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1 From the Sticht Center on Aging, Wake Forest University Health Sciences, Winston-Salem, NC (MC, MP, BWJHP, and HA); the Laboratory of Clinical Epidemiology, Italian National Research Council of Aging, Geriatric Department, Florence, Italy (BB); the Department of Gerontology and Geriatrics, University of Perugia, Perugia, Italy (AC); the Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ (GRW); the Human Nutrition Research Center on Aging, Tufts University, Boston (AM); the Epidemiology, Demography, and Biometry Program, National Institute on Aging, Bethesda, MD (JMG); the Longitudinal Studies Section, Clinical Research Branch, National Institute on Aging, Baltimore (LF).
2 Supported by Bristol-Myers Squibb Company (Princeton, NJ) and by an unrestricted grant from BRACCO Imaging SpA, Italy. The InCHIANTI study was supported as a "targeted project" (ICS 110.1/RS97.71) by the Italian Ministry of Health and in part by the US National Institute on Aging (NIA) (contracts 263 MD 9164 13 and 263 MD 821336). The work of MC was supported by the Wake Forest University Claude D Pepper Older Americans Independence Center (NIA grant P30-AG-021332-01). 3 Reprints not available. Address correspondence to M Cesari, Sticht Center on Aging, Wake Forest University Health Sciences, 1 Medical Center Boulevard, Winston-Salem, NC 27157. E-mail: mcesari{at}wfubmc.edu.
ABSTRACT
Background: Muscle strength and physical performance in old age might be related to the oxidative damage caused by free radicals.
Objective: The objective was to assess the correlation of plasma concentrations and daily dietary intakes of antioxidants with skeletal muscle strength and physical performance in elderly persons.
Design: This study is part of the Invecchiare in Chianti (InCHIANTI) study, which was conducted in 986 Italians aged =" BORDER="0">65 y. Physical performance was assessed on the basis of walking speed, ability to rise from a chair, and standing balance. Knee extension strength was assessed with a hand-held dynamometer. The European Prospective Investigation into Cancer and Nutrition (EPIC) questionnaire was used to evaluate the daily dietary intakes of vitamin C, vitamin E, ß-carotene, and retinol. Plasma - and -tocopherol concentrations were measured. Adjusted linear regression analyses were used to calculate regression coefficients per SD increase in plasma concentrations and daily dietary intakes.
Results: In adjusted analyses, plasma -tocopherol was significantly correlated with knee extension (ß = 0.566, P = 0.003) and the summary physical performance score (ß = 0.044, P = 0.008). Plasma -tocopherol was associated only with knee extension strength (ß = 0.327, P = 0.04). Of the daily dietary intake measures, vitamin C and ß-carotene were significantly correlated with knee extension strength, and vitamin C was significantly associated with physical performance (ß = 0.029, P = 0.04).
Conclusions: Plasma antioxidant concentrations correlate positively with physical performance and strength. Higher dietary intakes of most antioxidants, especially vitamin C, appear to be associated with higher skeletal muscular strength in elderly persons.
Key Words: Antioxidants • dietary intake • physical performance • elderly
INTRODUCTION
The assessment of physical function is becoming an increasingly important component in the evaluation of elderly persons, and standardized tests of physical performance are now widely used both in research and clinical geriatric settings. In fact, some of these performance measures, such as knee extension strength and performance—as assessed by walking speed, balance, and ability to rise from a chair (chair stands)—have been shown to be useful in the prediction of institutionalization, disability, and mortality (1–3).
Recently, some authors suggested that age-related physical decline might be related to oxidative damage perpetrated by free radicals (4). A free radical has been defined as "any species capable of independent existence that contains one or more unpaired electrons" (5). This situation is energetically unstable, making such species often highly reactive and short-lived. Free radicals disrupt the equilibrium of biological systems by damaging their major constituent molecules, leading eventually to cell death (6, 7). Oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging (8, 9). This hypothesis suggests a primary role for antioxidant agents in the attempt to avoid or reduce age-related physical decline. Antioxidants can be defined as substances whose presence significantly inhibits the rate of oxidation (10). The body contains an elaborate antioxidant defense (11), and vitamin C, vitamin E, ß-carotene, and retinol are the primary antioxidant vitamins (12). It has been suggested that DNA damage, one of the most sensitive biological markers of oxidative stress, might be caused by an imbalance between the excessive generation of free radicals and the deficient action of the antioxidant system (13).
