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Plasma antioxidant concentrations in a population of elderly women: Findings from the nun study
Nutrition Research, Vol. 16, Nos. 11112, pp. 1881~1890.1996 Copyright 0 19% Ekvier Science Inc. F’rinted in the USA. All rights reserved 0271.5317/96 $15.00 + .OO ELSEVIER
PH SO271-5317(96)00211-4
PLASMA ANTIOXIDANT CONCENTRATIONS IN A POPULATION ELDERLY WOMEN: FINDINGS FROM THE NUN STUDY
OF
David A. Snowdon Ph.D.2
Myron D. Gross Ph.D.1
IDivision of Epidemiology, School of Public Health, University of Minnesota, 1300 S. Second Street, Suite 300, Minneapolis, Minnesota 55454-1015. 612-624-5417 FAX: 612-625-8950. &Sanders-Brown Center on Aging, and the Department of Preventive Medicine, College of Medicine, University of Kentucky, Lexington, Kentucky.
Low plasma alpha-tocopherol and beta-carotene concentrations have been associated with an increased risk of numerous degenerative diseases, including cancer. Several characteristics that accompany aging, including changes in dietary habits and physiologic capacity, may place elderly populations at a high risk of low plasma antioxidant concentrations. Thus, the present study was undertaken to characterize plasma concentrations of alpha-tocopherol, beta-carotene and several other carotenoids in elderly subjects, to describe the relationship between age and plasma antioxidant concentrations in this population and to compare the concentrations of plasma antioxidants in middle-aged and elderly individuals. The study recruited 94 participants from the Nun Study, a longitudinal study of aging and Alzheimer disease. Women in this population were unique for their advanced age, 77-99 years old, and their comparability across age groups due to the absence of several potential confounders of plasma antioxidant concentrations. The population mean and standard deviation of several plasma carotenoids and alpha-tocopherol concentrations (@dl) were as follows: lycopene, 15.0+/- 10.0; beta-carotene, 30.0+/- 19.7; alpha-carotene, 15.0 +/- 9.6; zeaxanthin plus lutein, 22.0+/- 7.4; beta-cryptoxanthin, 14.0 +/- 8.6 and alpha-tocopherol, 980 +/- 310. Concentrations of all analytes, except lycopene, were similar to or higher than those reported for several middle-aged American populations. Lycopene concentrations were significantly lower in the population of sisters as compared with the middle-aged populations and tended to decrease across age groups within the population of sisters . Age appeared to be a relatively minor determinant of plasma alpha-tocopherol and the concentrations of carotenoids, other than lycopene, in this population. Importantly, the plasma concentrations of most carotenoids and alpha-tocopherol in this population of independent elderly women were apparently adequate on the basis of population comparisons. Further studies of this population may define determinants essential for the maintenance of antioxidant status in elderly populations. Copyright 0 1996 Elsevier Science 1~. Key words: women, carotenoids, elderly, tocopherol and plasma
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RODIJCTION Fat-soluble, dietary antioxidants may have a role in the prevention of conditions and diseases of aging. Comparative studies of mammalian species, including humans, have found a positive correlation between tissue concentrations of the specific antioxidants, vitamin E and beta-carotene, and life span (1). The antioxidants may prevent an accumulation of oxidative damage in the form of mutations and thereby prevent cellular malfunction leading to degenerative conditions and diseases (2,3,4,5). Numerous observational studies have found an inverse association between antioxidant exposure, dietary intake and plasma concentrations, and the incidence of degenerative diseases, including several cancers (6- 10) and cardiovascular disease (11-13). Some, but not all, of the early clinical studies have found a reduction in the incidence of certain cancers (14-17) and cardiovascular disease (18-19) with antioxidant supplementation. ln addition, antioxidants may aid in the prevention of cataracts (20), neurodegeneration (21) and Parkinson’s disease (22). These observations have the support of numerous animal (23), cellular (24-25) and mechanistic studies (26-27). As a consequence of these studies, there has been considerable interest in the carotenoid and alphatocopherol status of various populations. Vitamin E and beta-carotene concentrations are not regulated by homeostatic controls in humans and numerous factors can affect their plasma concentrations. A primary determinant of plasma concentrations is the dietary intake of fruits, vegetables and vitamin supplements(28) which are the major sources of fat-soluble, dietary antioxidants(29). Numerous other factors may significantly modify the relationship between dietary intake and plasma concentrations. These factors include smoking, alcohol consumption, gender, race, age, body mass index and plasma cholesterol concentrations(30-35). Because of the accumulation of oxidative damage and an increasing incidence of degenerative diseases with age, the effect of age on the plasma concentrations of fat-soluble antioxidants is of particular interest. Several studies have found low dietary intakes and plasma vitamin E and carotenoid concentrations in individuals over 40 years old (30-34). However, these results are controversial. A recent study found significant increases in plasma vitamin E and beta-carotene concentrations with age (35). The present study was undertaken to: 1) characterize plasma concentrations of alpha-tocopherol, beta-carotene and several other carotenoids in a unique population of elderly women, 2) evaluate the relationship between age and plasma antioxidant concentrations in this population and 3) compare the concentrations of plasma antioxidants in middle-aged and elderly individuals. The study population is unique in that it is characterized by advanced age (77-99 years old) and a high level of comparability between individuals due to the absence of several confounding factors. All of the women are Catholic sisters living in the same convent. These women eat food prepared in a central kitchen, do not smoke or drink significant amounts of alcoholic beverages and live in a common building. ln addition, all of the sisters receive nursing and medical care from the same medical staff. METHODS Study population The Nun Study is a longitudinal
AND MATW
study of aging and Alzheimer’s disease. Participants are Roman Catholic sisters in the School Sisters of Notre Dame religious congregation. American sisters in this international congregation reside throughout Eastern, Midwestern, and Southern regions of the United States. In 199193, American sisters born before 1917 were asked to join the study. The sisters were asked to participate in annual assessments of cognitive and physical function, to undergo a brief medical and neurological exam, and to have their blood drawn. All participants allowed investigators full access to their archival and medical records. Birth dates used to determine age in the present study were derived from the archival records. All participants also agreed to donate their brains at death for neuropathologic studies. Of the
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1027 eligible sisters, 678 (66%) agreed to participate in all phases of the study, a high rate of participation considering the brain donation requirement. Limited data available on the nonparticipating sisters included birth date, date of death, and country of birth. Participants did not differ from nonparticipants on mean age, percentage of annual mortality, or country of birth. In 1993, blood was drawn after an overnight fast from 95 sisters living at the Mankato, Minnesota Convent. Plasma antioxidant concentrations were determined in 94 of these sisters. These 94 women were white and predominantly of German heritage. None of the sisters drank significant amounts of alcohol (maximum intakes of two drinks in a week may occur, but only infrequently).
