a The Division of Human Nutrition and Epidemiology, Wageningen University and Research Center, Wageningen, The Netherlands.
b Department of Medicine, Division of General Internal Medicine, University Hospital Nijmegen, Nijmegen, The Netherlands.
Reprint requests to: FG de Waart, Wageningen University and Research Center, Division of Human Nutrition and Epidemiology, Dreijenlaan 1/Bodenr. 154, 6703 HA Wageningen, The Netherlands. E-mail: frouwkje. hans{at}consunet.nl
Abstract
Background Although ß-carotene has shown inverse associations with chronic diseases involving free radical damage in observational epidemiological studies less attention has been paid to five other major carotenoids also showing antioxidant activity in vitro.
Methods We studied the associations between 7.2-year mortality and serum levels of six carotenoids, and -tocopherol, measured in stored serum, sampled in 1991/1992 during a health survey among 638 independently living elderly subjects aged 6585 years. Proportional hazards regression was used to estimate hazard ratios of all-cause mortality for the lowest tertiles of serum vitamins with the highest tertiles, adjusting for possible confounding effects.
Results During a follow-up period of 7.2 years 171 elderly died. The adjusted hazard ratios for all-cause mortality for the lowest tertiles of vitamins compared with the highest tertiles were between 1.02 and 1.73. The strongest increase in mortality risk was seen for ß-cryptoxanthin (1.52, 95% CI : 1.00, 2.32), lutein (1.56, 95% CI : 1.05, 2.31) and zeaxanthin (1.32, 95% CI : 0.89, 1.97) and their sum (oxygenated carotenoids: 1.73, 95% CI : 1.12, 2.67). Tests for trend were significant (P < 0.05) for all-cause mortality risk and serum levels of total carotenoids, oxygenated carotenoids and ß-cryptoxanthin.
Conclusions Our findings suggest that serum levels of individual carotenoids, particularly the oxygenated species are inversely associated with all-cause mortality and should be considered as candidates for further investigations.
Keywords -tocopherol, carotenoids, all-cause mortality
Accepted 5 January 2000
Observational studies both on dietary intake of ß-carotene or ß-carotene rich products and serum or adipose tissue levels of ß-carotene products show inverse associations with cancer and cardiovascular disease.1 Observational studies on the five other major carotenoids show that -carotene intake was a better predictor for lung cancer than ß-carotene2 and that lycopene and its main source, tomato products, were inversely associated with prostate cancer3 and myocardial infarction.4 Lower levels of serum concentrations of lutein/zeaxanthin and ß-cryptoxanthin were observed in cases with asymptomatic atherosclerosis than in controls.5 Plasma levels of ß-cryptoxanthin were also lower in a high than in a low coronary heart disease (CHD) incidence area6 and in cases with upper aerodigestive tract cancer than in matched controls.7 The outcomes of these studies suggest that individual carotenoids, or the products from which they are derived, in isolation or in combination, are potentially protective against disease processes involving free radical damage.8
In order to add to the limited epidemiological evidence we studied the association between 7.2-year all-cause mortality and the individual carotenoid components ß-carotene, -carotene, lycopene, ß-cryptoxanthin, lutein and zeaxanthin among Dutch elderly. Furthermore,
-tocopherol (vitamin E) was included as an important antioxidant vitamin and also combined with the individual carotenoids in an antioxidant vitamin index.
Subjects and Methods
Population and design
The present study utilized data derived from a survey conducted in 1991/1992 on lifestyle and health among non-institutionalized Dutch elderly living in the city of Arnhem, aged 6585. Mortality follow-up data were collected consecutively. The selection of this study population has been described in detail elsewhere.9
Between 28 October 1991 and 6 April 1992 a random sample of the elderly, pre-stratified on sex and age, participated in a health survey, including home interviews (n = 1012) and a physical examination (n = 685) which included measurements of height, weight, blood pressure, electrocardiographic characteristics and spirometric function. In addition, non-fasting blood samples were taken. Of the 685 undergoing physical examination 641 donated a blood sample. For three people the amount of serum available for the vitamin analyses was too small. This left 638 subjects for these analyses.
