Journal of the American College of Nutrition, Vol. 22, No. 3, 232-238 (2003)
Published by the American College of Nutrition
Biochemical Antioxidant Levels Respond to Supplementation with an Enriched Drink in Frail Elderly People
Wendeline Wouters-Wesseling, PhD,
Lisette W. Wagenaar, MSc,
Lisette C.P.G.M. de Groot, PhD,
Jacques G. Bindels, PhD and
Wija A. van Staveren, PhD
Numico Research B.V., Department of Clinical Nutrition and Diets (W.W.-W., L.W.W., J.G.B.), Wageningen University, Wageningen, The Netherlands
Department of Human Nutrition and Epidemiology, Wageningen University (L.C.P.G.M.d.G., J.G.B., W.A.v.S.), Wageningen, The Netherlands
Address correspondence to: Wendeline Wouters-Wesseling, PhD, Numico Research B.V., P.O. Box 7005, 6700 CA Wageningen, THE NETHERLANDS. E-mail: Wendeline.Wouters-Wesseling{at}numico-research.nl
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ABSTRACT
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Objective: To investigate whether a drink enriched with essential vitamins and minerals can improve biochemical status of enzymatic and non-enzymatic antioxidants in frail elderly people.
Methods: A six-month randomized, double blind, placebo controlled intervention study. Frail elderly people 65 years of age or older, with a body mass index (BMI) below 25 kg/m2 and residing in a home for the elderly or in sheltered housing. Enriched (with essential vitamins and minerals in 30% to 150% of RDA and higher levels of antioxidants) drink (n = 28) or placebo (n = 27) to be taken twice a day in addition to the normal food consumed. Plasma levels of vitamin C, vitamin E, antioxidant capacity (TEAC), cysteine, uric acid and whole blood levels of total thiol and glutathione peroxidase (GSH-Px), dietary intake.
Results: Changes in vitamin E (16 ± 2 vs. 2 ± 1 mmol/L), vitamin C (37 ± 5 vs. 1 ± 5 mmol/L), TEAC (38 ± 15 vs. -10 ± 11 mmol/L Trolox eq) and cysteine (17 ± 10 vs. 0.4 ± 6 mmol/L) were significantly different between groups (p < 0.05). There was a trend towards significant changes in erythrocyte glutathione peroxidase (-0.2 ± 3 vs. -10 ± 7 U/mg Hb, p = 0.097). Baseline dietary intake of antioxidant vitamins was below 2/3 RDA for a substantial proportion (43% to 76%) of subjects.
Conclusions: Supplementation with an enriched drink can raise plasma levels of enzymatic and non-enzymatic antioxidants in frail elderly people.
Key words: elderly people, nutrition, antioxidant, supplementation
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INTRODUCTION
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According to the free radical theory of aging, free radicals play an important role in the aging process and contribute to many common diseases [1]. Data provided by cross-sectional, case-control and prospective epidemiological studies raise supportive arguments for the relation between the intake of antioxidant vitamins and trace elements, and the risk of pathologies [2–5]. Therefore, controlling free radicals may be important for health maintenance in elderly people.
The antioxidant defense system comprises a number of interconnected, overlapping components that include both enzymatic and non-enzymatic factors. Vitamin E, the major lipid-soluble antioxidant, protects against lipid peroxidation. Vitamin C can quench free radicals as well as singlet oxygen and can also regenerate the reduced antioxidant form of vitamin E. Together with uric acid, carotenoids, flavonoids and ubiquinol, these antioxidants make up the total antioxidative capacity (TEAC) in plasma. Some metals (zinc, copper, manganese, selenium) exert their actions as antioxidants via their incorporation into specific enzymes, e.g. superoxide dismutase (SOD), which catalyzes dismutation of the superoxide anion into hydrogen peroxide, and glutathione peroxidase (GSH-Px), which detoxifies hydrogen peroxide and converts lipid hydroperoxides into nontoxic alcohols. Glutathione assists in the synthesis of protein and DNA, the maintenance of intracellular thiol groups, the enzymatic reduction of dehydroascorbate, the transport of amino acids into cells and the elimination of toxic compounds [6]. Cysteine is a precursor for glutathione formation.
The dietary intake of the antioxidant micronutrients can modulate the activity of the defense system and thus impact on the degree of protection provided to the cell or tissue against oxidative reactions. The intake of the non-enzymatic antioxidants vitamin C and sometimes vitamin E is lower in elderly people than in younger adults [7], and plasma levels are low [7–9]. The most frequently investigated enzymatic antioxidant in elderly people is GSH-Px. Selenium is an important component of this enzyme, and plasma selenium status is reduced with aging [10] [11]. It seems that in elderly people the GSH-Px status is largely related to their health status [7,12–16], potentially through a relation with selenium status [11,17,18]. Also levels of glutathione have been found to be reduced in elderly people [13,19,20] and reduced levels have also been related to presence of illness [20,21].
