Abstract
Importance
Resveratrol, a polyphenol found in grapes, red wine, chocolate, and certain berries and roots, is considered to have antioxidant, anti-inflammatory, and anti-cancer effects in humans and is related to longevity in some lower organisms.
Objective
To determine whether resveratrol levels achieved with diet are associated with inflammation, cancer, cardiovascular disease, and mortality in humans.
Design
Prospective cohort study, the Invecchiare in Chianti (InCHIANTI) Study (“Aging in the Chianti Region”), 1998-2009.
Setting
Two villages in the Chianti area, Tuscany region of Italy.
Participants
Population-based sample of 783 community-dwelling men and women, ≥65 y
Exposure
24-h urinary resveratrol metabolites
Main outcomes and measures
Primary outcome measure was all-cause mortality. Secondary outcomes were markers of inflammation (serum C-reactive protein [CRP], interleukin [IL]-6, IL-1β, and tumor necrosis factor [TNF]-α), and prevalent and incident cancer and cardiovascular disease
Results
Mean (95% Confidence Interval) log total urinary resveratrol metabolite concentrations were 7.08 (6.69, 7.48) nmol/g creatinine. During nine years of follow-up, 268 (34.3%) of the participants died. From the lowest to the highest quartile of baseline total urinary resveratrol metabolites, the proportion of participants who died from all causes was 34.4, 31.6, 33.5, and 37.4%, respectively (P = 0.67). Participants in the lowest quartile had a hazards ratio for mortality of 0.80 (95% confidence interval 0.54, 1.17) when compared with those in the highest quartile of total urinary resveratrol in a multivariable Cox proportional hazards model that adjusted for potential confounders. Resveratrol levels were not significantly associated with serum CRP, IL-6, IL-1β, TNF-α, prevalent or incident cardiovascular disease or cancer. Conclusions: In older community-dwelling adults, total urinary resveratrol metabolite concentration was not associated with inflammatory markers, cardiovascular disease, or cancer, or predictive of all-cause mortality. Resveratrol levels achieved with a Western diet do not have a substantial influence on health status and mortality risk.
Keywords: cancer, cardiovascular disease, inflammation, longevity, resveratrol, InCHIANTI
Resveratrol (3,5,4′-trihydroxystilbene), a polyphenol found in grapes, red wine, peanuts, chocolate, and certain berries and Asiatic plant roots, has been shown to exert anti-inflammatory effects in vitro and following supplementation in animal models,1,2 and to increase lifespan and health in mice fed a high calorie diet.3-6 Studies performed in animal models have shown that resveratrol and other chemically related compounds inhibit sirtuin 1 (SIRT1) and mimic the effects of caloric restriction.6,7 In 1992, Siemann and Creasy postulated that the cardioprotective effects of red wine could be attributed to resveratrol.8 The so-called “French paradox” in which a low incidence of coronary heart disease occurs in the presence of a high dietary intake of cholesterol and saturated fat in France has been attributed to the regular intake of red wine9 and in particular, to resveratrol and other polyphenols contained in wine.10
Some preliminary evidence also suggests that resveratrol in humans may have anti-inflammatory effects, prevent cancer, and diminish arterial stiffness and improve endothelial reactivity in older women.4-6 In a randomized trial of 20 healthy adults, plasma concentrations of C-reactive protein (CRP) and tumor necrosis factor (TNF)-α decreased by about one-third during six weeks of supplementation with a plant extract containing resveratrol.11 In addition, peripheral blood mononuclear cell mRNA expression of interleukin (IL)-6 and TNF-α decreased in the group receiving resveratrol over the same intervention period. In a small crossover trial, a supplement containing resveratrol and polyphenols from muscadine grape suppressed the increase of IL-1β following a high-fat, high carbohydrate meal.12 A recent phase II study of SRT501, a micronized oral formulation of resveratrol that activates SIRT1, in multiple myeloma patients was halted early due to a high level of adverse side effects and renal failure.13
Although resveratrol has attracted a great deal attention due to its effects on inflammation, carcinogenesis, and longevity in vitro or in lower organisms, and in trials involving supra-physiological doses of resveratrol in humans, there is little epidemiological data to support a link between physiological levels of resveratrol achieved with the diet alone and health in humans. Some of the challenges in studying resveratrol in humans are the rapid uptake, metabolism, and excretion of resveratrol and the low concentrations found in plasma.4 Recently, mass spectrometric methods have been developed that allow insights into resveratrol metabolism in humans through the measurement of resveratrol metabolites in urine.14 We hypothesized that higher urinary concentrations of resveratrol metabolites were associated with reduced risk of all-cause mortality and associated with lower inflammation, and lower prevalence and incidence of cardiovascular disease and cancer. To address these hypotheses, we measured urinary metabolites of resveratrol in a population-based cohort study.
