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Journal of the American College of Nutrition, Vol. 17, No. 3, 244-249 (1998)
Published by the American College of Nutrition

Original Paper

Effects of Supplementation with a Combination of Antioxidant Vitamins and Trace Elements, at Nutritional Doses, on Biochemical Indicators and Markers of the Antioxidant System in Adult Subjects

Paul Preziosi, MD, Pilar Galan, MD, PhD, Bernard Herbeth, PhD, Pierre Valeix, PhD, Anne-Marie Roussel, PhD, Denis Malvy, MD, PhD, Agnes Paul-Dauphin, MD, Josiane Arnaud, PhD, Marie-Jeanne Richard, PhD, Serge Briancon, MD, Alain Favier, PhD and Serge Hercberg, MD, PhD

Institut Scientifique et Technique de la Nutrition et l’Alimentation, Conservatoire National des Arts et MáAaetiers, (P.P., P.G., P.V., S.H.), CHRU de Nancy, FRANCE
Paris; Centre de MáAaedecine PráAaeventive de Vandoeuvre-les-Nancy (B.H.), CHRU de Nancy, FRANCE
Laboratoire de Biochimie (A-M.R., J.A., M-J.R., A.F.), CHRU de Nancy, FRANCE
CHRU de Grenoble; INSERM U330 (D.M.), CHRU de Nancy, FRANCE
UniversitáAae de Bordeaux 2; and Ecole de SantáAae Publique (A.P-D., S.B.), CHRU de Nancy, FRANCE

Address reprint requests to: Serge Hercberg, MD, PhD, ISTNA/CNAM, 2 rue ContáAae, F-75003 Paris, FRANCE


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE:: To test the impact of supplementation with nutritional doses of antioxidant nutrients on biochemical indicators of vitamin and trace element levels.

DESIGN:: A randomized double-blind trial was performed comparing two groups receiving daily either a combination of vitamins (beta-carotene, 6 mg; vitamin C, 120 mg; and vitamin E, 30 mg) and trace elements (zinc, 20 mg; and selenium, 100 µg); or a placebo.

SUBJECTS:: 401 subjects (166 males aged 45 to 60 years and 235 females aged 35 to 60 years).

MEASURE OF OUTCOME:: Biological markers of vitamin and trace element status and free radical parameters were measured initially, 3 months, and 6 months after supplemention.

RESULTS:: Mean serum concentrations of alpha-tocopherol, vitamin C, beta-carotene, zinc and selenium increased significantly after 3 months of supplementation in the group receiving multivitamins associated with minerals. At baseline, 18.2% of the men and 5.1% of the women had low concentrations of serum vitamin C (<20 µmol/l); 2.4% of the men and 17% of the women presented low concentrations of serum retinol (<1.4 µmol/l); 18.7% of men and 10% of women had serum beta-carotene <0.30 µmol/l. None of the study subjects had serum alpha-tocopherol concentrations below the limit cut-off point (<9.3 µmol/l). Low serum zinc concentrations (<10.7 µmol/l) were found in 15.1% of men and 23.8% of women. Low serum selenium concentrations (<0.75 µmol/l) were found in 6% of men and 6.4% of women. A significant increase in plasma and red cell GPx activity was observed in groups receiving supplementation. No modifications were observed after 6 months of supplementation for malondyaldehyde.

CONCLUSION:: This study demonstrates the efficacy of an intake of antioxidant vitamins and trace elements, given at nutritional doses, on biochemical indicators of vitamin and trace elements status.