Several studies investigated the effects of micronutrient supplementation on plasma antioxidants in different groups of elderly people. Some studied plasma levels of vitamin E and/or C and observed an increase after supplementation with these vitamins [9,22–26]. Some studies also investigated the effect of a selenium supplement on glutathione peroxidase, mostly with positive results [10,23,25,26]. Only one study by Galan et al. [25] investigated the whole range of enzymatic and non-enzymatic antioxidants in the same study upon supplementation with a selection of vitamins and/or trace elements in hospitalized elderly people. They found increased levels of non-enzymatic antioxidants upon supplementation with vitamins and positive effects of trace element supplementation on GSH-Px levels.
Thus, the literature regarding the effects of supplementation of elderly people with a complete range of antioxidants on both enzymatic and non-enzymatic antioxidant levels is scarce. We therefore investigated whether supplementation with an enriched drink can improve biochemical status of enzymatic and non-enzymatic antioxidants in frail elderly people.
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MATERIALS AND METHODS
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Subjects
Frail elderly people 65 years of age or older were selected based on a body mass index (BMI) of less than 25 kg/m2 and residency in a home for the elderly or sheltered housing. Subjects were not eligible for the study when they had diagnosed cancer or chronic gastrointestinal disorders (Crohn’s disease, colitis ulcerosa, stoma), when they consumed diets that were not compatible with supplementation or when they were mentally unable to answer the study questions or to remember taking the supplement. The study protocol was approved by the Ethics Review Committee of Wageningen University and written informed consent was obtained before randomization.
Intervention
A randomized, double blind, placebo controlled intervention study was performed. At enrollment, subjects were stratified based on their BMI to receive a placebo or enriched drink. The enriched and placebo drinks were provided in two flavors in 125-mL tetrapacks. The drinks had to be taken in addition to the normal food consumed, two times a day, for six months. The enriched drink contained energy (100 kcal/100 mL), protein (3.5 g/100 mL of which 0.05 g cysteine), carbohydrate (11.4 g/100 mL), fat (4.5 g/100 mL) and micronutrients in amounts of approximately 30% to 150% of US RDA, with higher levels of antioxidants, i.e. vitamin C (225 mg, 375% RDA), vitamin E (55 mg, 550% RDA) and selenium (85 mg, 155% RDA) (Table 1). The placebo contained no energy and consisted of water, sweetener (sodium saccharine/acesulpham-k), cloudifier, thickener, flavoring and colorant. Subjects received the drinks at home during visits every two weeks. Compliance was verified every two weeks as part of home interviews and by counting leftover supplements. Compliance percentage was quantified as (number of supplements provided—number of supplements returned/(number of days participation in study * 2)) * 100%.
Measurements
Anthropometry.
At enrollment into the study, age and gender of participants were registered. After subjects had voided, body weight was measured to the nearest 0.1 kg on a calibrated weighing scale (Seca, Germany). As knee height is more stable than height in the elderly population, it was measured to the nearest 0.1 cm using a stadiometer. Body mass index (BMI) was estimated as weight/(knee height2 * 10) [27].
Dietary Intake.
At baseline a three day (two weekdays and one weekend day) estimated dietary record was filled out by the participants according to the method described by de Jong et al. [28]. This record was checked by a dietitian. Food intake was calculated estimated the amount taken by using standard portion sizes for each food with the computerized Dutch Food Composition Table of 1997 [29]. To calculate the percentage of subjects with inadequate vitamin intake, a cut-off point of 2/3 of US RDA was used (male 60 mg, female 50 mg of vitamin C; 10 mg of vitamin E; male 0.66 mg, female 0.53 mg of vitamin A and 6.6 mg vitamin D, respectively).
Blood Sampling.
Venous blood samples were collected in heparinized tubes at baseline and at the end of the study, in the subjects’ homes after an overnight fast while they were in a sitting position. Samples were cooled immediately after collection and 1 mL of whole blood was taken and stored at -80°C until analysis of total thiol and glutathione peroxidase. The remaining sample was centrifuged within four hours at 2500 g for 10 minutes at 4°C. After centrifugation, the plasma layer was removed and stored at -80°C for analysis of TEAC within one month and cysteine [30] at the end of the study (24 months).
Antioxidant Analysis.