We hypothesize that plasma antioxidants and the dietary intake of antioxidants may correlate with strength and physical performance. The purpose of this study was to assess the correlation of plasma antioxidant concentrations and daily dietary intakes of antioxidants with skeletal muscle strength and physical performance in elderly persons.
SUBJECTS AND METHODS
The present study is part of the Invecchiare in Chianti (InCHIANTI) study, a prospective population-based study of elderly people in Italy designed by the Laboratory of Clinical Epidemiology of the Italian National Research Council of Aging (Florence, Italy). The aims of the study are to identify risk factors for the onset of disability in elderly persons, to study physiologic subsystems critical for walking, and to define critical ranges for tests that evaluate the integrity of the physiologic subsystems that are important for walking. The study population included 1156 participants aged 65–102 y, who were randomly selected from residents in 2 towns of the Chianti geographic area (Greve In Chianti and Bagno a Ripoli, Tuscany, Italy) with the use of a multistage stratified sampling method (14). The data collection started in September 1998 and was completed in March 2000. A detailed description of the sampling procedure and data collection method was previously published (14). The Italian National Research Council of Aging Ethical Committee approved the study protocol.
The present analyses were conducted in 986 participants; we excluded subjects in whom daily nutrient intakes were not assessed or in whom tests of physical performance and of knee extension strength were not conducted (n = 136). We also excluded 34 participants who were vitamin supplement users. Sociodemographic characteristics showed that the excluded participants were elderly (79.8 compared with 75.3 y), more likely to be female (67.1% compared with 55.3%), and less educated (4.7 compared with 5.4 y) than were those eligible for the present analyses.
Physical performance tests
Knee extension strength was measured with a hand-held dynamometer (Nicholas Muscle Tester; Sammon Preston Inc, Chicago). Participants, lying in lateral decubitus (opposite to the examined limb) with the hip and knee in 45 ° and 60 ° flexed positions, respectively, were asked to perform the task twice with each leg. The average of the best results obtained for each leg was used for the present analyses. In our study we found a high correlation between the 2 strength measures done for each leg (r = 0.896, P < 0.001). This measurement of knee extension strength has been proven to be reliable on the basis of high test-retest and interrater reliabilities (0.85 and 0.74, respectively) (15).
Performance of the lower extremities was assessed with the use of 3 tests: walking speed, ability to stand from a chair, and ability to maintain balance in progressively more challenging positions. Walking speed was defined as the best performance (time in s) of two 4-m walks at usual pace along a corridor. For the chair-stand test, the participants were asked to rise and sit down 5 times as quickly as possible with their hands folded across the chest. The performance was expressed as total time (in s) to complete the test. For the standing-balance test, the subjects were asked to stand in 3 progressively more difficult positions for 10 s each: a side-by-side feet standing position, a semitandem position, and a full-tandem position. Good to excellent reliability of these tests has been shown, which supports the utilization of these measures in aging research (16).
An arithmetic summary performance score was calculated to obtain a continuous measure (17). The timed scores of the performance tests were rescaled to values ranging from 0 to 1, where 1 indicates the best performance and 0 the worst performance. The worst performers were participants who were unable to complete the task or those who had a performance above the 99th percentile (walking time: 15.5 s; chair-stand time: 27.3 s; standing-balance test: 30 s). The following formulas were applied to rescale measures: 1) walking speed, 1 - (15.5/speed in centimeters per second); 2) chair-stand test, 1 - (time in seconds/27.3); and 3) standing-balance test, time in seconds/30. A summary physical performance score ranging from 0 to 3 was calculated by adding these 3 rescaled scores. Such a summary score has been shown to have excellent predictive validity for mortality, institutionalization, hospitalization, and incident disability (1, 3, 18, 19). The internal consistency (Cronbach's ) of the summary scale in our study was 0.871.
