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The Effects of a Multivitamin/Mineral Supplement on Micronutrient Status, Antioxidant Capacity and Cytokine Production in Healthy Older Adults Consuming a Fortified Diet

Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts

Address reprint requests to: Jeffrey B. Blumberg, PhD, Antioxidants Research Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111. E-mail: blumberg{at}hnrc.tufts.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

 
Background: Inadequate micronutrient intake among older adults is common despite the increased prevalence of fortified/enriched foods in the American diet. Although many older adults take multivitamin supplements in an effort to compensate, studies examining the benefits of this behavior are absent.

Objective: To determine whether a daily multivitamin/mineral supplement can improve micronutrient status, plasma antioxidant capacity and cytokine production in healthy, free-living older adults already consuming a fortified diet.

Methods: An eight-week double-blind, placebo-controlled clinical trial among 80 adults aged 50 to 87 years (mean=66.5±8.6 years).

Results: Multivitamin treatment significantly increased (p<0.01, compared to placebo) plasma concentrations of vitamins D (77 to 100 nmol/L), E (27 to 32 µmol/L), pyridoxal phosphate (55.1 to 75.2 nmol/L), folate (23 to 33 nmol/L), B12 (286 to 326 pmol/L)), C (55 to 71 µmol/L), and improved the riboflavin activity coefficient (1.23 to 1.15), but not vitamins A and thiamin. The multivitamin reduced the prevalence of suboptimal plasma levels of vitamins E (p=0.003), B12 (p=0.004), and C (p=0.08). Neither glutathione peroxidase activity nor antioxidant capacity (ORAC) were affected. No changes were observed in interleukin-2, -6 or -10 and prostaglandin E₂, proxy measures of immune responses.

Conclusions: Supplementation with a multivitamin formulated at about 100% Daily Value can decrease the prevalence of suboptimal vitamin status in older adults and improve their micronutrient status to levels associated with reduced risk for several chronic diseases.

Key words: aging, antioxidant, multivitamin, supplementation

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

 
Aging is accompanied by a variety of physiological, psychological, economic and social changes that compromise nutritional status and/or affect nutritional requirements [1]. For these reasons, the diets of many older adults do not currently meet the recommended intake levels of several essential vitamins and minerals [2, 3]; thus, low micronutrient status is often reported in this population [4–6]. Nutritional status surveys of the elderly indicate a low to moderate prevalence of frank nutrient deficiencies, but an increased risk of malnutrition, along with evidence of subclinical deficiencies having a direct impact on physiologic function [7–10].

Overt micronutrient deficiencies have been reported as prevalent in nursing home populations, and recommendationsd have been proferred that all institutionalized older adults receive a multivitamin/mineral supplement for general nutritional prophylaxis [11–13]. Trials conducted in long-term hospitalized elders with modest doses of antioxidant vitamins have demonstrated their capacity to improve the status of vitamins C and E, ß-carotene and the activities of glutathione peroxidase and/or superoxide dismutase after six months [14–16]. In healthy, free-living older adults, clinical trials using multivitamin/mineral supplements have demonstrated improved nutrient status in as little as two months [17, 18]. Significant effects on immune response outcomes such as infectious disease episodes and delayed-hypersensitivity skin test responses have been noted after 12 months of intervention [19, 20].

The amount of any particular nutrient required to prevent a deficiency is inherently defined in the Recommended Dietary Allowances (RDA) [21]. This standard is being revised to include nutrient intakes associated with reductions in the risk for chronic disease, values often higher than those necessary to prevent deficiency [22, 23]. Many older adults are unable to consume sufficient, let alone optimal, levels of certain nutrients solely by diet, and a modified food guide pyramid for people over 70 years recommends the use of dietary supplements to bridge this gap [24].