Key words: vitamin C, vitamin E, beta-carotene, selenium, zinc, antioxidants, healthy subjects


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the last 20 years, many basic, clinical and epidemiological research has suggested a potential protective effect of antioxidant nutrients such as beta-carotene, vitamin C, vitamin E, selenium and zinc on the risk of cancer and cardiovascular diseases [1–5]. Data provided by cross-sectional, case-control and prospective epidemiological studies raise strong supportive arguments for the relationship between the intake of antioxidant vitamins and trace elements and the risk of pathologies [6–9]. However, the observational nature of these studies does not permit the establishment of causal relationships. Results of several randomized placebo-controlled trials recently published appear to be conflicting [10–13]. Some of these intervention studies have reported, in high risk populations (smokers or asbestos-exposed workers) receiving high doses of antioxidants, higher relative risk of some pathologies [11,12]. In these studies with a negative effect, the supplementation was responsible, after several months, for a marked increase in blood concentrations of indicators of vitamin status.

Thus it is advantageous to develop randomized, controlled trials in the general population which use a combination of nutrients at levels similar to those contained in a healthy diet and associated in observational studies, with the lowest risk of disease.

The objective of the present study is to assess, in subjects with slight-to moderate micronutrient deficiencies (as frequently observed in populations of developed countries), the impact of supplementation with antioxidant nutrients on biochemical indicators of vitamin and trace element levels to ensure that doses were adequate for correcting potential deficiencies, but not high enough to result in abnormally high concentrations of blood indicators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Population
A brief media campaign was organized to recruit volunteers for this study. Four hundred and one subjects (166 males aged 45 to 60 years and 235 females aged 35 to 60 years) were recruited for this test. To be eligible, the subjects had to declare themselves free of any severe pathology and not be taking supplements containing any of the studied vitamins or minerals. They were randomly selected in four French urban areas (Paris, n=149; Grenoble, n=107; Nancy, n=88 and Tours, n=57).

The research protocol was approved by the Committee on Human Experimentation of the Cochin Hospital of Paris. The study had a double-blind, placebo-controlled design. Participants were stratified by sex and age and randomly assigned to one of the two treatment groups using block randomization. They received one capsule per day for a period of 6 months.

Group S received 120 mg of ascorbic acid, 6 mg of beta-carotene (1000 retinol equivalents) and 30 mg of alpha tocopherol; 20 mg of zinc (as gluconate) and 100 µg of selenium (as yeast). Group P received the placebo (P group). Supplements and placebo appeared identical and were prepared especially for this study.

Biological Measurements and Laboratory Procedures
Biological indicators of vitamin and trace element status and markers of oxidative stress and defenses against oxidative stress were measured initially and at 3 and 6 months after the beginning of the supplementation. For each measurement, 35 ml of whole blood was withdrawn by venipuncture between 7 and 11 a.m. from fasting subjects.

Vitamin Determination.
Vitamin C status was evaluated by serum ascorbic acid determination using an automated method based on the continuous flow principle, segmented with air bubbles [14]. Serum retinol was measured by HPLC with normal phase (silicagel), isocratic elution with n-hexane/isopropanol (970:30) and detection by UV at 330 nm. Serum beta-carotene was measured by normal phase HPLC on Silicagel, isocratic elution with n-hexane/dioxane (990:10) and detection in visible light at 436 nm. Serum tocopherol was measured by normal phase HPLC on Silicagel, isocratic elution with n-hexane/ethyl acetate (930:70) and fluorescence detection with excitation at 298 nm and emission at 328 nm [15].

Selenium and Zinc Determination.
Blood was collected in trace element free BD vacutainer tubes (Becton & Dickinson, Meylan, France). Aliquots of serum were transferred to polystyrene tubes (CML, Nemours, France) using a polyethylene transfer pipet (Becton &Dickinson, Pont de Claix, France). Serum zinc concentrations were determined [16] by flame atomic absorption spectrometry (Perkin Elmer 460, Norwalk, CT). Serum selenium concentrations were determined on a Perkin Elmer 5100 (Norwalk, CT) equipped with an HGA 600 furnace, an EDL lamp and Zeeman background correction [17].