Plasma antioxidants
Plasma vitamin E (- and -tocopherol) concentrations were measured by reversed-phase HPLC as previously described (20). Briefly, 100 mL plasma was mixed with 100 mL ethanol; after being mixed by vortex, tocopherol was extracted into 500 mL hexane containing 0.002% butylated hydroxytoluene (Sigma, St Louis). Tocol, a gift from Hoffmann-La Roche (Nutley, NJ), was added to the mixture as an internal standard. Samples were centrifuged at 180 x g for 5 min at 4 °C. The supernatant fluid was collected and dried under a stream of nitrogen gas and was reconstituted in 100 mL methanol. Tocopherols were separated by HPLC with a 3-µm C18 reversed-phase column (Perkin-Elmer, Norwalk, CT). The mobile phase, delivered at a flow rate of 1.0 mL/min, consisted of 1% water in methanol containing 10 mmol lithium perchlorate/L. Samples were injected with an autosampler (1100 series; Hewlett-Packard, Palo Alto, CA). Eluted peaks were detected at an applied potential of +0.6 V by an LC 4B amperometric electrochemical detector (Bioanalytical System, West Lafayette, IN). Peaks were integrated with ChemStation software (Hewlett-Packard). Tocopherol concentrations were expressed in µmol/L. The reproducibility and accuracy of the procedures used were tested by analyzing representative samples in triplicate from a sample provide by the American Association for Laboratory Accreditation (Washington, DC), which contained known concentrations of -tocopherol. Intra- and interbatch CVs were 3% and 4.2%, respectively.
EPIC questionnaire
To determine daily nutritional intakes, we used the EPIC (European Prospective Investigation into Cancer and Nutrition) questionnaire (21, 22). This questionnaire was designed and validated in a multicenter study performed in 10 European countries, with the aim to evaluate the relation between diet and the onset of cancer. Participants were asked how many times in a year each food (or food category) and beverage were consumed. All of the answers provided by the participants were reported through specific software in a database, and the daily intakes of micro- and macronutrients were calculated. The software for the estimate of food composition was created and verified by the EPIC study group according to food-composition tables compiled in 1998 by researchers at the European Institute of Oncology.
In a previous study (23), data on dietary intake collected in the InCHIANTI study were compared with data collected in a nationwide study in which a direct method of measure based on 7-d weighted and registered food consumption was used. Despite different populations and different methods used for data collection, the distributions of dietary intake from the 2 studies were remarkably similar for most macro- and micronutrients, both in men and women, which suggests that the EPIC food-frequency questionnaire provides a good estimate of dietary intake. In the present analyses, we considered the EPIC questionnaire—derived daily intakes of 4 antioxidant agents: vitamin C, vitamin E, ß-carotene, and retinol.
Covariates
The covariates included sociodemographic variables (age, sex, site, smoking habit, and education), cognitive status (Mini-Mental State Examination score), plasma lipid concentrations (total cholesterol and triacylglycerols), comorbidity (self-reported diagnoses of hypertension, angina, myocardial infarction, stroke, cancer, diabetes, congestive heart failure, and chronic obstructive pulmonary disease), body mass index (computed as weight in kg/height in m2), the number of medications taken by participants at the baseline assessment, and physical activity (defined as a moderate-to-high intensity exercise performed for =" BORDER="0">1–2 h/wk or a light-intensity exercise performed for >4 h/wk). To adjust for overall food intake, we also adjusted our analyses for daily dietary energy intake, as assessed by the EPIC questionnaire. Participants were asked to report any medication taken in the last 2 wk. Drugs were coded according to the Anatomical Therapeutic and Chemical codes (24).
Statistical analyses
Because the plasma concentrations of -tocopherol and -tocopherol, and daily dietary intakes of vitamin C, vitamin E, ß-carotene, and retinol were not normally distributed, analyses were performed with the use of their log values. Spearman correlation tests were used to evaluate correlations of antioxidants plasma concentrations and daily dietary intakes with knee extension strength and summary physical performance score. All analyses were adjusted for age, sex, site, Mini-Mental State Examination score, smoking habit, education, body mass index, total cholesterol, triacylglycerol (log value), hypertension, angina or myocardial infarction, congestive heart failure, diabetes, stroke, number of medications taken, and daily dietary energy intake; all these variables showed a significant correlation with outcome measures. Linear regression analyses were used to identify regression coefficients for strength and performance outcomes per SD increase in plasma antioxidant concentrations and daily dietary intakes. To calculate adjusted knee extension strength and summary physical performance scores, analyses of covariance were performed for tertiles of antioxidants (plasma concentrations and dietary intakes) and daily energy intake. Sex interactions were assessed by adding the interaction term for sex x antioxidant concentration in the adjusted model.
RESULTS
The main characteristics of the participants are shown in Table 1. The mean (±SE) age of the participants was 75.3 ± 0.2 y, and 55.3% were female. The most common diagnoses reported by the participants were hypertension (44.4%), diabetes (11.6%), cancer (10.3%), and angina or myocardial infarction (7.6%).
