Plasma lycopene, other carotenoids, and retinol and the risk of cardiovascular disease in women

¹ From the Division of Preventive Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston (HDS, JEB, and JMG); the Department of Epidemiology, Harvard School of Public Health, Boston (HDS and JEB); the Massachusetts Veterans Epidemiology Research and Information Center, VA Boston Healthcare System, Boston (HDS and JMG); the Department of Ambulatory Care and Prevention, Harvard Medical School, Boston (JEB); and the Department of Medical Research, Our Lady of Mercy Medical Center, and Community and Preventive Medicine, New York Medical College, Bronx, NY (EPN).

² Supported by grants CA-47988 and HL-43851 from the National Institutes of Health, Bethesda, MD, and a grant from DSM Nutritional Products, Inc.

³ Address reprint requests to HD Sesso, Brigham and Women's Hospital, 900 Commonwealth Avenue East, Boston, MA 02215-1204. E-mail: hsesso{at}hsph.harvard.edu.

ABSTRACT  
Background: Growing evidence suggests that lycopene has significant in vitro antioxidant potential. Lycopene has rarely been tested in prospective studies for its role in cardiovascular disease (CVD) prevention.

Objective: We examined the association between plasma lycopene and the risk of CVD in middle-aged and elderly women.

Design: A prospective, nested, case-control study was conducted in 39 876 women initially free of CVD and cancer in the Women's Health Study. Baseline blood samples were collected from 28 345 (71%) of the women. During a mean of 4.8 y of follow-up, we identified 483 CVD cases and 483 control subjects matched by age, smoking status, and follow-up time. Plasma lycopene, other carotenoids, retinol, and total cholesterol were measured.

Results: In analyses matched for age and smoking, with adjustment for plasma cholesterol, the relative risks (RRs) and 95% CIs of CVD in increasing quartiles of plasma lycopene were 1.00 (referent), 0.78 (95% CI: 0.55, 1.11), 0.56 (0.39, 0.82), and 0.62 (0.43, 0.90). In multivariate models, the RRs were 1.00 (referent), 0.94 (0.60, 1.49), 0.62 (0.39, 1.00), and 0.67 (0.41, 1.11); those in the upper compared with the lower half of plasma lycopene had an RR of 0.66 (0.47, 0.95). For CVD, exclusive of angina, women in the upper 3 quartiles had a significant multivariate 50% risk reduction compared with those in the lowest quartile. The stepwise addition of individual plasma carotenoids did not affect the RRs.

Conclusions: Higher plasma lycopene concentrations are associated with a lower risk of CVD in women. These findings require confirmation in other cohorts, and the determinants of plasma lycopene concentrations need to be better understood.

Key Words: Lycopene • cardiovascular disease • women • prospective studies • nutrition

INTRODUCTION  
There is growing evidence from many studies that lycopene, a carotenoid without provitamin A activity and found in high concentrations in a small number of plant foods (tomato, watermelon, pink grapefruit, papaya, and apricot), has significant antioxidant potential in vitro, which suggests a role in preventing cardiovascular disease (CVD) (1). More than 80% of the lycopene intake in the American diet is from the consumption of tomato products, including ketchup, tomato juice, and tomato sauces (2). Lycopene may have a cholesterol synthesis–inhibiting effect that may enhance LDL degradation (3, 4).

Some (4, 5), but not all (6), dietary intervention studies involving either lycopene-containing foods or lycopene supplementation have shown potential short-term improvements in LDL oxidation. However, whether these apparent short-term benefits translate into long-term improvements in health, manifested by a reduction in the risk of CVD, remains unknown. Limited data suggest an inverse association between lycopene and CVD (7-13). Data concerning the relation between plasma lycopene concentrations and CVD risk are limited, particularly in women. Lingering questions remain about whether all carotenoids (9), specific carotenoids such as lutein/zeaxanthin (14), or carotenoids in general (15) are associated with a reduced risk of CVD.