Materials and Methods
Study population
The study participants consisted of men and women, aged 65 years and older, who participated in the Invecchiare in Chianti, “Aging in the Chianti Area” (InCHIANTI) Study, conducted in two small towns in Tuscany, Italy. The rationale, design, and data collection methods have been described elsewhere, and the main outcome of this longitudinal study is mobility disability.15 Briefly, in August 1998, 1270 people aged 65 years and older were randomly selected from the population registry of Greve in Chianti (pop. 11,709) and Bagno a Ripoli (pop. 4,704), and of 1,256 eligible subjects, 1,155 (90.1%) agreed to participate. Participants received an extensive description of the study and participated after written, informed consent was obtained. The study protocol complied with the Declaration of Helsinki and was approved by the Italian National Institute of Research and Care on Aging Ethical Committee and by the Institutional Review Board of the Johns Hopkins University School of Medicine.
InCHIANTI Study participants were evaluated again for a three-year follow-up visit from 2001-2003 (n = 926) and six-year follow-up visit from 2004-2006 (n = 844), and nine-year follow-up visit from 2007-2009 (n = 768).
Data collection
Information on demographic characteristics, life style factors and medication use were collected using standardized questionnaires. Smoking history was defined from self-report. Daily alcohol intake, expressed in g/day, and resveratrol intake, expressed in mg/day, were determined at each study visit based upon the European Prospective Investigation into Cancer and Nutrition food frequency questionnaire that had been validated in the Italian population.16 Education was recorded as years of school.
All participants were examined in a standardized manner by a study geriatrician. Diseases were ascertained according to standard, pre-established criteria and algorithms based upon those used in the Women's Health and Aging Study for diabetes mellitus, coronary heart disease, chronic heart failure, stroke, and cancer.17 The diagnostic algorithm for the diagnosis of diabetes was based upon the use of insulin, oral hypoglycemic agents, and a questionnaire administered to the primary care physician of the study participant.17 Systolic and diastolic blood pressures were calculated from the mean of three measures taken with a standard mercury sphygmomanometer during the physical examination. Weight and height were measured using a high-precision mechanical scale. Body mass index (BMI) was calculated as weight/height2 (kg/m2). Mini-Mental State Examination (MMSE) was administered at enrollment, and an MMSE score <24 was considered consistent with cognitive impairment.18 Chronic kidney disease was defined as estimated glomerular filtration rate of <60 mL/min/1.73 m2 using the four-variable Chronic Kidney Disease – Epidemiology Collaboration equation of Levey and colleagues.19
Mortality data were collected using data from the Mortality General Registry maintained by the Tuscany Region. Analyses include those who refused to participate in the follow-up after baseline or those who moved away but were known to be alive at the time of censoring of this analysis. Causes of death were not available for all participants who died because cause-specific data have not yet been released by the Tuscany regional authorities. Therefore, the analysis in the present study is based upon all-cause mortality.
