vitamin E and C supplementation in this population. Both asymptomatic and AIDS patients were included because in a previous study we did not detect a difference in lipid peroxidation or micronutrient deficiencies between these two subsets [11]. During the study period, antiviral drugs were kept stable and thus did not confound with the observed results.

At baseline, both groups were similar, although there was a trend towards a higher breath pentane and lower plasma MDA values in the placebo group. For breath pentane, we interpreted this as a bias against our results. In our previous studies [34,35], we found that the response to antioxidant supplementation was usually higher in those who had higher values of pentane at baseline. Therefore, in the vitamin-supplemented group, the effect expected should have been smaller, although it remained significant.

The reason for the lower plasma MDA values at baseline in the placebo group is unclear. Values in both groups were lower than those reported previously [8,12,13], probably because the method we used was more specific [24]. Other antioxidant micronutrients were measured in the plasma and found to be stable during the study. This excluded the effect of sudden dietary changes or use of other antioxidant supplements on our results.

The dose of vitamins used in the study was based mostly on our previous experience with smokers, a population also known to be oxidatively stressed. In these subjects, vitamin E 800 IU per day was found to significantly decrease lipid peroxidation [35]. In contrast, 500 mg vitamin C per day produced a significant effect only in smokers of more than 15 pack-years (unpublished). Observational studies with vitamin C over 750 mg and vitamin E 130 IU per day have also suggested a beneficial effect on risk of AIDS or disease progression [36,37]. Therefore, the dose of vitamins used in this study seemed appropriate. We also decided to use the combination of vitamin E and C because vitamin C regenerates vitamin E during the antioxidant defense process [22].

During this study, no concurrent multivitamin supplements were used in order to try to minimize confounding effect in the treatment or control arm, as previously reported [38]. Patients were monitored with food records and their intake of micronutrients met or exceeded the recommended daily allowances (vitamin E, 9–10 mg; vitamin C, 40–60 mg). In addition, at the beginning of the study, subjects received dietary counselling to maximize and standardize their intake of vitamins from food sources.

The methods of measuring lipid peroxidation are well recognized. For MDA determination, we used an HPLC procedure [24] that resolves the problem of specificity associated with the spectrophotometric method but markedly enhanced the sensitivity. Breath pentane output was used by our group in previous studies [34,35] and evolves from the peroxidation of ω-6 fatty acids. The volatile hydrocarbon gases are produced by the β-scission of polyunsaturated fatty acids and are passed from the lungs into the expired air [23]. In human studies, the measurement of these alkanes in the breath is non-invasive and has been used and validated as a measure of lipid peroxidation [23,39,40]. Because the intake of polyunsaturated fatty acids can influence lipid peroxidation [41], the subjects were given instructions about their dietary fat intake 2 weeks prior to the measurements. Smoking [42], liver disease [43] and alcohol consumption [44] can also affect these measurements. The subjects were screened prior to be enrolled and excluded if these confounders were present.

Increased oxidative stress found in HIV populations [7–10] may have some clinical significance. In vitro evidence implicates oxidative stress in the stimulation of HIV replication through activation of NF-κB to induce the expression and replication of HIV-1 in a human T-cell line. Addition of antioxidant vitamins blocked activation of NF-κB and inhibited HIV replication [4–6]. Similar effects may occur in vivo, as demonstrated by the trend towards a reduction in HIV viral load that was observed with vitamin supplementation. Potential synergistic activity between combination of antiviral therapy and antioxidant vitamin supplementation should be explored.

This study was unable to demonstrate any difference in the number of AIDS-defining, HIV-associated or nonassociated infections. However, we feel that this could be further investigated, especially since vitamin E is known to be an immunoenhancer and can improve immune function in other immunosuppressed populations [19].

There have been no other clinical trials using a combination of vitamin E and C or other antioxidant micronutrients in HIV populations. Trials with β-carotene studying the effect on immune functions were initially promising [45,46] but a more recent study was negative [38]. This negative result may have been attributed to the use of multivitamins in both arms. A non-randomized trial [47] performed in HIV-infected patients showed no significant effect from selenium and β-carotene supplementation on superoxide dismutase activity, an antioxidant enzyme, compared with baseline; but another enzyme playing a central role in ROS metabolism, glutathione peroxidase activity, increased. No clinical outcomes were measured.

The results of this study may have some implications for maternal and child health [48–50], especially in developing countries [51], since increased oxidative stress has been associated with adverse pregnancy and birth outcomes [48,49]. Furthermore, the effect of antioxidant supplementation on mother-to-infant transmission could be investigated since, in the present study, viral load reduction was similar to that seen with zidovudine, a drug known to reduce HIV transmission from mother to infant [50]. Finally, HIV-infected smokers may benefit even more from antioxidant supplementation since smoking alone has been associated with an increase in ROS production and a decrease in antioxidant plasma concentrations [34,35].

In conclusion, this study showed that vitamin E and C supplementation significantly decreases oxidative stress in HIV-infected individuals. Furthermore, with this vitamin supplementation there was a trend towards a reduction in viral load suggesting that there may be some clinical benefit worthy of larger clinical trials. Since combination antiretroviral therapies containing protease inhibitors are limited for economic reasons to only about 10% of HIV-infected individuals in the world, consideration of the potential for this antioxidant therapy remains important for the developing world [52]. It could have great benefit, perhaps similar to the effect of vitamin A supplementation on childhood mortality in developing countries [52,53].

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Keywords:

Lipid peroxidation; antioxidants; vitamins; α-tocopherol; ascorbic acid; HIV; AIDS; viral load

© 1998 Lippincott Williams & Wilkins, Inc.