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TABLE 1. . Participant characteristics1
Mean (±SE) plasma concentrations of
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TABLE 2. . Spearman correlations between physical performance and muscular strength tests and plasma concentrations and dietary intakes of antioxidants
Adjusted regression coefficients per SD increase in plasma antioxidants for knee extension strength and physical performance are described in Table 3. -Tocopherol was significantly correlated with both performance measures (knee extension: ß = 0.566, SE = 0.193, P = 0.003; physical performance: ß = 0.044, SE = 0.017, P = 0.008). -Tocopherol was associated only with knee extension strength (ß = 0.327, SE = 0.165, P = 0.04). No significant sex interaction was found between plasma antioxidants and knee extension strength or physical performance (all P values > 0.05). Daily dietary intakes of vitamin C and ß-carotene were significantly correlated with knee extension strength (vitamin C: ß = 0.383, SE = 0.162, P = 0.02; ß-carotene: ß = 0.311, SE = 0.159, P = 0.05). Only the daily intake of vitamin C was significantly associated with physical performance (ß = 0.029, SE = 0.014, P = 0.04). No significant sex interaction was found between dietary intake and physical performance (all P values > 0.05).
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TABLE 3. . Adjusted regression coefficients for knee extension strength and summary physical performance scores per SD increase (log) in plasma antioxidants and in dietary intakes of antioxidants1
Adjusted means for knee extension strength and summary physical performance score according to tertiles of plasma antioxidant concentrations and daily dietary intakes were assessed. Greater knee extension strength was found at higher concentrations of -tocopherol (lowest compared with highest tertile: P = 0.003 and P for trend = 0.01). Higher plasma concentrations of -tocopherol were found at higher physical performance scores (lowest compared with highest tertile: P = 0.009 and P for trend = 0.003). Similar results were found for higher daily intakes of vitamin C and greater knee extension strength (lowest compared with highest tertile: P = 0.008 and P for trend = 0.03) and physical performance (lowest compared with highest tertile: P = 0.03 and P for trend = 0.08). Higher daily intakes of ß-carotene tended to be associated with greater knee extension strength (lowest compared with highest tertile: P = 0.05 and P for trend = 0.06).
DISCUSSION
This study evaluated the relation between antioxidants and objective performance assessments in elderly persons. We investigated whether plasma concentrations and daily dietary intakes of antioxidants were associated with physical performance and muscular skeletal strength in an elderly population. To our knowledge, this is the first study that evaluated simultaneously the effects of dietary antioxidant intakes and plasma antioxidant concentrations on physical performance in an elderly population. Previous studies were focused on younger samples, especially athletes, and principally aimed at assessing whether antioxidant supplementation reduces the rate of oxidation or helps promote faster recovery from the damage generated by exercise (11, 12, 25–27). Our analyses suggest a relation between plasma concentrations of antioxidants and physical performance and strength. A significant positive association, especially on muscular skeletal strength, was also found for the daily dietary intakes of several antioxidants, including vitamin C and ß-carotene.
Free radical oxidative stress has been implicated in the pathogenesis of a variety of diseases, and natural antioxidant defenses have been found to be defective in many diseases (10). A free radical is a molecule that contains an unpaired electron in its outer orbit and that can exist independently (5, 12). Stability is achieved by the removal of electrons from surrounding molecules to produce an electron pair. The remainder of the attached molecule then possesses an unpaired electron and therefore becomes a free radical. In this way, the presence of a single radical may initiate a chain sequence of electron transfer (redox) reactions. Several studies suggest an important role of oxidation in physiologic decline with normal aging (8, 9, 13). Oxidative stress can come from a variety of sources, from abnormal metabolic pathways to inflammatory cells (eg, macrophages and neutrophils producing hydrogen peroxide and hypochlorus acid as means of bacterial killing) (28), smoking, and radiation (10).
Our results confirmed previous findings that suggest a strong correlation of plasma antioxidant concentrations with physical performance and strength. In fact, we found a significantly high correlation of -tocopherol, the predominant form of circulating vitamin E (29), with physical performance and strength tests. We also reported a strong correlation between skeletal muscular strength and -tocopherol, a vitamin E form indicated by some studies to be a more effective trap for lipophilic-free radicals than -tocopherol (29). In our study, we found no significant sex interaction between performance and antioxidants.