Dietary supplement use by Americans over 50 years ranges from 31% to 56% [25]. The most often consumed supplement is a multivitamin preparation [26]. The effects of multivitamin supplementation in this population have not been examined since the advent of mandatory folate fortification of flour, voluntarily fortification of common foods such as vitamin C in milk, calcium and vitamin E in orange juice, and ‘superfortified’ breakfast cereals. Thus we examined whether a multivitamin/mineral supplement formulated at about the Daily Value (DV) can improve micronutrient status, antioxidant capacity and immune function (via cytokine production as a proxy) in healthy, older adults consuming a fortified diet.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

 
Subjects
Healthy, free-living older adults 50 years residing in the Greater Boston area were recruited by newspaper advertisements, direct mailings and clinic postings. Volunteers were excluded if they were smokers, used dietary supplements regularly for three months prior to screening, were taking medications known to interfere with folate metabolism, had established diseases of the gastrointestinal tract, liver and/or kidney, or any disability which would impede full participation in the study. On the basis of these criteria, 272 men and women were eligible for an initial blood screening visit. Since homocysteine is a functional indicator of low vitamin status, and the mean homocysteine concentration at the initial screening visit was 7.8±2.0 µmol/L, participants with a total plasma homocysteine concentration above the mean ( 8.0 µmol/L) were invited to participate in the clinical trial. Of the 92 participants eligible for the trial, six had blood chemistry measures outside standard reference ranges, three developed medical conditions undetected during the initial screening visit, one lost interest, one was unwilling to refrain from dietary supplements, and one had gastrointestinal complaints, leaving a total of 80 subjects who completed the study. The age range of study subjects was 50 to 87 years (mean=66.5±8.6 years).

The study design was approved by the Human Investigation Research Committee of Tufts University and the New England Medical Center. All subjects signed a written informed consent agreement before participating.

Experimental Design
The protocol was designed as a double-blind, placebo-controlled clinical trial of an effervescent multivitamin/mineral preparation formulated at about 100% DV for most nutrients (Table 1). After gender stratification, subjects were randomized to receive either supplement or placebo. The placebo was composed of the same, non-nutritive base ingredients found in the supplement, i.e., citric acid, sodium bicarbonate, sweeteners, flavoring and coloring agent. During the seven days prior to the intervention, all subjects were given placebo to test their ability to comply with the protocol and required to give two overnight fasting blood samples (on day -7 and day 0) to determine baseline values for the micronutrients of interest. Fasting blood samples were again collected for analyses on days 49 and 56. The duplicate ‘before’ and ‘after’ measures were intended to ensure precision in the results, especially for those nutrients where the magnitude of change was anticipated to be small.

Micronutrient Analyses
All micronutrient analyses were performed using validated methods for assessing nutrient status (Table 2). The enzyme activity coefficient assays for thiamin, riboflavin and vitamin B6 measure activity coefficient or A.C. units.

Cytokine Production and Prostaglandin E₂ Activity
Cytokines IL-2, 6, 10, and prostaglandin E₂ (PGE₂) were assayed using commercially available immunoassay kits (Quantikine, R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Statistical Analyses
All statistical analyses were performed with SPSS v8.0 (SPSS, Inc., Chicago IL). Prior to formal analysis, a logarithmic transformation was applied to concentrations of folate, vitamin B6 and vitamin B12 in order to achieve homogeneity of variance and linearity of regressions, but untransformed values were used to construct tables and graphs of summary statistics. Tests of repeated measures ANOVA were used to determine statistically significant changes in plasma nutrient concentrations, ORAC values and cytokine production between placebo and supplemented groups. Student’s t test was used to compare baseline characteristics between the placebo and supplemented groups. The Wilcoxon-Mann-Whitney test was used to determine whether nutrient concentration changes from suboptimal to optimal categories were different in the placebo and supplemented groups. Summaries are expressed as means ± standard deviation (SD), and two-sided observed significance levels (p values) <0.05 are considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

 
Baseline characteristics of the study participants are presented in Table 3. No significant differences were observed between the placebo and supplemented groups with respect to age, gender and BMI. Although all participants were apparently healthy, the number of most frequently reported chronic illnesses were distributed evenly between the two groups. Baseline plasma micronutrient concentrations were also not significantly different between groups (Table 4).