For vitamin E analysis 10 mL of Trolox (12.5 mg/100 mL Trolox [Aldrich; 23,881-3] 25% ethanol solution) and 10 mL 0.05 mM butylated hydroxytoluene (Sigma; B-1378) were added to 0.5 mL plasma and stirred. Vitamin E was measured using a plasma ethanol dilution of 1/10 and a triple hexane extraction by reversed-phase HPLC [31]. For vitamin C and uric acid analysis, 0.5 mL plasma was added to 0.5 mL of a solution of 10 g metaphosporic acid (Merck; 1.00546) and 15.4 mg dithiothreitol (ICN; 1000596) per 100 mL. Samples were centrifuged for five minutes at 3000 g at 4°C. Supernatant was stored at -80°C for analysis within six months. Vitamin C was analyzed by HPLC [32,33] and uric acid according to Margolis & Duewer [34]. Glutathione was measured within one month of sampling in whole blood as total thiol [35]. Glutathione peroxidase was analyzed within one month according to Belsten et al. [36]. To correct for erythrocyte concentration, data for total thiol and glutathione peroxidase are expressed per mg hemoglobin (Hb [37]).
Plasma total antioxidant capacity (TEAC) was measured within one month of sampling [38]. TEAC was expressed as Trolox equivalent antioxidant capacity (units), which is defined as the equivalent antioxidant status of a mM concentration of a water soluble vitamin E analogue (Trolox) solution.
For all analyses the within run variation was <5%.
Statistical Analysis
Normal distribution of variables was tested by the Shapiro-Wilk normality test, using Z scores. Per protocol comparisons between groups were made using Student’s t tests for normally distributed data and using Mann-Whitney U test for data that were not normally distributed. Two-sided p-values less than 0.05 were considered significant for baseline comparisons. One-sided p-values less than 0.05 were considered significant for comparisons of changes (6 months—baseline). All calculations were carried out using the Statistical Package SPSS for Windows, Version 10.0 (SPSS Inc., Chicago, USA).
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RESULTS
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Study Sample
Out of the 104 included subjects, 68 completed the study period, and blood samples to perform analysis of antioxidants were available for 55 participants. Reasons for loss to follow-up were supplement taste (7 vs. 11), illness (3 vs. 2) and effort to participate (5 vs. 5) (placebo vs. supplement group), and for lack of samples were refusal of blood draw or insufficient amount available. At baseline age, BMI, gender distribution and vitamin C level were similar for both the participants that completed the study and the dropouts (data not shown). However, vitamin E levels were significantly lower in the group that completed the study (28.3 ± 8.1 vs. 33.5 ± 9.6 mmol/L, p = 0.014). Baseline characteristics of the participants who completed the study are presented in Table 2. No significant differences were found between the two groups. Average compliance with the supplementation was 96% (range 54% to 130%) and did not differ between supplement and placebo group. Antioxidant intake did not differ between groups, and in both groups a large proportion (43% to 76%) of subjects had calculated intakes below 2/3 of US RDA.
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Table 2. Baseline Characteristics (Mean ± SD) of Frail Elderly Participants of the Nutritional Supplementation Study
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Antioxidant Levels
In Table 3 the changes in non-enzymatic and enzymatic antioxidants after intervention are presented for both groups. None of the subjects had plasma vitamin E levels below normal values, whereas a large proportion (about 40%) had low plasma vitamin C levels. After supplementation, no subjects in the supplemented group had plasma levels below the reference values for vitamin C, whereas in the placebo group a large proportion of deficient subjects remained.
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Table 3. Plasma Antioxidant Levels (Mean ± SD) and Changes (Mean ± SE) after Six Months’ Supplementation in Frail Elderly People
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Changes in vitamin E, vitamin C, TEAC and cysteine were significantly different between groups. There was a trend towards significance in changes in erythrocyte glutathione peroxidase. No difference was observed for uric acid and total thiol.
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DISCUSSION
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In this six-month intervention trial with an enriched drink among frail elderly people, we observed a significant difference in changes in plasma vitamin E, vitamin C, TEAC and cysteine levels in favor of the supplement group.
For our supplementation trial, we aimed to study a group of frail elderly people who are at risk of nutritional deficiencies as they will benefit most from nutritional supplementation. The intake of vitamin E in our study was below RDA for a large proportion of subjects. However, serum levels were adequate, but slightly lower than measured in healthy free living elderly people [14,39]. There may have been a degree of selection bias with regard to lower serum vitamin E levels in those that completed the study; however there were no differences among other parameters for those who completed the study versus those who did not. Vitamin C intake was also low and plasma levels were very low, a little higher than in institutionalized elderly people [9,25], but lower than in apparently healthy free living elderly people [26,39]. From the low levels of vitamin E and C, energy intake and BMI, we conclude we indeed studied a group of frail elderly people that was aimed for.