Therefore, we evaluated baseline concentrations of plasma lycopene, other carotenoids, and retinol in middle-aged and elderly women who were initially free of CVD in the Women's Health Study (WHS) to examine their relation with the subsequent risk of CVD in female health professionals in the United States.

SUBJECTS AND METHODS  
Study population
The WHS is an ongoing randomized, double-blind, placebo-controlled 2 x 2 factorial trial of the relation between low-dose aspirin and vitamin E and the primary prevention of CVD and cancer (16). The ß-carotene component of the trial was terminated in 1996 after a median follow-up of 2.1 y, in part because of the lack of effect of ß-carotene on cancer incidence in the Physicians' Health Study (17, 18). A total of 39 876 female US health professionals, who were aged =" BORDER="0">45 y in 1992, were postmenopausal or not intending to become pregnant, and had no history of myocardial infarction (MI), stroke, transient ischemic attack, or cancer (except for nonmelanoma skin cancer) were enrolled in the study. Fasting baseline blood samples were collected from 28 345 (71%) participants and stored in liquid nitrogen until analyzed.

A prospective, nested, case-control design was used to identify 483 case-control pairs of WHS participants. Cases included women who provided baseline blood samples and subsequently experienced a cardiovascular event, defined as CVD death, nonfatal MI, nonfatal stroke, percutaneous transluminal coronary angioplasty, coronary artery bypass graft, or angina pectoris. Study physicians conducted blinded reviews of all cases. CVD death was documented on the basis of convincing evidence of a cardiovascular mechanism from death certificates and medical records. The diagnosis of MI was confirmed on the basis of World Health Organization criteria (19). A stroke was defined as a typical neurologic deficit, sudden or rapid in onset, that lasted >24 h. Revascularization procedures were confirmed by hospital records. Angina was self-reported on questionnaires and confirmed in a small proportion of cases through hospital records.

Each case of CVD was matched with a control subject according to age (±1 y), smoking status (never, former, and current), and follow-up time (±6 mo). In addition, each woman selected as a control must have provided a baseline blood sample and remained free of CVD during the follow-up period. The Institutional Review Board at Brigham and Women's Hospital approved all procedures, and written informed consent was obtained from all participants.

Blood assays and covariates
All investigators and laboratory personnel were blinded to the subject's case-control status. Blood samples from all subjects were handled identically and blindly through all stages of the blood collection, storage, retrieval, and analytic processes. The storage time for plasma in liquid nitrogen freezers was 7 y until the assays were conducted. Baseline plasma blood samples from cases and controls were thawed and assayed for total lycopene, other carotenoids, and retinol at Our Lady of Mercy Medical Center, Bronx, NY. All analytes were quantitated by reversed-phase HPLC after extraction with the use of standard methods (20). Internal standards (echinenone for carotenoids and retinyl proprionate for retinol) were used to correct for recoveries of all samples that were analyzed, and the laboratory has participated in the US Quality Assurance Program to ensure consistent methodology with other laboratories. Plasma total cholesterol also was assayed by enzymatic, endpoint spectroscopy with the use of commercially available diagnostic kits (Sigma-Aldrich Chemical Co, St Louis) and conventional methods (21, 22). Plasma total cholesterol was assayed because plasma lipoproteins are nonspecific carriers for all the carotenoids in plasma and, to date, total cholesterol appears to be the best way to control for confounding effects due to differences in lipoprotein concentrations between subjects (23). The laboratory maintains its own sets of internally and externally prepared control specimens that are assayed in every run. From these control specimens, the laboratory accuracy was within 7% for each measured carotenoid and retinol, whereas the day-to-day and within-day precision (CV) for these analytes was 5%.