Plasma antioxidantanalysis Antioxidant concentrations were determined from blood collected in red dye-coated EDTA tubes (to minimize carotenoid exposure to light). Samples were placed on ice and plasma obtained by centrifugation. Aliquots of plasma were flushed with nitrogen and stored in amber Wheaton vials at -700 C. The carotenoids (lycopene, beta-carotene, alpha-carotene, zeaxanthin and lutein combined, and betacryptoxanthin) were determined by the HPLC-based method described by Bieri et al. (36). Alphatocopherol was determined by the HPLC-based method as described by Craft et al. (37). Quality control procedures included routine analysis of plasma control pools containing low and high concenWations of each analyte. Calibration was done using crystalline standards with purities greater than 95%. All preparations of carotenoid standards were quantitated by light absorption spectrophotometry, using extinction coefficients as reported by Stacewicz-Sapuntzakis and colleagues (38). Statisticalanalyses Standard statistical methods were used to calculate means, standard deviations and percentiles and to perform T-tests (39). Least squares regression was used to model the relationship between age and the plasma antioxidants. Two-tailed p-values were derived from each statistical test. Other studies indicate a high correlation between plasma cholesterol and alpha-tocopherol concentrations (34). This relationship undoubtiy stems from the role of lipoproteins as the major carriers of plasma alpha-tocopherol. The effect of lipids on alpha-tocopherol values is taken into account for the current study by a separate analysis wherein alpha-tocopherol values are adjusted for cholesterol. Adjustment of alpha-tocopherol values for cholesterol concentrations did not materially change the results or conclusions of this study. Therefore, results are reported without the adjustment for cholesterol values. While the correlations between the major plasma carotenoids and cholesterol are generally lower than those for alpha-tocopherol, the effect of lipids on carotenoid values is taken into account in the same manner as described for alpha-tocopherol. Adjustment of carotenoid values for cholesterol concentrations did not materially change the results or conclusions of this study. Therefore, results are reported without the adjustment for cholesterol values. ULTS The mean age and plasma antioxidant concentrations of the population are shown in Table 1. Mean concentrations of individual carotenoids ranged from 14-30 yg/dl. The mean concentration of alphatocopherol was 0.98 mg/dl. Minimum and maximum values of each carotenoid concentration differed by at least 8.6-fold and indicated significant interindividual variation. A smaller range of values was found for alpha-tocopherol (range=O.44-1.80 mg/dl). Coefficients of variation ranged from 31.6-66.6% for plasma concentrations of carotenoids and alpha-tocopherol. The 94 women had an age range of 77-99 years old. The mean plasma concentrations of most carotenoids and alpha-tocopherol in the population of sisters were similar to higher than those in middle-aged populations. The concentrations of carotenoids in two middle-aged populations are shown in Table 2. Lutein plus zeaxanthin and beta-carotene were similar or marginally higher in the population of sisters than they were in middle-aged populations. Betacryptoxanthin and alpha-carotene were significantly higher in the population of sisters as compared with the other populations. On the other hand, lycopene concentrations were significantly lower in the population of sisters than they were in the two middle-aged populations.
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M.D. GROSS and D.A. SNOWDON TABLE 1 Mean age and Antioxidant Concentration of 94 Participants in the Nun Study
.
rstnbme Variable’ Mean Std Dev Min 50th 25th 75th Age 86.0 6.0 77 81 85 90 Lycopene 15.4 10.0 0 8 14 19 Beta carotene 30.4 19.7 3 16 26 39 Alpha carotene 15.0 9.6 3 9 14 20 Zeaxanthin & lutein 21.6 7.4 5 16 21 26 Beta cryptoxanthin 14.2 8.6 2 9 12 18 Total carotenoids 96.6 42.7 21 66 90 121 Alpha tocopherol 0.98 0.31 0.44 0.76 0.96 1.11 ?? Carotenoids were measured as @dl, and alpha- tocopherol as mg/dl.