Laboratory studies
Twenty-four hour urine samples were collected from participants at baseline. Urine samples were then aliquoted and immediately stored at -80° C. Of the 1155 adults, ≥65 y, who enrolled in the study, 783 had 24-h urine samples available for measurements of resveratrol. Resveratrol conjugates derived from gut and microbial metabolism were measured in 24-h urine samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS).14 Briefly 1 mL of urine with the internal standard was loaded into a previously equilibrated Oasis HLB solid-phase extraction 96-well plate (30 mg; Waters). Urinary resveratrol metabolites were eluted with acidified methanol solution and ethyl acetate. After evaporation, the samples were reconstituted with 100 μL of the mobile phase and then analyzed in the LC (Perkin-Elmer s200) coupled to a triple-quadrupole mass spectrometer (API3000; Applied Biosystems) as described elsewhere.14 The overall time taken per sample was around 14 min, including the clean-up by solid-phase extraction, optimized runtime by liquid chromatography, and mass spectrometry detection.14 Intra- and inter-batch coefficients of variation were <10.5% and <10.7%, respectively. Because we were uncertain whether all the participants collected urine for a full 24-h period, all results for urinary resveratrol metabolites were corrected for creatinine. Urinary creatinine was measured by the modified Jaffe method,20 and results for 24-h urinary resveratrol metabolites are reported as nanomoles per gram of creatinine.
Serum C-reactive protein (CRP), interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were measured in duplicate by high-sensitivity enzyme-linked immunosorbent assays (ELISA) using commercial kits (Bioscource International, Camarillo, CA) as described in detail elsewhere.21 Commercial enzymatic tests (Roche Diagnostics) were used for measuring serum total cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol concentrations. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula.22 Plasma glucose concentration was measured by the glucose oxidase method (Beckman Instruments, Inc., Fullerton, CA). Normal, impaired, and diabetic fasting glucose were defined as fasting plasma glucose ≤99 mg/dL, 100-125 mg/dL, and >125 mg/dL, respectively.23
Statistical analysis
Variables are reported as means (standard deviations) or as percentages. Variables that were highly skewed (i.e., markers of inflammation) were log-transformed to achieve a normal distribution. Characteristics of subjects were compared across quartiles of urinary resveratrol metabolites using Kruskal-Wallis tests for continuous variables and chi-square tests for categorical variables. Spearman correlation was used to examine the relationship between alcohol intake and urinary resveratrol metabolites. Cox proportional hazards models were used to examine the relationship between quartile of total urinary resveratrol metabolites and all-cause mortality, incident cardiovascular disease, and incident cancer over nine years of follow-up. Multivariable Cox proportional hazards models were adjusted for age, sex, BMI, and then other variables that were significant in the univariate analyses. All analyses were performed using SAS (v. 9.1.3, SAS Institute, Inc., Cary, NC) with a type I error of 0.05.
Results
Overall, mean (95% CI) log total urinary resveratrol metabolite concentrations were 7.08 (6.69, 7.48) nmol/g creatinine. Less than 1% of study participants reported using any type of nutritional supplement. The characteristics of the participants across quartiles of total urinary resveratrol metabolite concentrations are shown in Table 1. There were significantly more males in the highest quartiles of total urinary resveratrol metabolites. Alcohol consumption, current smoking, and physical activity were higher among participants in the highest quartile of total urinary resveratrol metabolites compared with the lower quartiles. The proportion of participants with abnormal fasting plasma glucose was significantly different across quartiles, with the highest proportion of subjects with diabetic fasting glucose in the lowest and highest quartiles. The proportion of participants with cognitive impairment (MMSE<24) was significantly lower in the participants in the highest quartile of total urinary resveratrol metabolites. There were no significant differences across the quartiles of total urinary resveratrol metabolite concentrations by age, education, BMI, CRP, IL-6, IL-1β, TNF-α, mean arterial pressure, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, or by prevalence of hypertension, heart failure, peripheral artery disease, stroke, cancer, and chronic kidney disease. The prevalence of coronary artery disease and diabetes were higher among those in the lowest quartile of total urinary resveratrol metabolites. The Spearman correlation between alcohol consumption in grams per day and total urinary resveratrol metabolite concentrations was 0.67 (P <0.0001).