	Fat-Soluble Vitamins

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

Although the prevalence of suboptimal vitamin A status (<2.5 µmol/L) [29] in the group receiving the multivitamin (78%) was not reduced by the supplement, mean dietary intake was 12,137 IU (>2X RDA), and all subject baseline levels were well above the cutoff point for the category associated with low risk of deficiency (>1.05 µmol/L) [22]. After the intervention, one placebo group subject had a drop in plasma retinol sufficient to reclassify him into the moderate risk category (0.35-1.05 µ mol/L). Supplementation reduced the prevalence of low vitamin D status (<37.5 nmol/L) [30] from 7% to 0% and suboptimal -tocopherol (<30 µmol/L) [29] from 73% to 49%. In the placebo group, the prevalence of low vitamin D status was reduced from 13% to 8%. The prevalence of suboptimal -tocopherol increased from 80% to 82% in the placebo group. Statistical significance was achieved only for -tocopherol (Wilcoxon-Mann-Whitney test, p=0.003). The change in plasma retinol concentration after supplementation ranged from -0.4 to +0.5 µmol/L; the change in 25(OH)D ranged from -6.2 to +61.2 nmol/L; and the change in -tocopherol ranged from -0.7 to +20.1 µmol/L.

	Water-Soluble Vitamins

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

 
After eight weeks of treatment, supplemented subjects showed significant improvement in the plasma concentrations of vitamins B6, B12, C, riboflavin, pyridoxal phosphate and folate (Table 4). Activity coefficient assays for riboflavin and vitamin B6 decreased 7% and 6% respectively, and no significant change in the status of thiamin was detected. Activity coefficient generating assays for thiamin, riboflavin and vitamin B6 are inversely proportional to their respective changes in plasma concentration. Plasma concentrations of pyridoxal phosphate, folate, vitamins B12 and C were increased by 36%, 42%, 14% and 29%, respectively. No significant changes in water-soluble vitamin status were found in the placebo group, although vitamin B12 decreased 4%. Dietary intake of the water-soluble vitamins did not change in either group during the intervention.

Supplementation reduced the prevalence of suboptimal plasma vitamin C concentrations (<50 µmol/L) [29] from 29% to 5%, suboptimal folate (<15 nmol/L) [31] from 15% to 5%, suboptimal vitamin B12 (<258 pmol/L) [32] from 42% to 27% and low vitamin B6 (<20 nmol/L) [33] from 7% to 0%. In the placebo group, the prevalence of suboptimal vitamin C status was reduced from 41% to 33% and suboptimal vitamin B12 increased from 67% to 80%. Only the change in vitamin B12 achieved statistical significance (p=0.004), although the vitamin C change approached it (p=0.08). The change in plasma vitamin C concentration after supplementation ranged from -21.6 to +78.9 µmol/L, the change in folate concentration ranged from -8 to +25 nmol/L, the change in PLP concentration ranged from -136.4 to +105.0 nmol/L, and the change in vitamin B12 concentration ranged from -24 to +141 pmol/L.

	Antioxidant Capacity

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

 
As shown in Table 6, neither plasma nor red blood cell glutathione peroxidase activity changed in either the supplement or placebo group after eight weeks of treatment. No significant differences in ORAC were detected in either group after treatment.

	Cytokine Production and PGE2

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

 
No significant differences in the production of cytokines IL-2, 6, 10 and PGE₂ activity were detected in either group after eight weeks of treatment (Table 7).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

Chavance et al. [18] examined older adults in France given either multivitamin or placebo for four months. After two months plasma folate increased 33 nmol/L compared to the 10 nmol/L change observed here; however, baseline concentrations were lower in the French subjects (16 vs. 23 nmol/L) who were not consuming fortified foods. Baseline vitamin C status was lower in the French group (30 vs. 55 µmol/L), and the change in vitamin C status after two months was also lower (12 vs. 16 µmol/L); however, their formulation contained half the amount of vitamin C (120 mg). The plasma -tocopherol concentrations in the French cohort were slightly higher at baseline (32 vs. 27 µmol/L); however, their change in status after two months was similar to ours (+6 vs. +5 µmol/L). After four months only plasma vitamin C and folate continued to rise after the first two-month interval in the French subjects. All other plasma micronutrients remained close to their concentrations determined at two months.