Max 99 59 99 53 43 52 235 1.80
TABLE 2
Comparison of 94 Nun Study Participantsto Middle-Aged PopulatllnS
AlltiiXidC3.M
Sisters*’
Middle-aged I#
Middle-aged2 M
Middle-aged3m
Zeaxanthin 8 Lutein 21.6 (7.4) 18.9 (7.4)“’ 19.2 (9.1) 8-Cryptoxanthin 14.2 (8.6) 10.0 (5.4). Lycopene 15.4 (10.0) 40.9 (17.2)’ 18.8 (1.9)’ Alpha-Carotene 15.0 (9.6) 6.5 (5.4)’ 3.8 (2.4)’ O-Carotene 30.4 (19.7) 31.2 (19.9) 23.7 (14.7) 0.98 (.310) 1.13 (361 j’ Alpha-Tocopherol 0.94 (.320) Indicates a statrstrcally signrficant drfference meen the population of sisters and a middle-aged population at the P
Consistent with the higher concentration of lycopene in two middle-aged populations (34,39) as compared with Nun study participants, similar concentrations of lycopene have been reported for other middle-aged and young adult populations (2840). Plasma alpha-tocopherol concentrations were similar among the middle-aged populations and significantly higher in the middle-aged populations as compared to the population of sisters. Age-specific distributions of each analyte are given in Table 3. The means for each plasma antioxidant were very similar across age groups. In a similar manner, indicators of variation (standard deviations and ranges) were quite stable. Only lycopene concentrations had a suggestive trend with age, decreasing in a relatively consistent manner across four age groups.
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TABLE 3 Age-Specific Distribution of Plasma Antioxidants in 94 Participants of the Nun Study*
Min 0 4 0 5 0
25th 7 10 8 10 7
50th 15 14 16 15 13
75th 23 17 19 18 23
Max 59 49 32 24 40
18.7 16.2 21 .o 23.7 23.7
6 3 8 13 9
17 16 13 18 12
29 25 20 28 24
41 36 39 37 52
71 73 99 97 79
18 14 15 17
12.5 6.9 11.6 9.2
3 3 3 4
IO 9 7 10
17 12 13 15
23 17 19 20
52 34 53 38
75-79 80-84 85-89 90-94 95+
19 22 22 23 21
7.1 8.4 8.1 6.7 4.3
5 7 10 13 16
15 16 17 19 18
18 23 23 21 21
24 28 27 25 21
34 43 38 37 32
Beta cryptoxanthin
75-79 80-84 85-89 90-94 95+
15 15 15 12 13
12.3 9.0 8.3 4.4 6.3
2 3 6 6 5
6 9 9 7 8
12 13 12 11 11
16 19 18 16 19
52 42 39 19 25
Total carotenoids
75-79 80-84 85-89 90-94 95+
100 94 96 101 96
51.4 39.3 45.4 38.7 45.3
21 30 39 54 43
65 56 60 76 51
99 91 89 88 86
126 122 120 134 127
235 175 213 197 185
Antioxidant# Lycopene
Age 75-79 80-84 85-89 90-94 95+
Mean 17 16 15 14 15
Beta carotene
75-79 80-84 85-89 90-94 95+
31 28 29 35 34
Alpha carotene
75-79 80-84 85-89 90-94 95+
Zeaxanthin & lutein
Alpha tocopherol
Std Dev 14.1 9.3 8.9 6.1 12.3
75-79 1.04 0.29 0.72 1.19 0.82 0.95 80-84 0.92 0.25 0.50 0.75 0.97 1.08 85-89 0.99 0.32 0.53 0.77 1.18 0.93 90-94 0.97 0.35 0.67 0.70 0.84 1.02 1.02 0.42 0.44 95+ 1.02 0.63 1.26 “Sixteen women were 75 to 79 years old, 32 were 80 to 84 years old, 21 were 8589 years old, 14 were years old, and 11 were 95 years and older. Warotenoids were measured as pg/dl and alpha tocopherol as mg/dl.
1.70 1.72 1.72 1.80 1.70 90 to 94
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and D.A. SNOWDON
A least squares regression analysis of age and antioxidant concentration is shown in Table 4. No differences in plasma antioxidant concentrations were found across age groups: none of the regression equations had a significant slope.
TABLE 4 Change in Plasma Antioxidants per Ten Year Increase in Age as Determined by Least Squares Regression for 94 Participants of the Nun Study.
AntioXidant
Change in Antioxidant Concentration per Ten Year Increase in age (95% Cl)
Lycopene -1.6 (-5.0, 1.9) Beta carotene 2.5 (-4,3, 9.3) Alpha carotene -1 .O (-4.4, 2.3) Zeaxanthin & lutein 0.7 (-1.9, 3.3) Beta cryptoxanthin -1.7 (-4.7, 1.3) Total carotenoids -1 .I (-15.9, 13) Alpha tocopherol 0.003 (-0.103, 0) ?? Carotenoids were measured as ug/dl, and alpha-tocopherol as mgldl.