Table 1. Baseline characteristics of 783 adults, ≥65 years, in the InCHIANTI Study by quartiles of total urinary resveratrol metabolites.
| Characteristic1,2 | Quartiles of Total Urinary Resveratrol Metabolites (nmol/g creatinine) | P3 | ||||
|---|---|---|---|---|---|---|
| <1554 (n = 195) | 1554 – 4996 (n = 196) | 4996 – 15010 (n = 196) | >15010 (n = 196) | |||
| Age, y | 75.2 (7.5) | 74.3 (7.0) | 74.5 (7.0) | 73.8 (6.2) | 0.52 | |
| Sex | Male | 28.7 | 33.2 | 48.2 | 68.7 | <0.0001 |
| Female | 71.3 | 66.8 | 51.8 | 31.3 | ||
| Education, years | 5.0 (2.6) | 5.1 (3.1) | 5.6 (3.8) | 5.8 (3.4) | 0.14 | |
| Alcohol intake, g/day | 2.4 (5.8) | 8.8 (15.9) | 16.3 (16.0) | 31.1 (24.7) | <0.0001 | |
| Consumes alcohol (%) | 37.4 | 66.8 | 87.8 | 99.0 | <0.0001 | |
| Current smoker (%) | 7.7 | 12.8 | 8.6 | 25.1 | <0.0001 | |
| Body mass index (kg/m2) | 27.5 (4.2) | 27.7 (4.1) | 27.5 (3.8) | 27.3 (4.0) | 0.48 | |
| Physical activity (%) | Inactive | 24.6 | 17.9 | 17.9 | 15.0 | 0.002 |
| Low | 44.6 | 51.3 | 41.8 | 36.8 | ||
| Moderate-High | 30.8 | 30.8 | 40.3 | 48.2 | ||
| Fasting plasma glucose (%) | Normal | 72.8 | 80.1 | 75.1 | 69.7 | 0.05 |
| Impaired | 15.9 | 12.8 | 18.8 | 15.9 | ||
| Diabetic | 11.3 | 7.1 | 6.1 | 14.4 | ||
| Log C-reactive protein (μg/mL) | 1.09 (1.07) | 1.08 (0.98) | 0.97 (1.04) | 0.99 (1.01) | 0.52 | |
| Log interleukin-1β (pg/mL) | -2.15 (1.30) | -2.19 (1.14) | -2.24 (1.22) | -2.25 (0.92) | 0.24 | |
| Log interleukin-6 (pg/mL) | 1.08 (0.55) | 1.06 (0.50) | 1.16 (0.58) | 1.17 (0.57) | 0.18 | |
| Log tumor necrosis factor-α (pg/mL) | 1.47 (0.59) | 1.43 (0.52) | 1.45 (0.54) | 1.54 (0.78) | 0.59 | |
| Mean arterial pressure (mm Hg) | 105 (11) | 106 (11) | 105 (10) | 106 (12) | 0.78 | |
| Total cholesterol (mg/dL) | 215 (37) | 223 (41) | 219 (42) | 220 (38) | 0.28 | |
| High density lipoprotein cholesterol (mg/dL) | 56 (14) | 56 (15) | 55 (16) | 58 (15) | 0.45 | |
| Low density lipoprotein cholesterol (mg/dL) | 133 (31) | 142 (36) | 138 (37) | 138 (34) | 0.16 | |
| Triglycerides (mg/dL) | 130 (79) | 132 (63) | 128 (73) | 123 (57) | 0.20 | |
| Mini-Mental State Exam Score <24 (%) | 32.8 | 31.1 | 31.0 | 16.4 | 0.0007 | |
| Hypertension (%) | 48.2 | 50.5 | 44.2 | 47.7 | 0.65 | |
| Coronary artery disease (%) | 6.7 | 1.5 | 7.1 | 3.6 | 0.03 | |
| Heart failure (%) | 6.7 | 3.1 | 4.6 | 3.6 | 0.32 | |
| Peripheral artery disease (%) | 6.2 | 4.6 | 4.1 | 7.7 | 0.40 | |
| Stroke (%) | 4.1 | 3.1 | 5.6 | 3.1 | 0.53 | |
| Diabetes mellitus (%) | 15.9 | 10.2 | 9.1 | 18.5 | 0.02 | |
| Cancer (%) | 7.7 | 6.1 | 7.6 | 3.4 | 0.30 | |
| Chronic kidney disease (%) | 27.2 | 23.0 | 20.3 | 16.4 | 0.07 | |
| Died during follow-up, overall (%) | 34.4 | 31.6 | 33.5 | 37.4 | 0.67 | |
Data are given as mean (standard deviation) or percentage, as indicated.