Written informed consent was obtained from the subjects prior to the physical examination.
Mortality follow-up
Data on death were obtained from the municipal register of Arnhem. After the collection of baseline data, information on death and migrations was reported to our department every 6 months. Survival status for those who moved from Arnhem during follow-up (n = 70) was checked starting from February 1998 with the municipal authorities of the new residence. Vital status could be determined for all but one. Survival time in years began with the date of the blood collection in 1991/1992 and continued until date of death, date of checking vital status, date of loss to follow-up or 23 March 1999 whichever came first.
Total mortality of the 638 subjects after a mean follow-up time of 7.2 years was 27% (n = 171; males n = 108, females n = 63).
Serum antioxidant vitamins and cholesterol
Non-fasting serum samples had been stored for a mean time of 6 years at 80°C before vitamin analyses. Following extraction,10 concentrations of the carotenoids -carotene, ß-carotene, lycopene, lutein, zeaxanthin and ß-cryptoxanthin and of
-tocopherol were measured in serum, by reverse-phase HPLC (adapted from Hess et al.11 and Craft and Wise12). Detection after separation was carried out using two UV detectors, one for determination of carotenoids (UV 2000) and one (UV 1000) for determination of
-tocopherol. The coefficient of variation (CV) of pooled serum measured in duplicate in every run (n = 17) was 12.3% for
-carotene, 10.2% for ß-carotene, 23.9% for lycopene, 10.7% for ß-cryptoxanthin, 14.9% for zeaxanthin, 7.2% for lutein, and 5.1% for
-tocopherol.
Total cholesterol was determined by an enzymatic method (CHOD-PAP method13).
Other characteristics
The interview, which was mainly pre-coded, included questions on physical activity, drinking and smoking habits, chronic diseases, use of health care, medication, supplements and personal characteristics. Physical activity was assessed by a validated questionnaire on household activities, sports and other physically active leisure time activities, developed for the free-living elderly. From the responses a total activity score was calculated using an intensity code based on net energetic costs of the specific activities.14 The presence of diseases was assessed using a list of chronic diseases and conditions. The use of prescribed medication was asked with a reference period of 3 months prior to the interview. Cardiovascular disease (CVD) was considered present in subjects consulting a physician for heart disease, stroke or peripheral vascular disease 3 months prior to interview or in subjects using drugs for CVD; hypertension in subjects reporting the use of antihypertensive medication, or having a systolic blood pressure 160 mmHg and/or a diastolic blood pressure
95 mmHg; lung disease in subjects reporting chronic obstructive pulmonary disease (COPD) or asthma or the use of drugs for asthma or COPD; diabetes mellitus in subjects reporting the use of drugs for diabetes mellitus.
Alcohol consumption was coded as current drinking yes or no and for the drinkers as number of glasses per week. Pack years of cigarette smoking were calculated for present and past smokers as the number of cigarettes smoked times the number of years divided by 20. Never smokers and cigar and pipe smokers were classified as subjects with 0 pack years.
The physical examination included measurements of height, weight and blood pressure. Systolic and diastolic blood pressure were measured twice in supine position with a random-zero sphygmomanometer (Hawksley, England). The mean of the two measurements was used in the analyses.
Data analyses
Age-adjusted mean characteristics were compared between survivors and deceased by ANCOVA. Age-adjusted differences between the vital status groups were tested by using logistic regression with vital status as dependent and prevalence characteristics and age as independent variables. Plasma -tocopherol levels were adjusted for plasma cholesterol by calculating their residuals from linear regression models with plasma cholesterol as the dependent variable.15 For ease of interpretation, descriptives are expressed as unadjusted data.
All serum antioxidant vitamin concentrations are presented as median with their 90% ranges. Differences in vitamin concentrations between survivors and deceased were tested with the Mann-Whitney U-test.