On the WHS baseline questionnaire, women also provided data on age (in y), weight and height (converted to body mass index; in kg/m²), smoking status (categorized as never, former, or current), alcohol use (categorized as rarely or never, 1–3 drinks/mo, 1–6 drinks/wk, and =" BORDER="0">1 drink/d), frequency of exercise (categorized as rarely or never, <1 time/wk, 1–3 times/wk, and =" BORDER="0">4 times/wk), parental history of MI at <60 y (no, yes), history of hypertension (no, yes), history of diabetes (no, yes), history of hypercholesterolemia (no, yes), and postmenopausal hormone use (categorized as never, former, or current). Women also completed a 131-item validated semiquantitative food-frequency questionnaire developed by Willett et al (24), from which the dietary intake of specific nutrients included lycopene (µg/d), total fiber (g/d), folate (µg/d), and saturated fat intake (g/d), which were each adjusted for total energy intake with the use of the residual method (25). The total fruit and vegetable intake was summed and expressed as servings/d.

Data analyses
Women were first compared according to case-control status by using mean values or proportions of baseline coronary disease risk factors and biochemical markers. Measurements of plasma lycopene concentrations were divided into fourths on the basis of overall distribution in the 483 controls. Coronary disease risk factors were also compared according to quartiles of plasma lycopene among the control population to assess potential confounding with the use of analysis of variance for continuous variables, Cochran-Armitage trend tests for dichotomous variables, or chi-square tests for categorical variables. In this same group of women, age-adjusted Spearman correlation coefficients were used to assess the associations between plasma lycopene, its primary food sources (tomatoes, tomato juice, tomato sauce, and pizza; all expressed as servings/d), and dietary lycopene.

We used conditional logistic regression analyses to compute RRs and 95% CIs for future CVD risk for increasing quartiles of plasma lycopene concentrations, with the lowest quartile as the referent. Models were first adjusted for randomized treatment assignments and plasma total cholesterol concentrations; the next model added body mass index, exercise, parental history of MI before the age of 60 y, hypertension, diabetes, postmenopausal hormone use, and hypercholesterolemia. Another multivariate model added dietary factors, including intakes of fruit and vegetables, alcohol, fiber, folate, and saturated fat. A fourth model included dietary lycopene intake to examine whether any association between plasma lycopene and CVD may be independent of its dietary component.

Linear trend tests across quartiles of plasma lycopene concentrations were tested by using the median concentrations for each quartile as an ordinal variable. Post hoc tests comparing quartiles 3 and 4 versus quartiles 1 and 2 for CVD, and quartiles 2–4 versus quartile 1 for important vascular events, were also conducted in light of an apparent threshold of plasma lycopene above which there was a possible reduced risk of CVD. Because of concern that discrepant results may exist for angina pectoris, we repeated the models with 339 case-control pairs after excluding 144 cases of angina pectoris. We also examined whether other plasma carotenoids besides lycopene or retinol added individually to a multivariate model attenuated the association between plasma lycopene and the risk of CVD. We then compared multivariate models for plasma lycopene with those for retinol, ß-cryptoxanthin, and lutein/zeaxanthin, with each biochemical marker divided into fourths among the controls and entered into a separate model. All analyses were conducted with the use of SAS (version 8; SAS Institute Inc, Cary, NC).

RESULTS  
Of the 483 cases identified with CVD, there were 109 cases of MI, 112 cases of stroke (86 ischemic, 24 hemorrhagic, and 2 other), 85 cases of revascularization, 33 cases of CVD death, and 144 cases of angina pectoris. The mean (±SD) follow-up time was 4.8 ± 2.4 y for cases and controls matched for follow-up time. Baseline lifestyle, clinical, and dietary risk factors for cases and controls at the start of the study are compared in Table 1. As expected, women who developed CVD tended to weigh more, and more of these women had a history of hypertension, diabetes mellitus, and hypercholesterolemia than did those women who remained free of CVD. Women who developed CVD also had higher total cholesterol concentrations and were more likely to have a parent with a history of MI before the age of 60 y. Age and smoking status were identical in the cases and controls because of our matching criteria. Small differences in dietary factors between the cases and controls were observed.