Value 0.37 0.46 0.55 0.60 0.28 0.89 0.95
PISCUSSION Few studies have measured plasma antioxidant concentrations of elderly individuals, and the results have not been consistent. Knekt et al. (3 1) found a decrease in plasma vitamin E concentrations with age, in males older than 49 years old. Hallfrisch et al. (35) found increases in plasma vitamin E and beta-carotene with age in elderly subjects. However, dietary and plasma data are not consistent for vitamin E in the Hallfrisch et al. study; the apparent relationship between dietary intakes and plasma concentrations for elderly women (>60 years old) is negative. Garry et al. (30) and Tucker et al. (32) found that elderly individuals consume significantly less than the RDA for tocopherol. Ascherio et al. (34) found decreases in zeaxanthin and lycopene with age. These reports suggest that age may be a significant determinant of plasma antioxidant concentrations among elderly individuals, and that age may have an inverse relationship with plasma carotenoid and alpha-tocopherol concentrations. However, the effect of age is controversial and several lifestyle factors may be confounders of the results from the noted reports. The current study reports plasma concentrations of alpha-tocopherol, beta-carotene and several other carotenoids in a unique population of elderly women, and evaluates the relationship between age and plasma antioxidant concentrations in this population. The absence of several known determinants of plasma antioxidants provides for an evaluation of concentrations without multiple adjustments for certain lifestyle factors. Thus, age effects, if present, may be more apparent in this population than in many other elderly populations. The results of our study indicate that low plasma carotenoid and tocopherol concentrations, with the possible exception of lycopene concentrations, are not an inevitable consequence of aging. On the contrary, our population of nonsmoking, nondrinking women, who consume a typically American-type diet, have relatively high concentrations of plasma antioxidants. Concentrations of all analytes, except lycopene, for our study population were similar to or higher than those reported for several middle-aged American populations. Thus, our results indicate that independent, elderly populations can have high
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plasma antioxidant concentrations. While the precise determinants of plasma antioxidants in this population are not known, several factors may contribute to the relatively high concentrations of plasma antioxidants. These include the absence of lifestyle factors (e.g. smoking) that are associated with low plasma antioxidant concentrations, the consistent availability of foods high in the dietary antioxidants, and a consistent pattern of meals; the sisters dine as a group with routine assistance provided for any impaired individuals. Interestingly, the antioxidant concentrations remain stable across age groups within this population. Although lycopene tended to decrease with age, no age-plasma antioxidant relationships were statistically significant. Importantly, a Cox proportional hazards regression analysis (data not shown) of the relationship between plasma antioxidants (with the exception of lycopene) and the risk of total mortality did not reveal a selective survival to old age of individuals with high plasma antioxidants. Thus, the results for each antioxidant with the exception of lycopene are not consistent with an effect of age on plasma concentrations of fat-soluble, dietary antioxidants. Age appears to be, at best, a relatively weak determinant of most plasma carotenoid and tocopherol concentrations in this population, Unlike other plasma carotenoid concentrations in our population, lycopene concentrations were substantially lower in the population of sisters than in middle-aged American populations (34). The average concentration in middle-aged populations is approximately 30 l.@dl as compared with 15.0 clg/dl for our population, a two-fold lower value. These findings are consistent with the results of other studies which have reported an age-dependent decline of lycopene (34). The lack of a strong relationship between age and lycopene in our population may be the result of a narrow age range and the prevalence of certain conditions of aging. A significant relationship exists between physical function and plasma lycopene concentrations in our population (41). Also, our preliminary analysis of the relationship between plasma lycopene concentrations and mortality found a significant negative association. Individuals with low lycopene concentrations had a significantly higher risk of mortality. The relationship is not materially different after adjustment for plasma cholesterol concentrations. This relationship of high lycopene concentrations to survival may blunt and mask an apparent effect of age in that those individuals with relatively high lycopene concentrations appear to live to the most advanced ages. In the future, we plan to expand our antioxidant investigation to the larger population of participants in the Nun study. Several possible mechanisms for lower lycopene concentration at advanced ages are a reduction of dietary intake, decreases in absorption, and an increase in the metabolism of lycopene. While the mechanism for a decline of lycopene concentrations with age is not known, several observations suggest that factors other than dietary intake may have a role. First, dietary lycopene is found almost exclusively in tomatoes and tomato products (29), food components in widespread use throughout the United States and readily available to most populations, including our elderly population. Moreover, the relatively high concentration of other plasma antioxidants in our population suggests an adequate intake of fruits and vegetables. Second, plasma lycopene concentrations are relatively resistant to dietary intake as compared with other carotenoids. While radical dietary changes (switch to purified diets) will shift lycopene concentrations, populations with twofold to threefold differences in fruit and vegetable intake have similar plasma lycopene concentrations (28). Third, plasma cholesterol is a major determinant of plasma lycopene concentrations. Cholesterol concentrations generally increase with age until approximately age 65, afterwhich small declines can occur with age. However, cholesterol concentrations remain high at advanced ages. Thus, an age-related decline of lycopene intake is not a likely mechanism for the decline of lycopene with age. Studies of dietary intake and lycopene metabolism are necessary for clarification of determinants of lycopene concentrations. From the standpoint of disease prevention, studies of elderly populations with relatively high plasma antioxidants may be of particular interest. These populations are at a lower risk of several major degenerative diseases. Thus, identification of the determinants of plasma carotenoid and tocopherol concentrations may provide further information of the factors important in disease prevention.
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Identification of determinants requires an evaluation of demographic, lifestyle, genetic and dietary factors. Further studies are needed to define the major determinants of plasma antioxidants for our population. In particular, detailed nutritional information is not available for the population of sisters and attempts should be made to evaluate the role of carotenoid intake. The estimation of carotenoid intake has been limited until recently by the lack of a database for the carotenoid composition of foods. The recent completion of a massive study on the carotenoid content of foods provides a basis for the estimation of carotenoid intake (29). We intend to collect dietary data and evaluate nutritional factors as determinants of plasma antioxidants in future studies.
This study would not have been possible without the spirited support of the members, leaders, and health care providers of the School sisters of Notre Dame religious congregation. The following people were especially helpful in the present study: Sisters Marlene Manney, Rita Schwalbe, and Gabriel Mary Spaeth; Drs. John Belcher and Christine Tully; and Lydia Greiner, Jeannie Heskett, and Christian Prouty. This study was funded by grants from the National Institute on Aging (grant no. ROl AGO9862 and K04 AG00553).
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for
publication
July
8,
1996.