Conversion to SI units as follows: ethanol 1 gm = 0.0217 moles; total cholesterol, HDL cholesterol, LDL cholesterol in mg/dL × 0.0259 = mmol/L; triglycerides mg/dL × 0.0113 = mmol/L; C-reactive protein in mg/L × 9.524 = μmol/L; interleukin-6 in pg/mL × 0.131 = μmol/L
Kruskal-Wallis test for continuous variables and chi-square test for categorical variables.
We compared the characteristics of the 782 participants with resveratrol measurements with the 273 participants who had no resveratrol measurements at baseline. The participants with no resveratrol measurements had a significantly higher proportion with cognitive impairment (MMSE score <24), stroke, lower physical activity, and mortality compared to participants who had resveratrol measurements. They also had lower total cholesterol, and higher IL-1β and higher TNF-α compared with the participants who had resveratrol measurements. There were no significant differences in age, education, BMI, smoking, chronic disease, or other variables as shown in Table 1 between those with and without resveratrol measurements.
During nine years of follow-up, 268 (34.2%) of the participants died. There were no significant differences in the proportion of participants who died across quartiles of total urinary resveratrol metabolite concentrations. The baseline characteristics of the participants by vital status during follow-up are shown in Table 2. Participants who died were older, more likely to be male, with lower education, lower BMI, physically inactive, with diabetic fasting plasma glucose, and with higher CRP, IL-6, and TNF-α concentrations, higher mean arterial pressure, and lower total, HDL, and LDL cholesterol. A higher proportion of those who died had MMSE<24%, heart failure, peripheral artery disease, stroke, diabetes, and chronic kidney disease. There were no significant differences between participants who lived or died by alcohol intake, current smoking, gut resveratrol metabolites, microbial resveratrol metabolites, total urinary resveratrol metabolites, IL-1β, triglycerides, coronary artery disease, or cancer.
Table 2. Baseline characteristics of 783 adults, ≥65 years, in the InCHIANTI Study, by vital status during 9 years of follow-up.
| Characteristic | Died (n = 268) | Alive (n = 515) | P | |
|---|---|---|---|---|
| Age, y | 79.3 (7.2) | 71.9 (5.3) | <0.0001 | |
| Sex | Male | 51.1 | 41.4 | 0.01 |
| Female | 48.9 | 58.6 | ||
| Education, years | 4.6 (2.9) | 5.8 (3.4) | <0.0001 | |
| Alcohol intake, g/day | 13.4 (16.7) | 15.3 (21.6) | 0.91 | |
| Current smoker (%) | 16.0 | 12.2 | 0.14 | |
| Body mass index (kg/m2) | 26.9 (4.1) | 27.8 (4.0) | 0.002 | |
| Physical activity (%) | Inactive | 34.6 | 10.7 | <0.0001 |
| Low | 41.0 | 45.0 | ||
| Moderate-High | 24.4 | 44.3 | ||
| Fasting plasma glucose (%) | Normal | 72.0 | 75.7 | 0.04 |
| Impaired | 14.6 | 16.5 | ||
| Diabetic | 13.4 | 7.8 | ||
| Log gut resveratrol metabolites (nmol/g creatinine) | 2.29 (8.93) | 1.51 (9.06) | 0.08 | |
| Log microbial resveratrol metabolites (nmol/g creatinine) | 5.72 (7.16) | 6.59 (5.90) | 0.91 | |
| Log total urinary resveratrol metabolites (nmol/g creatinine) | 6.69 (6.40) | 7.29 (5.21) | 0.51 | |
| Log C-reactive protein (μg/mL) | 1.23 (1.10) | 0.92 (0.97) | 0.0002 | |
| Log interleukin-1β (pg/mL) | -2.28 (1.21) | -2.17 (1.12) | 0.13 | |
| Log interleukin-6 (pg/mL) | 1.30 (0.59) | 1.02 (0.51) | <0.0001 | |
| Log tumor necrosis factor-α (pg/mL) | 1.57 (0.58) | 1.43 (0.63) | <0.0001 | |
| Mean arterial pressure (mm Hg) | 107 (11) | 105 (11) | 0.01 | |
| Total cholesterol (mg/dL) | 211 (41) | 224 (38) | <0.0001 | |
| High density lipoprotein cholesterol (mg/dL) | 55 (17) | 56 (14) | 0.07 | |
| Low density lipoprotein cholesterol (mg/dL) | 130 (35) | 141 (34) | <0.0001 | |
| Triglycerides (mg/dL) | 129 (70) | 128 (68) | 0.64 | |
| Mini-Mental State Exam Score <24 (%) | 41.8 | 20.6 | <0.0001 | |
| Hypertension (%) | 50.8 | 46.0 | 0.21 | |
| Coronary artery disease (%) | 4.9 | 4.7 | 0.91 | |
| Heart failure (%) | 9.0 | 2.1 | <0.0001 | |
| Peripheral artery disease (%) | 12.7 | 1.9 | <0.0001 | |
| Stroke (%) | 7.8 | 1.9 | <0.0001 | |
| Diabetes mellitus (%) | 16.8 | 11.7 | 0.05 | |
| Cancer (%) | 7.8 | 5.4 | 0.19 | |
| Chronic kidney disease (%) | 31.3 | 16.7 | <0.0001 | |
Data are given as mean (standard deviation) or percentage, as indicated.