In order to improve both non-enzymatic and enzymatic antioxidant levels we used a complete supplement containing all essential vitamins and trace elements at moderate doses, with higher levels of antioxidants. The compliance with the supplement and placebo was adequate, indicated by counting the leftover packages and also by the rise in plasma levels for vitamin C and vitamin E. To ensure availability of the product during the study period, subjects received a slightly larger amount of product than required, which explains the presences of compliance rates over 100%. The antioxidants we measured are very commonly used to assess antioxidant status [40]. These have been reported to be reduced in elderly people and reflect the status of essential nutrients as provided by our supplement.
The increase in plasma levels of vitamin E and C has also been reported by other supplementation studies in elderly people living in homes for the elderly [23] or free living elderly people [22,26]. We measured TEAC as an indicator of total antioxidative capacity in the plasma. The majority of the TEAC is composed of uric acid. Uric acid tends to increase with age, which may cover part of the antioxidant deficiency in old age [41]. As expected, but not demonstrated before, the increases in non-enzymatic antioxidant levels and the lack of change in uric acid in our study were reflected in an increase in TEAC and thus may reflect a relevant change. McKay et al. [26] did not find an effect of a multivitamin supplement on oxygen radical absorbance capacity (ORAC) assay. They ascribe the lack of effect to the ORAC assay and to the large contribution of uric acid. However, they did not measure the stability of uric acid over the study period. Another explanation for the lack of change is that they studied free living elderly people who had relatively high vitamin E and C levels at start and who may therefore have had only small increases upon supplementation.
Our enriched drink contained a protein source that was five times higher in cysteine (11 mg/g) than the standard casein (2 mg/g) used in nutritional supplements. This was reflected in the significant increase we found in plasma cysteine levels. Because cysteine is an important precursor for glutathione formation, we had also expected to find a rise in total thiol levels. A study in glutathione depleted patients showed that supplementation with a whey based (cystein rich) diet increased plasma glutathione, whereas a casein based diet did not [42]. Michels et al. [43] found similar results in HIV patients. We did not observe this effect on glutathione with our cysteine rich drink. This may be explained by the fact that the measurement of total thiol is not specific enough, as it takes into account not only glutathione but also other sulfur compounds present in the blood. Another explanation could be that the rise in cysteine was too small to affect glutathione formation.
The difference between groups we found in GSH-Px levels should largely be ascribed to the decrease in the placebo group. This can be confirmed by studies that have shown that GSH-Px levels decrease with aging [13,15,16] or with impaired health status [17]. Supplementation may have prevented a decline in GSH-Px levels in these frail elderly people over a six-month time period. We did not measure selenium status in this study. If selenium status was adequate at the start of the study period, then this might explain the lack of increase of GSH-Px levels in the supplement group. However, Clausen et al. [23] reported an increase in GSH-Px after supplementation with high doses of selenium, vitamin C and vitamin E in elderly people living in homes for the elderly with an adequate selenium status. In contrast, McKay et al. [26] found in free living elderly people no effect of multivitamin supplementation on GSH-Px; plasma selenium levels were not measured. Thus other factors besides selenium status may influence the response of GSH-Px to supplementation.
Since our study was focused on the biochemical status of antioxidants, it is important to note that the health consequences of the rise in antioxidant blood levels in our study remain unknown. Positive effects of raising antioxidant status on immune function [44] and memory [45] have been reported in the literature, and we have found similar effects in our study [46]. However, due to the nature of our supplement, we cannot attribute these effects solely to the biochemical effects on antioxidant levels we observed.
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CONCLUSION
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We conclude that supplementation with an enriched drink with antioxidants can raise plasma levels of enzymatic and non-enzymatic antioxidants in frail elderly people.
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ACKNOWLEDGMENTS
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The study was sponsored by Numico Research B.V. We thank the laboratory of Prof. A. Bast (Department of Toxicology, Maastricht University, The Netherlands) for performing the TEAC analyses and Martin Balvers, Harja Pol, Erik Hogenkamp, and Jeroen van den Berg (Numico Research) for performing the remaining antioxidant analyses. We are grateful to Jeannette Gallas and the other nurses for their excellent assistance in obtaining blood samples and to all participants for their contribution to the study.
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FOOTNOTES
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The study was sponsored by Numico Research B.V.
Received May 17, 2002.
Accepted February 24, 2003.
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TOP
ABSTRACT
INTRODUCTION