PH SO271-5317(96)00211-4
PLASMA ANTIOXIDANT CONCENTRATIONS IN A POPULATION ELDERLY WOMEN: FINDINGS FROM THE NUN STUDY
OF
David A. Snowdon Ph.D.2
Myron D. Gross Ph.D.1
IDivision of Epidemiology, School of Public Health, University of Minnesota, 1300 S. Second Street, Suite 300, Minneapolis, Minnesota 55454-1015. 612-624-5417 FAX: 612-625-8950. &Sanders-Brown Center on Aging, and the Department of Preventive Medicine, College of Medicine, University of Kentucky, Lexington, Kentucky.
Low plasma alpha-tocopherol and beta-carotene concentrations have been associated with an increased risk of numerous degenerative diseases, including cancer. Several characteristics that accompany aging, including changes in dietary habits and physiologic capacity, may place elderly populations at a high risk of low plasma antioxidant concentrations. Thus, the present study was undertaken to characterize plasma concentrations of alpha-tocopherol, beta-carotene and several other carotenoids in elderly subjects, to describe the relationship between age and plasma antioxidant concentrations in this population and to compare the concentrations of plasma antioxidants in middle-aged and elderly individuals. The study recruited 94 participants from the Nun Study, a longitudinal study of aging and Alzheimer disease. Women in this population were unique for their advanced age, 77-99 years old, and their comparability across age groups due to the absence of several potential confounders of plasma antioxidant concentrations. The population mean and standard deviation of several plasma carotenoids and alpha-tocopherol concentrations (@dl) were as follows: lycopene, 15.0+/- 10.0; beta-carotene, 30.0+/- 19.7; alpha-carotene, 15.0 +/- 9.6; zeaxanthin plus lutein, 22.0+/- 7.4; beta-cryptoxanthin, 14.0 +/- 8.6 and alpha-tocopherol, 980 +/- 310. Concentrations of all analytes, except lycopene, were similar to or higher than those reported for several middle-aged American populations. Lycopene concentrations were significantly lower in the population of sisters as compared with the middle-aged populations and tended to decrease across age groups within the population of sisters . Age appeared to be a relatively minor determinant of plasma alpha-tocopherol and the concentrations of carotenoids, other than lycopene, in this population. Importantly, the plasma concentrations of most carotenoids and alpha-tocopherol in this population of independent elderly women were apparently adequate on the basis of population comparisons. Further studies of this population may define determinants essential for the maintenance of antioxidant status in elderly populations. Copyright 0 1996 Elsevier Science 1~. Key words: women, carotenoids, elderly, tocopherol and plasma
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RODIJCTION Fat-soluble, dietary antioxidants may have a role in the prevention of conditions and diseases of aging. Comparative studies of mammalian species, including humans, have found a positive correlation between tissue concentrations of the specific antioxidants, vitamin E and beta-carotene, and life span (1). The antioxidants may prevent an accumulation of oxidative damage in the form of mutations and thereby prevent cellular malfunction leading to degenerative conditions and diseases (2,3,4,5). Numerous observational studies have found an inverse association between antioxidant exposure, dietary intake and plasma concentrations, and the incidence of degenerative diseases, including several cancers (6- 10) and cardiovascular disease (11-13). Some, but not all, of the early clinical studies have found a reduction in the incidence of certain cancers (14-17) and cardiovascular disease (18-19) with antioxidant supplementation. ln addition, antioxidants may aid in the prevention of cataracts (20), neurodegeneration (21) and Parkinson’s disease (22). These observations have the support of numerous animal (23), cellular (24-25) and mechanistic studies (26-27). As a consequence of these studies, there has been considerable interest in the carotenoid and alphatocopherol status of various populations. Vitamin E and beta-carotene concentrations are not regulated by homeostatic controls in humans and numerous factors can affect their plasma concentrations. A primary determinant of plasma concentrations is the dietary intake of fruits, vegetables and vitamin supplements(28) which are the major sources of fat-soluble, dietary antioxidants(29). Numerous other factors may significantly modify the relationship between dietary intake and plasma concentrations. These factors include smoking, alcohol consumption, gender, race, age, body mass index and plasma cholesterol concentrations(30-35). Because of the accumulation of oxidative damage and an increasing incidence of degenerative diseases with age, the effect of age on the plasma concentrations of fat-soluble antioxidants is of particular interest. Several studies have found low dietary intakes and plasma vitamin E and carotenoid concentrations in individuals over 40 years old (30-34). However, these results are controversial. A recent study found significant increases in plasma vitamin E and beta-carotene concentrations with age (35). The present study was undertaken to: 1) characterize plasma concentrations of alpha-tocopherol, beta-carotene and several other carotenoids in a unique population of elderly women, 2) evaluate the relationship between age and plasma antioxidant concentrations in this population and 3) compare the concentrations of plasma antioxidants in middle-aged and elderly individuals. The study population is unique in that it is characterized by advanced age (77-99 years old) and a high level of comparability between individuals due to the absence of several confounding factors. All of the women are Catholic sisters living in the same convent. These women eat food prepared in a central kitchen, do not smoke or drink significant amounts of alcoholic beverages and live in a common building. ln addition, all of the sisters receive nursing and medical care from the same medical staff. METHODS Study population The Nun Study is a longitudinal
AND MATW
study of aging and Alzheimer’s disease. Participants are Roman Catholic sisters in the School Sisters of Notre Dame religious congregation. American sisters in this international congregation reside throughout Eastern, Midwestern, and Southern regions of the United States. In 199193, American sisters born before 1917 were asked to join the study. The sisters were asked to participate in annual assessments of cognitive and physical function, to undergo a brief medical and neurological exam, and to have their blood drawn. All participants allowed investigators full access to their archival and medical records. Birth dates used to determine age in the present study were derived from the archival records. All participants also agreed to donate their brains at death for neuropathologic studies. Of the
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1027 eligible sisters, 678 (66%) agreed to participate in all phases of the study, a high rate of participation considering the brain donation requirement. Limited data available on the nonparticipating sisters included birth date, date of death, and country of birth. Participants did not differ from nonparticipants on mean age, percentage of annual mortality, or country of birth. In 1993, blood was drawn after an overnight fast from 95 sisters living at the Mankato, Minnesota Convent. Plasma antioxidant concentrations were determined in 94 of these sisters. These 94 women were white and predominantly of German heritage. None of the sisters drank significant amounts of alcohol (maximum intakes of two drinks in a week may occur, but only infrequently).