Kruskal-Wallis test for continuous variables and chi-square test for categorical variables.
The relationship between total urinary resveratrol metabolites and all-cause mortality was examined using multivariable Cox proportional hazards models (Table 3). Total urinary resveratrol metabolites concentration was not significantly associated with mortality in models adjusting for age, sex, BMI, serum levels of lipids, chronic diseases, and other variables. The relationship between total urinary resveratrol metabolites and mortality did not change in additional models that included markers of inflammation in addition to the covariates used in the final models (data not shown). Sensitivity analyses were conducted to take into consideration mortality in the first year following resveratrol measurements, as those who died within the first year may have been ill at the time of resveratrol measurements, and potential effects of excessive alcohol consumption, since resveratrol was strongly associated with alcohol intake, as shown previously. In an alternative analysis, 12 participants who died within one year of enrollment were excluded. Total urinary resveratrol metabolites were not significantly related to all-cause mortality in multivariable Cox proportional hazards models adjusting for the same covariates as the models in Table 3 (data not shown). In another analysis, 40 participants who consumed more than four drinks per day (1 drink = 14 g ethanol, or >56 g/day) were excluded. Total urinary resveratrol metabolites were not significantly related to all-cause mortality in multivariable Cox proportional hazards models adjusting for the same covariates as the models in Table 3 (data not shown).
Table 3. Relationship between total urinary resveratrol metabolites and all-cause mortality in separate multivariable Cox proportional hazards models.
| Covariates in models | Quartiles of Total Urinary Resveratrol Metabolites (nmol/g creatinine)1 | P2 | |||
|---|---|---|---|---|---|
| <1554 | 1554 – 4996 | 4996 – 15010 | >15010 | ||
| Age, sex. | 0.83 (0.58, 1.17) | 0.95 (0.67, 1.36) | 0.75 (0.53, 1.05) | 1.00 | 0.55 |
| Age, sex, education, BMI, physical activity, total cholesterol, HDL cholesterol, MMSE score. | 0.74 (0.51, 1.08) | 0.90 (0.62, 1.30) | 0.71 (0.49, 1.02) | 1.00 | 0.30 |
| Age, sex, education, BMI, physical activity, total cholesterol, HDL cholesterol, MMSE score, mean arterial pressure, and chronic diseases.3 | 0.80 (0.54, 1.17) | 1.03 (0.70, 1.51) | 0.84 (0.58, 1.22) | 1.00 | 0.43 |
Hazards ratios and 95% confidence intervals shown for each quartile of urinary resveratrol metabolites relative to the highest quartile (reference).
P-value for trend across quartiles.
Chronic diseases include coronary heart disease, heart failure, stroke, peripheral artery disease, diabetes, cancer, and chronic kidney disease.