Mann et al. [17] examined older American adults administered either multivitamin or placebo for four months. After two months no significant changes were noted in the status of vitamins A and E. Their baseline -tocopherol levels were higher (26–31 µmol/L) than observed in this study and improved after four months with 30 IU to 33–34 µmol/L. Although the supplement used by Mann et al. [17] contained more vitamin C (300 mg), the mean plasma vitamin C concentration was lower at two months, but the same by four months, as achieved with this protocol.

In addition to reducing the prevalence of poor nutrient status, the multivitamin appears to have increased several vitamins into a more optimal range relevant to reducing the risk of chronic disease. Gey et al. [29] defined the ‘optimal’ plasma concentrations for vitamins A, C, and E through cross-cultural studies examining plasma nutrient levels and risk for cardiovascular diseases and cancer. Values 20% to 50% lower than the target thresholds for either vitamin C (>50 µmol/L), E (>30 µmol/L) or retinol (>2.5 µmol/L) were found to approximately double the relative risk of CVD and cancer, respectively [34, 35]. Although the mean plasma vitamin A concentration in the present study is considered less than optimal, Garry et al. [36] suggests that years of regular supplement use are required to cross that threshold. The mean plasma vitamin C concentration after multivitamin supplementation increased within the boundaries of the third quintile of plasma vitamin C described in the Sayhoun et al. survey of 747 older Americans [37]. Their third quintile was associated with a 0.51 relative risk of heart disease and 0.64 relative risk of overall mortality versus those in the lowest quintile. Elevating the mean vitamin E concentration to >28 µmol/L shifted subjects from the fourth to fifth quintiles of plasma vitamin E, as described by Riemersma et al. [38] for a population of 504, which was associated with a 64% reduced risk of angina pectoris. Similarly, this shift was sufficient to place our subjects in the group at lowest risk of CHD mortality as determined in the Vitamin Substudy of the WHO/MONICA project [34].

Improving 25-hydroxyvitamin D (25(OH)D) concentrations above the level associated with subclinical deficiency (<37.5 nmol/L) may reduce the risk of developing skeletal fractures due to secondary hyperparathyroidism, lower serum calcium and phosphate levels, higher serum alkaline phosphatase and osteoporosis [39–41]. However, parathyroid hormone (PTH) levels become minimal when 25(OH)D concentrations exceed 100 nmol/L [42, 43]. In studies showing osteoporosis fracture prevention with vitamin D and calcium supplementation, mean 25(OH)D concentrations exceeded 100 nmol/L [42, 44, 45]. Multivitamin supplementation was able to elevate the mean plasma 25(OH)D concentration to the 100 nmol/L level.

Improving the status of folate, vitamins B6 and B12 is effective in reducing plasma homocysteine [46–53], which, in turn, is associated with a reduced risk for vascular disease [54, 55]. The multivitamin intervention increased plasma levels of these B-vitamins sufficiently to have a significant effect in lowering total plasma homocysteine concentrations [56]; however, low plasma levels of folate, B6 and B12 are also associated with an increased risk for heart disease independent of plasma homocysteine concentration [57, 58]. Multivitamin supplementation reduced the prevalence of suboptimal plasma vitamin B12 (>258 pmol/L, p=0.004), but did not shift the mean to the lowest risk quartile (i.e., >335 pmol/L) for coronary atherosclerosis as described by Siri et al. [57]. Eliminating suboptimal plasma pyridoxal phosphate concentrations may have an impact on the risk for atherosclerosis considering the report by Robinson et al. [58] of a 76% increased risk for vascular disease at suboptimal PLP levels.