Plasma antioxidantanalysis Antioxidant concentrations were determined from blood collected in red dye-coated EDTA tubes (to minimize carotenoid exposure to light). Samples were placed on ice and plasma obtained by centrifugation. Aliquots of plasma were flushed with nitrogen and stored in amber Wheaton vials at -700 C. The carotenoids (lycopene, beta-carotene, alpha-carotene, zeaxanthin and lutein combined, and betacryptoxanthin) were determined by the HPLC-based method described by Bieri et al. (36). Alphatocopherol was determined by the HPLC-based method as described by Craft et al. (37). Quality control procedures included routine analysis of plasma control pools containing low and high concenWations of each analyte. Calibration was done using crystalline standards with purities greater than 95%. All preparations of carotenoid standards were quantitated by light absorption spectrophotometry, using extinction coefficients as reported by Stacewicz-Sapuntzakis and colleagues (38). Statisticalanalyses Standard statistical methods were used to calculate means, standard deviations and percentiles and to perform T-tests (39). Least squares regression was used to model the relationship between age and the plasma antioxidants. Two-tailed p-values were derived from each statistical test. Other studies indicate a high correlation between plasma cholesterol and alpha-tocopherol concentrations (34). This relationship undoubtiy stems from the role of lipoproteins as the major carriers of plasma alpha-tocopherol. The effect of lipids on alpha-tocopherol values is taken into account for the current study by a separate analysis wherein alpha-tocopherol values are adjusted for cholesterol. Adjustment of alpha-tocopherol values for cholesterol concentrations did not materially change the results or conclusions of this study. Therefore, results are reported without the adjustment for cholesterol values. While the correlations between the major plasma carotenoids and cholesterol are generally lower than those for alpha-tocopherol, the effect of lipids on carotenoid values is taken into account in the same manner as described for alpha-tocopherol. Adjustment of carotenoid values for cholesterol concentrations did not materially change the results or conclusions of this study. Therefore, results are reported without the adjustment for cholesterol values. ULTS The mean age and plasma antioxidant concentrations of the population are shown in Table 1. Mean concentrations of individual carotenoids ranged from 14-30 yg/dl. The mean concentration of alphatocopherol was 0.98 mg/dl. Minimum and maximum values of each carotenoid concentration differed by at least 8.6-fold and indicated significant interindividual variation. A smaller range of values was found for alpha-tocopherol (range=O.44-1.80 mg/dl). Coefficients of variation ranged from 31.6-66.6% for plasma concentrations of carotenoids and alpha-tocopherol. The 94 women had an age range of 77-99 years old. The mean plasma concentrations of most carotenoids and alpha-tocopherol in the population of sisters were similar to higher than those in middle-aged populations. The concentrations of carotenoids in two middle-aged populations are shown in Table 2. Lutein plus zeaxanthin and beta-carotene were similar or marginally higher in the population of sisters than they were in middle-aged populations. Betacryptoxanthin and alpha-carotene were significantly higher in the population of sisters as compared with the other populations. On the other hand, lycopene concentrations were significantly lower in the population of sisters than they were in the two middle-aged populations.
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M.D. GROSS and D.A. SNOWDON TABLE 1 Mean age and Antioxidant Concentration of 94 Participants in the Nun Study
.
rstnbme Variable’ Mean Std Dev Min 50th 25th 75th Age 86.0 6.0 77 81 85 90 Lycopene 15.4 10.0 0 8 14 19 Beta carotene 30.4 19.7 3 16 26 39 Alpha carotene 15.0 9.6 3 9 14 20 Zeaxanthin & lutein 21.6 7.4 5 16 21 26 Beta cryptoxanthin 14.2 8.6 2 9 12 18 Total carotenoids 96.6 42.7 21 66 90 121 Alpha tocopherol 0.98 0.31 0.44 0.76 0.96 1.11 ?? Carotenoids were measured as @dl, and alpha- tocopherol as mg/dl.
Max 99 59 99 53 43 52 235 1.80
TABLE 2
Comparison of 94 Nun Study Participantsto Middle-Aged PopulatllnS
AlltiiXidC3.M
Sisters*’
Middle-aged I#
Middle-aged2 M
Middle-aged3m
Zeaxanthin 8 Lutein 21.6 (7.4) 18.9 (7.4)“’ 19.2 (9.1) 8-Cryptoxanthin 14.2 (8.6) 10.0 (5.4). Lycopene 15.4 (10.0) 40.9 (17.2)’ 18.8 (1.9)’ Alpha-Carotene 15.0 (9.6) 6.5 (5.4)’ 3.8 (2.4)’ O-Carotene 30.4 (19.7) 31.2 (19.9) 23.7 (14.7) 0.98 (.310) 1.13 (361 j’ Alpha-Tocopherol 0.94 (.320) Indicates a statrstrcally signrficant drfference meen the population of sisters and a middle-aged population at the P
Consistent with the higher concentration of lycopene in two middle-aged populations (34,39) as compared with Nun study participants, similar concentrations of lycopene have been reported for other middle-aged and young adult populations (2840). Plasma alpha-tocopherol concentrations were similar among the middle-aged populations and significantly higher in the middle-aged populations as compared to the population of sisters. Age-specific distributions of each analyte are given in Table 3. The means for each plasma antioxidant were very similar across age groups. In a similar manner, indicators of variation (standard deviations and ranges) were quite stable. Only lycopene concentrations had a suggestive trend with age, decreasing in a relatively consistent manner across four age groups.