Red Blood Cell Selenium Dependent Glutathione Peroxidase (GPx), Superoxide Dismutase (SOD), Total (GSHT) and Oxidized Glutathione and Malondialdehyde (MDA) Measurements.
GPx was measured by a modified method of Gunzler et al [18] using tert-butyl hydroperoxide (Sigma Chemical Co, France) as substrate instead of hydrogen peroxide; results were expressed as µmol of NADPH (Boehringer-Mannhein, Germany) oxidized per minute per g of hemoglobin. Erythrocyte Cu-Zn SOD activity was measured after hemoglobin precipitation by monitoring the autoxidation of pyrogallol according to the technique described by Marklund and Marklund [19]. GPx and SOD assay were adapted to the Hitachi 704 automated analyser (Boehringer Mannheim, Meylan, France). For whole blood reduced (GSH) and oxidized (GSSG) glutathione determination, just after venipuncture, 400 µl of whole blood were transferred to a tube containing 3600 µl 6% (v/v) of metaphosphoric acid in water. Total glutathione (GSH+GSSG) was determined by a modified method of et al [20] in the acidic protein-free supernatant. An aliquot of the deproteinized extract was neutralized with a solution containing 0.4 M N-morpholoinopropanesulfonic acid and 2 mM EDTA adjusted to pH 6.75 with KOH 1M. Glutathione was determined using enzymatic cycling of GSH by means of NADPH and glutathione reductase coupled with DTNB. To assay oxidized glutathione, GSH was masked by adding 10 µl of 2-vinyl-pyridine to 500 µl of deproteinized extract adjusted to pH 6 with triethanolamine. Plasma lipid peroxidation was determined by measuring thiobarbituric acid reactants (TBARs) and expressed as µmol of malondialdehyde. We used the Sobioda MDA kit (Grenoble, France), as described by Richard et al [21].

Statistical Analysis
Data were collected on an Apha-VMS system using SAS for statistical analysis. Since some variable distributions were markedly positively skewed, logarithm transformation of these variables was used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Table 1 summarizes the biological characteristics of the subjects upon admission to the study. Serum concentrations of beta-carotene and vitamin C were significantly higher in women than in men; conversely, serum retinol concentrations were lower in women. No difference was found between men and women for plasma vitamin E, zinc and selenium concentrations, nor for red blood GPx, SOD, GSHT, GSSG and MDA.


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  		Table 1. Serum Concentrations (Mean±SD) of Vitamins and Trace Elements and Markers of Oxidative Stress and Defenses Against Oxidative Stress in Men and Women

 
At baseline, 18.2% of the men and 5.1% of the women had low concentrations of serum vitamin C (<20 µmol/l); 8.5% of men and 1.3% of women had particularly low serum vitamin C <2 µmol/l; 2.4% of men and 17% of women presented low concentrations of serum retinol (<1.4 µmol/l); 18.7% of men and 10% of women had serum beta-carotene <0.30 µmol/l. None of the study subjects had a serum alpha-tocopherol concentration below the limit cut-off point (<9.3 µmol/l). Low serum zinc concentrations (<10.7 µmol/l) were found in 15.1% of men and 23.8% of women. Low serum selenium concentrations (<0.75 µmol/l) were found in 6% of men and 6.4% of women.

Table 2 shows laboratory variables before and after 3 and 6 months of supplementation in the supplemented group and in the placebo group. At baseline, mean values and prevalences of low concentrations for biochemical markers of vitamin and trace element status and indicators of oxidative stress and antioxidant enzymes did not differ between the two groups for any of the parameters tested.


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  		Table 2. Comparison of Serum Concentrations (Mean±SD) for Vitamin and Trace Elements and Markers of Oxidative Stress and Defenses Against Oxidative Stress in Supplemented and Placebo Groups at Baseline and after 3 and 6 Months

 
After 3 months, serum concentrations were significantly higher in the supplemented group for beta-carotene, vitamins C and E, zinc and selenium. This difference between the two groups persisted after 6 months of supplementation, but serum concentrations did not differ in the supplemented group between 3 and 5 months. Supplementation did not have any effect on retinol concentrations at 3 or 6 months.