In order to corroborate the relationship between urinary resveratrol metabolites and all-cause mortality further, we also examined the relationship of dietary intake of resveratrol with all-cause mortality. In the 783 participants, the mean (95% CI) of log dietary intake of resveratrol in was -2.42 (-2.55, -2.28) mg/day. The Spearman correlation between dietary intake of resveratrol and total resveratrol metabolites was 0.67 (P <0.0001). The relationship between dietary intake of resveratrol and all-cause mortality was examined using a multivariable Cox proportional hazards model with the same covariates as in the final model in Table 3. Compared with the highest quartile of resveratrol intake, the HR (95% CI) for all-cause mortality in the lowest, second, and third quartiles of resveratrol was 1.17 (0.75, 1.81), 1.29 (0.84, 1.99), and 1.42 (0.97, 2.09), respectively, after adjusting for age, sex, education, BMI, physical activity, total energy intake, total cholesterol, HDL cholesterol, MMSE score, mean arterial pressure, and chronic diseases.
Since dietary intake of resveratrol could change over time, we assessed dietary resveratrol intake in 608, 517, and 434 participants seen at the 3-, 6-, and 9-year follow-up visits, respectively. The mean (95% CI) log dietary intake of resveratrol at the 3-, 6-, and 9-year follow-up visits was -2.71 (-2.87, -2.55), -2.82 (-2.99, -2.64), and -2.66 (-2.81, -2.50) mg/day, respectively. The intra-class correlation of dietary intake of resveratrol over the 9-year follow-up period was 0.52. The Spearman correlations between dietary resveratrol intake at the 3-, 6-, and 9-year follow-up visits and urinary resveratrol concentrations at baseline were 0.59 (P <0.0001), 0.56 (P <0.0001), and 0.45 (P <0.0001), respectively.
Of 639 participants who were free of cardiovascular disease at enrollment, 174 (27.2%) developed cardiovascular disease during follow-up. The proportion of participants with incident cardiovascular disease from the lowest to the highest quartile of resveratrol was 22.3, 29.6, 28.4, and 28.0%, respectively (P = 0.44). Of 734 participants who were free of cancer at enrollment, 34 (4.6%) developed cancer during follow-up. The proportion of participants with incident cancer from the lowest to the highest quartile of resveratrol was 4.4, 4.9, 5.0, and 4.3%, respectively (P = 0.98).
Discussion
The present study shows, contrary to all our hypotheses, that urinary resveratrol metabolites are not predictive of longevity in older community-dwelling adults, and, in addition, are not significantly associated with markers of inflammation, prevalent or incident cardiovascular disease or cancer. Resveratrol levels achieved with the diet do not show any apparent protective association with disease and markers of disease in humans and are not associated with lifespan. To our knowledge, this is the first large, observational epidemiological study to examine the relationship between urinary resveratrol from normal dietary intake and health outcomes in humans. To our knowledge, no urinary resveratrol concentration has been established as an effective threshold in humans. Therefore, we analyzed urinary resveratrol concentrations as quartiles, as has been done in a previous study of resveratrol and health outcomes.24
The strengths of the study are the population-based sampling, the strict criteria for assessment of chronic diseases in the cohort, low use of nutritional supplements (<1%), the measurement of multiple biomarkers for inflammation, the high follow-up rates, and the availability of nine years of monitoring of vital events. In addition, urinary resveratrol metabolites were measured in urine samples that had been collected over a 24 h period. Urinary resveratrol levels in the present study are similar to those reported elsewhere.25 The lack of an association between resveratrol, health, and longevity might be due to variability in resveratrol intake in a population that has a large variability in exposure to resveratrol, inter-individual variation and variability of host-gut microbiota,26,27 which might imply that a much larger sample size was needed to detect the association.
Although annual sales of resveratrol supplements have reached $30 million in the United States alone,28 there is limited and conflicting human clinical data demonstrating any metabolic benefits of resveratrol as well as there are no data concerning its safety in high doses and for long term supplementation in older subjects, who are often multimorbid and polymedicated. Supra-physiological doses of resveratrol (100 mg) from Asiatic roots have been shown to decrease circulating levels or expression of CRP, IL-6, TNF-α, and IL-1β in human trials.11,12 In addition, recent trials show that one year of supplementation with lower doses of resveratrol (8 mg) decreased levels of CRP, TNF-α, IL-6/IL-10 ratio and increased the anti-inflammatory cytokine IL-10 in 75 subjects with cardiovascular risk factors.29 Another study of resveratrol, 150 mg/day, for 30 days to healthy obese men did not result in significant changes to circulating CRP, IL-6, IL-1β, and IL-18, but there was a reduction in TNF-α concentrations.30
In the present study of community-dwelling older adults, there were no significant associations between urinary resveratrol metabolites and serum CRP, IL-6, IL-1β, or TNF-α. A previous study showed that resveratrol supplementation decreased fasting plasma glucose in adults with type 2 diabetes31 and in healthy obese men.30 Resveratrol supplementation decreased LDL cholesterol in patients recovering from myocardial infarction.32 In 75 patients with cardiovascular disease, supplementation with grape extract for six months decreased oxidized LDL and ApoB levels.33 In the present study, there was no significant relationship between urinary resveratrol metabolites and total cholesterol, HDL cholesterol, LDL cholesterol, or triglycerides.