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TABLE 3 Age-Specific Distribution of Plasma Antioxidants in 94 Participants of the Nun Study*
Min 0 4 0 5 0
25th 7 10 8 10 7
50th 15 14 16 15 13
75th 23 17 19 18 23
Max 59 49 32 24 40
18.7 16.2 21 .o 23.7 23.7
6 3 8 13 9
17 16 13 18 12
29 25 20 28 24
41 36 39 37 52
71 73 99 97 79
18 14 15 17
12.5 6.9 11.6 9.2
3 3 3 4
IO 9 7 10
17 12 13 15
23 17 19 20
52 34 53 38
75-79 80-84 85-89 90-94 95+
19 22 22 23 21
7.1 8.4 8.1 6.7 4.3
5 7 10 13 16
15 16 17 19 18
18 23 23 21 21
24 28 27 25 21
34 43 38 37 32
Beta cryptoxanthin
75-79 80-84 85-89 90-94 95+
15 15 15 12 13
12.3 9.0 8.3 4.4 6.3
2 3 6 6 5
6 9 9 7 8
12 13 12 11 11
16 19 18 16 19
52 42 39 19 25
Total carotenoids
75-79 80-84 85-89 90-94 95+
100 94 96 101 96
51.4 39.3 45.4 38.7 45.3
21 30 39 54 43
65 56 60 76 51
99 91 89 88 86
126 122 120 134 127
235 175 213 197 185
Antioxidant# Lycopene
Age 75-79 80-84 85-89 90-94 95+
Mean 17 16 15 14 15
Beta carotene
75-79 80-84 85-89 90-94 95+
31 28 29 35 34
Alpha carotene
75-79 80-84 85-89 90-94 95+
Zeaxanthin & lutein
Alpha tocopherol
Std Dev 14.1 9.3 8.9 6.1 12.3
75-79 1.04 0.29 0.72 1.19 0.82 0.95 80-84 0.92 0.25 0.50 0.75 0.97 1.08 85-89 0.99 0.32 0.53 0.77 1.18 0.93 90-94 0.97 0.35 0.67 0.70 0.84 1.02 1.02 0.42 0.44 95+ 1.02 0.63 1.26 “Sixteen women were 75 to 79 years old, 32 were 80 to 84 years old, 21 were 8589 years old, 14 were years old, and 11 were 95 years and older. Warotenoids were measured as pg/dl and alpha tocopherol as mg/dl.
1.70 1.72 1.72 1.80 1.70 90 to 94
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and D.A. SNOWDON
A least squares regression analysis of age and antioxidant concentration is shown in Table 4. No differences in plasma antioxidant concentrations were found across age groups: none of the regression equations had a significant slope.
TABLE 4 Change in Plasma Antioxidants per Ten Year Increase in Age as Determined by Least Squares Regression for 94 Participants of the Nun Study.
AntioXidant
Change in Antioxidant Concentration per Ten Year Increase in age (95% Cl)
Lycopene -1.6 (-5.0, 1.9) Beta carotene 2.5 (-4,3, 9.3) Alpha carotene -1 .O (-4.4, 2.3) Zeaxanthin & lutein 0.7 (-1.9, 3.3) Beta cryptoxanthin -1.7 (-4.7, 1.3) Total carotenoids -1 .I (-15.9, 13) Alpha tocopherol 0.003 (-0.103, 0) ?? Carotenoids were measured as ug/dl, and alpha-tocopherol as mgldl.