Compared to baseline, plasmatic GPx activity was significantly higher in the supplemented group at 3 and 6 months, as were red cell GPx activity at 6 months (Table 3). No significant difference existed at 3 and 6 months for concentrations of SOD, GSH, GSSG, GSHT and MDA. The percentage of subjects with low serum concentrations decreased in the group receiving supplementation (Fig. 1): from 12% to 1% for beta-carotene; from 10% to 1% for vitamin C; from 2.5% to 0% for selenium; and from 18 to 9% for zinc.


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  		Table 3. Effect of Supplementation on Plasmatic and Red Cell GPx

 


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  		Fig. 1. Frequency of low values for serum concentrations of vitamin C, beta-carotene, zinc and selenium at inclusion (BASELINE) and after 3 months (T3) and 6 months (T6).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, mean values observed for initial serum vitamin and trace element concentrations were similar to those previously described for French adult populations [2223]. The effect of supplementation of antioxidant micronutrients during a 6-month period on serum concentrations of biochemical markers assessing vitamin and trace element status was significant for all of the studied nutrients, vitamin C, E, beta-carotene, zinc and selenium. We did not find any low concentrations after 6 months of supplementation with the tested doses of vitamins and trace elements. The effectiveness of supplementation began at 3 months and persisted through 6 months. It is important to note that these positive biological effects were observed with daily multi-vitamin and mineral supplements containing one to two times the recommended dietary allowances. These doses corresponded to levels of dietary antioxidant micronutrients associated, in observational studies, with the lowest risk of cancer and cardiovascular diseases.

Moreover, two intervention studies recently developed in Finland (ATBC [11]) and in the USA (CARET [12]) have shown an apparent negative effect of beta-carotene supplementation on the lung cancer incidence rate in high-risk subjects. In these two studies of men with a high risk of lung cancer (heavy smokers and/or asbestos-exposed workers), initial serum beta-carotene medians were similar (0.32 µmol/l) and much lower than those in our study (0.59 µmol/l) in men from our sample. Our values are consistent with those observed in the cohort of subjects participating in the Physician Health Study [24], in whom no positive or negative effects were observed after supplementation for 12 years with relatively high doses of beta-carotene.

In CARET, as in ATBC, the relatively high levels of supplementation were associated with a substantial increase in the plasma concentrations of beta-carotene. For example, the initial concentrations of plasma carotene were multiplied by 18 in ATBC and by 12 in CARET. The final concentrations were higher than those associated, in observational studies, with the lower risk of diseases. They were also higher than those observed in our study. Conversely, in the Physician Health Study, initial values were multiplied only by four, reaching 2.25 µmol/l compared to 1.84 µmol/l in our study. The latter is very close to those described in the only trial showing a positive effect of supplementation on mortality and cancer incidence. That study, carried out for more than 5 years in China [24], provided nutritional doses of beta-carotene (associated with selenium and vitamin E). Our data thus indicate that short-term supplementation (6 months), with moderate doses of antioxidant vitamins and trace elements in presumably healthy subjects, clearly though moderately alter vitamin and mineral status so that blood levels reach concentrations consistent with a positive effect.

Concerning the effect of antioxidant vitamin and trace element supplementation on indicators of peroxidation, we did not find any effect on plasma malondialdehyde. However, we observed a significant increase in plasmatic and red cell GPx in the groups receiving trace elements associated with vitamins. This improvement may be related to the selenium contained in supplementation, given the fact that GPx is a seleno-dependent enzyme.

This study demonstrates the efficacy of an intake of antioxidant vitamins and trace elements, given at nutritional doses, on biochemical indicators of vitamin and trace element status. Supplementation quickly improved these biochemical indicators, but after 6 months, they remained at a reasonable level without reaching concentrations as high as those observed in the Finnish and American intervention studies, which tested relatively high doses of antioxidants, and ended up with higher risk of pathology.

Received June 1, 1997. Accepted October 1, 1997.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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