On the other hand, there are trials with resveratrol that reported negative results, A trial of resveratrol-enriched grape extract supplementation for one year in hypertensive men with type 2 diabetes mellitus showed no impact of resveratrol on blood pressure, glucose, and lipids, however, there was a significant reduction in serum IL-6 and alkaline phosphatase levels and reduction of the expression of pro-inflammatory cytokines CCL3, IL-1β, and TNF-α.34 Resveratrol supplementation did not change body composition, resting metabolic rate, plasma lipids, or inflammatory markers in a randomized, double-blind, placebo-controlled trial in nonobese women with normal glucose tolerance.33 In addition, resveratrol did not affect its putative molecular targets, including SIRT1, in either skeletal muscle or adipose tissue. In a randomized, double-blind, placebo-controlled trial, high-dose resveratrol supplementation had no effect on glucose metabolism, insulin sensitivity, resting energy expenditure, or inflammatory markers in obese men.35
Resveratrol is only one of many polyphenols that are found in red wine and grapes. In the present study, urinary resveratrol levels were significantly associated with alcohol intake. The study population is located in the wine-growing Chianti region of Tuscany. The moderately high correlation between alcohol intake and urinary resveratrol is most likely attributed to a correlation between wine intake and resveratrol. A previous study has shown that urinary resveratrol levels are a valid biomarker of wine consumption.25 Human studies of the oral absorption of 14C- resveratrol show that the elimination half-life of total resveratrol metabolites is about 6-15 h after oral doses.36 Resveratrol metabolites can be detected in the urine of humans who consume one glass of wine per week if the last drink were consumed three days previously, or in those who consume three glasses of wine per week if the last drink were consumed five days previously.25
In conclusion, this prospective study of nearly eight-hundred older community-dwelling adults shows no association between urinary resveratrol metabolites and longevity. This study suggests that dietary resveratrol from Western diets in community-dwelling older adults does not have a substantial influence on inflammation, cardiovascular disease, cancer, or longevity.
Acknowledgments
This work was supported by National Institutes of Health Grants R01 AG027012, R01 HL094507, the Italian Ministry of Health (ICS110.1/RF97.71), the Spanish Government from INGENIO CONSOLIDER program FUNC-FOOD (CSD2007-00063) and AGL2009 AGL2009-13906-C02-01, NIA contracts 263 MD 9164, 263 MD 821336, N.1-AG-1-1, N.1-AG-1-2111, and N01-AG-5-0002, the Intramural Research Program of NIA, National Institutes of Health, Baltimore, Maryland. The funders had no role in data collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Footnotes
The authors have no conflicts of interest.
Author Contributions: Dr. Semba had full access to all of the data in the study and takes responsibility for the integrity and accuracy of the data analyses.
Design and conduct of the study: Semba, Ferrucci, Bartali, Cherubini, Bandinelli, Andres-Lacueva
Acquisition of data: Ferrucci, Urpí-Sarda, Zamora-Ros, Bandinelli, Andres-Lacueva
Collection, management, analysis, and interpretation of the data: Semba, Ferrucci, Bartali, Urpí-Sarda, Zamora-Ros, Sun, Cherubini, Bandinelli, Andres-Lacueva
Preparation, review, and approval of the manuscript: Semba, Ferrucci, Bartali, Urpí-Sarda, Zamora-Ros, Sun, Cherubini, Bandinelli, Andres-Lacueva
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