View this table:
TABLE 1. Baseline characteristics of 483 women who subsequently developed cardiovascular disease (cases) and an equal number of women who remained free of cardiovascular disease (controls)

 
The mean plasma lycopene concentration in women who developed CVD was 16.2 ± 7.6 µg/dL and in the controls was 16.9 ± 8.2 µg/dL. Plasma lycopene concentrations ranged from 2.3 to 81.4 µg/dL in the 483 controls free of CVD and were significantly correlated with dietary lycopene concentrations (r = 0.14, P = 0.004), although the magnitude of this age-adjusted Spearman correlation coefficient was low and suggested little or no correlation. Of the major lycopene food sources, tomato sauce was the only food source with a significant age-adjusted Spearman correlation coefficient (r = 0.16, P = 0.001) with plasma lycopene in the controls. Intake of other dietary factors—cheese, refined grains, sodium, and red meat—were not correlated with plasma lycopene concentrations.

Baseline characteristics of the subjects according to quartiles of plasma lycopene in the 483 women who remained free of CVD are shown in Table 2. Women with higher plasma lycopene concentrations tended to be younger, consume more alcohol, and weigh less. In contrast, only women in the highest quartile of plasma lycopene were more likely to be former smokers. Total cholesterol increased with increasing quartiles of plasma lycopene (P < 0.001), as did the proportion of women with a history of hypercholesterolemia (P = 0.04). No appreciable differences in dietary factors were observed, other than lycopene intake (P = 0.03), which was higher in higher quartiles of plasma lycopene. The proportion of women randomly assigned to active treatment were equally distributed within each quartile of plasma lycopene (all P > 0.05).

View this table:
TABLE 2. Comparison of baseline characteristics of 483 controls according to quartile of plasma lycopene

 
In the analyses matched for age and smoking and with adjustment for randomized treatment assignments and plasma total cholesterol concentrations, the risk of total CVD appeared to decrease with higher concentrations of plasma lycopene (Table 3). The RRs of total CVD for women in the lowest to highest quartiles of plasma lycopene were 1.00 (referent), 0.78, 0.56, and 0.62. Exclusive of cases of angina, the risk reductions for CVD were slightly stronger, with a 52% risk reduction among women in the second through fourth quartiles. For total CVD, despite a borderline significant multivariate linear trend (P = 0.05), the pattern in RRs compelled us to do a post hoc comparison of women in the upper compared with the lower half of plasma lycopene concentrations. We found that the women in the upper half of plasma lycopene had a significant 34% reduction in the risk of total CVD. Similarly, for women with CVD (exclusive of angina), an L-shaped association was apparent with a consistent reduction in risk starting at the second quartile. In fact, women in the upper 3 quartiles of plasma lycopene had a significant 50% reduction in the risk of CVD (exclusive of angina) compared with those in the lowest quartile of plasma lycopene. Multivariate models that included dietary lycopene did not appreciably confound the RRs for plasma lycopene.

View this table:
TABLE 3. Relative risks (RRs) and 95% CIs of cardiovascular disease (CVD) and important vascular events (myocardial infarction, stroke, and CVD death, exclusive of angina) according to quartiles of plasma lycopene

 
Plasma lycopene was most strongly correlated with plasma ß-carotene (Spearman r = 0.34), followed by ß-cryptoxanthin (r = 0.33), lutein/zeaxanthin (r = 0.29), -carotene (r = 0.28), and retinol (r = 0.07). We also individually and separately added other plasma carotenoids and retinol to the multivariate model and found no differences in the RRs, which suggested that these correlated nutritional biomarkers did not explain the reductions in total CVD risk for plasma lycopene shown in Table 3.