Value 0.37 0.46 0.55 0.60 0.28 0.89 0.95
PISCUSSION Few studies have measured plasma antioxidant concentrations of elderly individuals, and the results have not been consistent. Knekt et al. (3 1) found a decrease in plasma vitamin E concentrations with age, in males older than 49 years old. Hallfrisch et al. (35) found increases in plasma vitamin E and beta-carotene with age in elderly subjects. However, dietary and plasma data are not consistent for vitamin E in the Hallfrisch et al. study; the apparent relationship between dietary intakes and plasma concentrations for elderly women (>60 years old) is negative. Garry et al. (30) and Tucker et al. (32) found that elderly individuals consume significantly less than the RDA for tocopherol. Ascherio et al. (34) found decreases in zeaxanthin and lycopene with age. These reports suggest that age may be a significant determinant of plasma antioxidant concentrations among elderly individuals, and that age may have an inverse relationship with plasma carotenoid and alpha-tocopherol concentrations. However, the effect of age is controversial and several lifestyle factors may be confounders of the results from the noted reports. The current study reports plasma concentrations of alpha-tocopherol, beta-carotene and several other carotenoids in a unique population of elderly women, and evaluates the relationship between age and plasma antioxidant concentrations in this population. The absence of several known determinants of plasma antioxidants provides for an evaluation of concentrations without multiple adjustments for certain lifestyle factors. Thus, age effects, if present, may be more apparent in this population than in many other elderly populations. The results of our study indicate that low plasma carotenoid and tocopherol concentrations, with the possible exception of lycopene concentrations, are not an inevitable consequence of aging. On the contrary, our population of nonsmoking, nondrinking women, who consume a typically American-type diet, have relatively high concentrations of plasma antioxidants. Concentrations of all analytes, except lycopene, for our study population were similar to or higher than those reported for several middle-aged American populations. Thus, our results indicate that independent, elderly populations can have high
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plasma antioxidant concentrations. While the precise determinants of plasma antioxidants in this population are not known, several factors may contribute to the relatively high concentrations of plasma antioxidants. These include the absence of lifestyle factors (e.g. smoking) that are associated with low plasma antioxidant concentrations, the consistent availability of foods high in the dietary antioxidants, and a consistent pattern of meals; the sisters dine as a group with routine assistance provided for any impaired individuals. Interestingly, the antioxidant concentrations remain stable across age groups within this population. Although lycopene tended to decrease with age, no age-plasma antioxidant relationships were statistically significant. Importantly, a Cox proportional hazards regression analysis (data not shown) of the relationship between plasma antioxidants (with the exception of lycopene) and the risk of total mortality did not reveal a selective survival to old age of individuals with high plasma antioxidants. Thus, the results for each antioxidant with the exception of lycopene are not consistent with an effect of age on plasma concentrations of fat-soluble, dietary antioxidants. Age appears to be, at best, a relatively weak determinant of most plasma carotenoid and tocopherol concentrations in this population, Unlike other plasma carotenoid concentrations in our population, lycopene concentrations were substantially lower in the population of sisters than in middle-aged American populations (34). The average concentration in middle-aged populations is approximately 30 l.@dl as compared with 15.0 clg/dl for our population, a two-fold lower value. These findings are consistent with the results of other studies which have reported an age-dependent decline of lycopene (34). The lack of a strong relationship between age and lycopene in our population may be the result of a narrow age range and the prevalence of certain conditions of aging. A significant relationship exists between physical function and plasma lycopene concentrations in our population (41). Also, our preliminary analysis of the relationship between plasma lycopene concentrations and mortality found a significant negative association. Individuals with low lycopene concentrations had a significantly higher risk of mortality. The relationship is not materially different after adjustment for plasma cholesterol concentrations. This relationship of high lycopene concentrations to survival may blunt and mask an apparent effect of age in that those individuals with relatively high lycopene concentrations appear to live to the most advanced ages. In the future, we plan to expand our antioxidant investigation to the larger population of participants in the Nun study. Several possible mechanisms for lower lycopene concentration at advanced ages are a reduction of dietary intake, decreases in absorption, and an increase in the metabolism of lycopene. While the mechanism for a decline of lycopene concentrations with age is not known, several observations suggest that factors other than dietary intake may have a role. First, dietary lycopene is found almost exclusively in tomatoes and tomato products (29), food components in widespread use throughout the United States and readily available to most populations, including our elderly population. Moreover, the relatively high concentration of other plasma antioxidants in our population suggests an adequate intake of fruits and vegetables. Second, plasma lycopene concentrations are relatively resistant to dietary intake as compared with other carotenoids. While radical dietary changes (switch to purified diets) will shift lycopene concentrations, populations with twofold to threefold differences in fruit and vegetable intake have similar plasma lycopene concentrations (28). Third, plasma cholesterol is a major determinant of plasma lycopene concentrations. Cholesterol concentrations generally increase with age until approximately age 65, afterwhich small declines can occur with age. However, cholesterol concentrations remain high at advanced ages. Thus, an age-related decline of lycopene intake is not a likely mechanism for the decline of lycopene with age. Studies of dietary intake and lycopene metabolism are necessary for clarification of determinants of lycopene concentrations. From the standpoint of disease prevention, studies of elderly populations with relatively high plasma antioxidants may be of particular interest. These populations are at a lower risk of several major degenerative diseases. Thus, identification of the determinants of plasma carotenoid and tocopherol concentrations may provide further information of the factors important in disease prevention.
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Identification of determinants requires an evaluation of demographic, lifestyle, genetic and dietary factors. Further studies are needed to define the major determinants of plasma antioxidants for our population. In particular, detailed nutritional information is not available for the population of sisters and attempts should be made to evaluate the role of carotenoid intake. The estimation of carotenoid intake has been limited until recently by the lack of a database for the carotenoid composition of foods. The recent completion of a massive study on the carotenoid content of foods provides a basis for the estimation of carotenoid intake (29). We intend to collect dietary data and evaluate nutritional factors as determinants of plasma antioxidants in future studies.
This study would not have been possible without the spirited support of the members, leaders, and health care providers of the School sisters of Notre Dame religious congregation. The following people were especially helpful in the present study: Sisters Marlene Manney, Rita Schwalbe, and Gabriel Mary Spaeth; Drs. John Belcher and Christine Tully; and Lydia Greiner, Jeannie Heskett, and Christian Prouty. This study was funded by grants from the National Institute on Aging (grant no. ROl AGO9862 and K04 AG00553).
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40. Peng YM, Peng YS, Lin Y, Moon T, Roe DJ, Ritenbaugh C. Concentrations of plasma-tissue-diet relationship of carotenoids, retinoids, and tocopherol in humans. Nutr Cancer 1995; 23:233-246. 41. Snowdon DA, Gross MD, Butler SM. Antioxidants and reduced functional capacity in the elderly: Findings from the Nun Study. J Gerontol 1996; 51A:MlO-16. Accepted
for
publication
July
8,
1996.