We also examined the effects of the dietary intake of major antioxidants on physical performance and strength. Vitamin C is a water-soluble vitamin that represents a first-line antioxidant defense in plasma. It is a powerful inhibitor of lipid peroxidation and regenerates vitamin E in lipoproteins and membranes. ß-Carotene is a lipid-soluble antioxidant and a precursor of retinol that works synergistically with vitamin C. Vitamin E is a major lipid-soluble chain-breaking antioxidant that prevents lipid peroxidation in lipoproteins and biological membranes. Its antioxidant function is strongly supported by regeneration promoted by vitamin C (10, 12).
Our findings suggest an association between higher daily dietary intakes of vitamin C and ß-carotene and skeletal muscle strength. Whether higher antioxidant intakes are beneficial in promoting better physical performance and muscular strength is controversial. Although the findings of some studies do not support a beneficial effect of increased antioxidant intakes on physical performance (11, 12, 30), other studies have shown improvements in the recovery from exercise with antioxidant intake and a preventive role of antioxidant supplements against oxidative damage (10, 27, 31).
In our study we showed a nonsignificant correlation between the dietary intake of vitamin E and -tocopherol but a significant correlation with -tocopherol. The nonsignificant association between vitamin E and -tocopherol may have been due to the participants' high consumption of olive oil, which was the major source of vitamin E in the study. Dietary intake of vitamin E includes the sum intake of all tocopherol and tocotrienols with vitamin activity. Therefore, such a summary measure is probably not precise and specific and may explain the nonsignificant association between the dietary intake of vitamin E and physical performance and strength measures.
Even though antioxidant supplementation has been suggested as a mean to perform better and recover more quickly and fully from vigorous exercise, the theoretical basis for why antioxidants may enhance performance is not clear. An improved antioxidant status is one adaptation to regular exercise training (32), and an acute increase in plasma antioxidant activity has been observed after exercise (33). This finding suggests that antioxidant status may be important in healthy muscle function, especially during the recovery phase after exercise. To our knowledge, no evidence is available that documents the suppression of oxidative damage in muscle by antioxidant supplements, but it seems reasonable that an adequate antioxidant intake is needed to maintain healthy muscular activity (10, 34).
Several limitations of our study should be mentioned. The cross-sectional design of the study did not permit us to evaluate the effect of daily nutrient intakes on changes in physical performance. Further longitudinal studies are needed to evaluate the role that antioxidant intakes may have on physical performance in the elderly. The study population was characterized by high nutritional indexes and tended to be overweight. These characteristics did not allow us to assess the effect of very poor nutrition. The general overweight status of participants may have influenced our findings. To better identify the effects of antioxidants on physical performance, it may be useful to evaluate a population in which under- and normal-nourished persons are used as comparison groups. Finally, another limitation of our study was its limited assessment of biological measures. The evaluation of both dietary intake estimates and biological measures would have been the ideal combination, especially given the significant association found between dietary intakes of vitamin C and ß-carotene and knee extension strength.
In conclusion, our study indicates a significant positive correlation between plasma antioxidant concentrations and physical performance and strength. Higher dietary intakes of antioxidants, especially of vitamin C, in the elderly were also found to be associated with greater skeletal muscular strength. However, the potentially positive effects of antioxidant intakes on performance and strength need to be confirmed in future prospective studies and clinical trials.
ACKNOWLEDGMENTS
The EPIC-Italy food-frequency questionnaire was developed as a cooperative project supported by the Italian Association for Cancer Research. We are particularly grateful to Domenico Palli and Simonetta Salvini (Molecular and Nutritional Epidemiology Unit, CSPO, Scientific Institute of Tuscany, Florence, Italy) and Vittorio Krogh (National Cancer Institute, Milan, Italy) for their help in processing and developing the InCHIANTI nutritional database.
MC helped design the study, analyze the data, and write the manuscript. MP and BWJHP helped design the study, write the manuscript, and critically review the manuscript. BB helped collect and analyze the data and critically review the manuscript. AC and AM helped collect the data and critically review the manuscript. GRW and HA helped critically review the manuscript. JMG helped design the study and critically review the manuscript. LF helped design the study, collect and manage the data, and critically review the manuscript. None of the authors had any financial or personal conflict of interest. Bristol-Myers Squibb sponsored the data analyses but had no influence on the design of the study, the data collection, the analyses and interpretation of the data, or the decision to publish the results.
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