We then compared multivariate models for plasma lycopene with retinol and other carotenoids for the risk of CVD (Table 4). Increasing quartiles of plasma retinol, ß-cryptoxanthin, and lutein/zeaxanthin were not associated with the risk of CVD. Results for other plasma markers related to the WHS randomized treatments (-carotene and ß-carotene) further showed a distinct inverse association limited to lycopene on CVD among the carotenoids investigated (data not shown).

View this table:
TABLE 4. Multivariate relative risks (RRs) and 95% CIs of cardiovascular disease (CVD) in a comparison of quartiles of plasma lycopene with other plasma carotenoids and retinol in separate models

 
Other potential coronary disease risk factors collected in the WHS that may be potential confounders (blood pressure, multivitamin use, and other dietary factors) did not significantly affect our findings. Because of the concern that plasma lycopene may have differential effects on the risk of stroke and nonstroke, we ran separate models for these 2 endpoints. The multivariate RRs were similar, which suggested similar effects for plasma lycopene for stroke and nonstroke CVD endpoints. All tests for interaction between plasma lycopene and potential modifiers of interest (baseline age, smoking status, dietary lycopene intake, and each randomized treatment) were not significant (all P > 0.05), because the RRs were not different within each stratum (data not shown).

DISCUSSION  
Our prospective data indicate an apparent L-shaped association between increasing quartiles of plasma lycopene and a lower risk of CVD in middle-aged and elderly women that appears to be independent of major lifestyle, clinical, and dietary risk factors. Women with concentrations of plasma lycopene greater than the median (=" BORDER="0">16.5 µg/dL) had a possible 34% reduction in total CVD compared with those in the lowest quartile after multivariate adjustment. The magnitude of effect was stronger after women with angina were excluded. Additional adjustment for other plasma carotenoids and retinol did not explain the association between plasma lycopene and CVD. Individual results for other plasma carotenoids and retinol showed that plasma lycopene might have a distinct inverse association with the risk of CVD that warrants further investigation.

Although most research on lycopene has focused on a potential reduction in the risk of prostate cancer (26), limited epidemiologic data support a preventive effect of lycopene against CVD. The broad range of plasma lycopene concentrations in the WHS is at the low end of the range of lycopene measurements reported in previous observational studies (9, 10, 27-35). This may reflect differences in assay methods, diet, and baseline disease status or between serum and plasma concentrations of lycopene across study populations. This raises questions about the absence of any specific evidence of a possible threshold or L-shaped effect for plasma lycopene in other studies. Two reports suggest that intimal wall thickness and the risk of MI are lower in persons with higher adipose tissue concentrations of lycopene (7, 8). Another study of Finnish men and women noted similar significant decreases in intimal wall thickness in men but not in women (34). Other studies report possible inverse associations between serum lycopene concentrations and a reduced risk of MI (9, 35), CVD (11, 12, 35), carotid atherosclerosis (10, 13), and aortic atherosclerosis (36).

Despite early promise from initial epidemiologic studies, little is known regarding the specific biological mechanisms responsible for the lycopene-associated reduction in CVD. Lycopene is one of the most potent singlet oxygen quenchers, which suggests that it may have comparatively stronger antioxidant properties than other major plasma carotenoids (37). Lycopene may also contribute to the regulation of cholesterol metabolism in cell cultures because the addition of lycopene to macrophage cell lines has been shown to augment the activity of macrophage LDL receptors (3). In 2 intervention studies, lycopene reduced LDL oxidation (4, 5). However, lycopene supplementation over several weeks did not reduce LDL cholesterol (4). Lycopene and other carotenoids may also be associated with an acute phase response in atherosclerosis. Of the carotenoids, only lycopene modulated adhesion molecule expression in human aortic endothelial cell cultures, which suggests a role in mitigating atherogenesis (38). In the cross-sectional third National Health and Nutrition Examination Survey, nonsmoking subjects in the upper 15% of the C-reactive protein distribution had significantly lower serum lycopene concentrations (28). Another study of elderly nuns also found that elevated C-reactive protein concentrations were associated with lower plasma lycopene concentrations (39).