Supplemental vitamin E treatment has been reported to enhance cell-mediated and humoral immune responses [59]. Chandra et al. [20] found that after one year of multivitamin supplementation, older adults had significantly enhanced in vitro lymphocyte proliferative responses to mitogens, interleukin 2 (IL-2) production and IL-2 receptor release, natural killer cell activity, antibody responses to influenza vaccine, and a lower incidence of infectious diseases. Immunological responses were greatest among the subjects with the lowest nutrient status at baseline. In contrast to our subjects, this cohort presented with a marked prevalence of deficiencies, e.g., in vitamin C (23%), vitamin A (13%), ß-carotene (17%), and vitamin E (8%). Bogden et al. [19] determined delayed hypersensitivity skin test responses after one year of multivitamin supplementation in older adults and found no change at six months, but significant increases after 12 months. After four months of multivitamin supplementation, Chavance et al. [18] observed no significant difference in the incidence of reported infectious episodes between supplement and placebo groups. Direct comparisons are not possible between these studies because of the different outcome parameters and duration of treatment. However, it is noteworthy that multivitamin interventions shorter than six months appear not to impact immune function.

Chao et al. [60] observed no change in total ORAC in young men assigned to one of four antioxidant treatments (2000 RE vitamin A as ß-carotene, 500 mg vitamin C, 440 mg -TE vitamin E, or all combined with 100 µg selenium and 30 mg zinc) or placebo, and subjected to strenuous activity for 28 days despite increases in measures of oxidative stress (breath pentane, serum blood lipid peroxide, urine malondialdehyde and 8-hydroxydeoxyguanosine) in all groups. In contrast, Cao et al. found that non-protein plasma ORAC increased in older women one to two hours after consuming 1250 mg vitamin C [61]. Within a biological system, the total ORAC assay measures the total antioxidant capacity of all known nonenzymatic water- and lipid-soluble antioxidants, including ß-carotene, glutathione, methionine, uric acid, bilirubin, phenolic acids, flavanols, flavonols, flavones, isoflavones, flavanones, anthocyanins, in addition to vitamin C and -tocopherol [28, 62, 63]. Plasma proteins and lipoproteins account for about 85% to 90% of the overall peroxyl-radical trapping capacity, while one half of the non-protein ORAC value in humans is attributed to uric acid [64]. The change in plasma antioxidant vitamins in this study is either insufficient to affect ORAC due to their low contribution to the non-protein plasma oxidant capacity in fasting plasma, or uric acid status may have masked any changes in ORAC at the antioxidant concentrations achieved here. The activity of glutathione peroxidase, another component of antioxidant defense mechanisms, was unaffected by the multivitamin. This response might not be unexpected as the subjects were not selenium deficient, and the supplement contained only 20 µg of selenium. Similarly, Meydani et al. found no change in glutathione peroxidase activity with vitamin E supplementation [65].

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

 
Supplementation with a multivitamin supplement can improve micronutrient status in healthy, older Americans to levels above those obtained with a fortified diet. This increase in nutritional status reduces the prevalence of suboptimal plasma vitamin concentrations and will shift blood levels of key nutrients into ranges associated with reduced risk for several chronic diseases.

	ACKNOWLEDGMENTS

 
We thank all of our volunteers as well as the nurses and staff of the Metabolic Research Unit, Dietary Assessment Unit, and Nutrition Evaluation Laboratory at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University for their invaluable efforts. We also thank Leslie Abad for her technical assistance.

	FOOTNOTES

 
Research support was provided by the U.S. Department of Agriculture (USDA) Agricultural Research Service under Cooperative Agreement No. 58-1950-001 and a grant from the Pharmavite Corporation (Mission Hills, CA). The contents of this publication do not necessarily reflect the views or policies of the USDA nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.

Received March 7, 2000. Accepted August 3, 2000.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS Fat-Soluble Vitamins Water-Soluble Vitamins Antioxidant Capacity Cytokine Production and PGE2 DISCUSSION CONCLUSION REFERENCES

 

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