Dietary, lifestyle, and other factors that influence plasma lycopene concentrations must be considered given the possible inverse association with CVD. In our study, dietary and plasma lycopene concentrations were weakly but significantly correlated, whereas other studies reported stronger correlations (29). Some small-scale dietary intervention studies indicate that lycopene supplements or tomato products may increase plasma lycopene concentrations in healthy subjects (4-6). Correlations among tomato products with plasma lycopene were highest for tomato sauce, which is consistent with the lipophilic properties of lycopene that require the simultaneous ingestion of fats for optimal absorption (40, 41). No other dietary factors were strongly associated with plasma lycopene. Therefore, beyond modest effects for tomato sauce in our data, no clear dietary pattern emerged that greatly affected plasma lycopene concentrations. Other factors related to lycopene absorption and metabolism may also confound the association between plasma and dietary lycopene. For example, small, dense LDL—a potential independent coronary risk factor (42)—carries proportionally less lycopene than do LDL particles of other sizes (43).

Some potential limitations of the present study also warrant discussion. First, we relied on a single baseline measurement of plasma lycopene, which raises the possibility of regression to the mean that may bias our RRs toward the null hypothesis, thus underestimating the observed risk reductions. Second, we have not directly assessed the long-term stability of plasma lycopene stored at -140 °C in liquid nitrogen–chilled freezers since 1993. However, studies that have compared the long-term stability of plasma lycopene and other carotenoid measurements support the stability of the biochemical markers used in this study (44, 45). Third, because we observed a potential threshold effect in reducing the risk of CVD, we performed post hoc tests that compared the upper with the lower half of plasma lycopene concentrations for the risk of total CVD and the upper 3 quartiles with the lowest quartile for CVD (exclusive of angina). Although such tests yielded results that support the possibility of a threshold effect, they must be interpreted with caution because we had not anticipated the observed pattern of RRs that emerged in our results.

Next, we relied on a composite endpoint for total CVD that included MI, stroke, revascularization, CVD death, and angina. Heterogeneity in the results by specific CVD endpoints cannot be ruled out because we had limited power to assess any single outcome. The exclusion of angina case-control pairs strengthened the inverse association, which suggested that plasma lycopene may prevent more serious CVD outcomes. Another potential limitation was that we lacked information on whether ß-carotene or vitamin E treatment affected carotenoid concentrations. However, because the randomized treatment status was evenly distributed within each quartile of plasma lycopene, potential confounding was minimized. Finally, residual confounding may also be of concern, as it would in any epidemiologic investigation. However, in addition to matching for age, smoking, and follow-up time, we comprehensively controlled for other coronary disease risk factors.

In conclusion, higher plasma lycopene concentrations may be associated with a lower risk of CVD in middle-aged and elderly women. Alternatively, plasma lycopene may simply be a marker for another dietary or nutritional component that travels with lycopene in foods. It remains unclear whether the inverse association between plasma lycopene and CVD risk follows a strict linear dose-response trend or is L-shaped, as observed in the present study. Further research on the potential mechanistic actions of lycopene in the prevention of CVD and other chronic diseases is warranted.

ACKNOWLEDGMENTS  
We acknowledge the crucial contributions of the entire staff of the WHS, including the leadership of David Gordon, Susan Burt, Mary Breen, Marilyn Chown, Lisa Fields-Johnson, Georgina Friedenberg, Inge Judge, Jean MacFadyen, Geneva McNair, David Potter, Claire Ridge, and Harriet Samuelson. We are also indebted to the 39 876 dedicated and committed participants of the WHS.

HDS helped conceive and design the study, analyze and interpret the data, draft the article, and obtain funding. JEB helped critically revise the article, collect and assemble the data, and obtain funding. EPN helped critically revise the article and collect and assemble the data. JMG helped conceive and design the study, interpret the data, critically revise the article, and obtain funding. The authors had no financial or personal interests related to this research.

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