Total carotenoid concentration was calculated by summing the absolute individual serum carotenoid concentrations. Oxygenated carotenoids were combined by summing serum concentrations of ß-cryptoxanthin, lutein and zeaxanthin and the hydrocarbon carotenoids were combined by summing ß-carotene, -carotene and lycopene. An index combining both the carotenoids and
-tocopherol concentrations was made by summing individual standardized scores (Z-scores) calculated for each log transformed vitamin by subtracting its group mean from the individual values and then dividing by the standard deviation.
Proportional hazards regression methods16 were used to estimate the hazard ratios and corresponding 95% CI for all-cause mortality for tertiles of serum antioxidant vitamin concentrations with the highest tertile as reference. Tests for trend were performed by fitting the variables in their continuous form in the proportional hazards model. ß-Cryptoxanthin, zeaxanthin, lutein and the combination of oxygenated carotenoids were log transformed as this gave a better fit of the estimated models, as judged by the log likelihood statistic value.
The proportionality assumption was judged by visual inspection of the log-log curves of tertiles of the serum vitamin concentrations.
Models with interaction terms for gender or smoking status and the individual serum antioxidant vitamins revealed no interactions between gender or smoking status and antioxidant vitamins, so no interaction terms were included in the multivariable models. In a multivariate model adjustments were made for important confounders17 as age, gender, pack years of cigarette smoking, alcohol consumption, serum cholesterol, body mass index, physical activity and antioxidant supplement use. Furthermore we adjusted for important predictors of total mortality; the presence of CVD, hypertension, lung disease or cancer in the multivariate models.
All analyses were performed with the statistical package SAS 18. A P-value < 0.05 was considered as statistically significant.
Results
Male and female survivors were as expected significantly younger than the deceased (Table 1). Furthermore, age was significantly associated with most of the other characteristics in Table 1
. Therefore, differences were tested after adjustment for age. Some age-adjusted differences in characteristics between survivors and deceased were more pronounced in males than females, e.g. significantly lower systolic blood pressure, higher activity scores, and a lower prevalence of lung disease and hypertension in survivors than in deceased.
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The individual antioxidant carotenoids were highly correlated (P = 0.0001) with the sum of these individual carotenoids ranging from Spearman correlations r = 0.50 for lutein to r = 0.80 for ß-carotene. Serum concentrations of cholesterol-adjusted -tocopherol showed a Spearman correlation of r = 0.18 (P = 0.0001) with total carotenoids. Among each other the highest Spearman correlations for the carotenoids were seen for ß- and
-carotene and for lutein and zeaxanthin, r = 0.78 and r = 0.67 respectively. Lowest correlations were observed for lycopene with the other carotenoids ranging from r = 0.14 with zeaxanthin to r = 0.41 for ß-carotene.
In Figure 1 survival curves (Kaplan-Meier) show that the subjects in the lowest tertile of oxygenated carotenoids and cholesterol-adjusted
-tocopherol have a lower survival than in the highest tertile. For the hydrocarbon carotenoids this is not observed, caused mainly by a drop in the survival for the highest tertile after 7 years.
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In this study we observed that the sum of serum levels of six carotenoids, of oxygenated carotenoids and the individual carotenoid ß-cryptoxanthin were inversely associated with 7.2-year all-cause mortality. For the sum of oxygenated carotenoids, ß-cryptoxanthin and lutein the subjects in the lowest tertile showed a significant increase in all-cause mortality compared with the highest tertile ranging from 56% for lutein to 73% for the sum of oxygenated carotenoids.
Discrepancies between study findings for serum levels of total carotenoids and mortality may partly be explained by differences in component carotenoids.19,20 In our study comprising all six major carotenoids an inverse association with all-cause mortality was observed. In a prospective nested-case control study20 including only lycopene and ß- and -carotene, no association was found between quartiles of the sum of these carotenoids and risk of non-fatal myocardial infarction or death from CHD among smoking and non-smoking males. In a study by Sahyoun et al.19 it is not clear which carotenoids were included in the total carotenoids variable. They19 found in a cohort of 725 elderly aged
60 years an inverse association between plasma carotenoid levels and 12-year all-cause mortality after adjustment for age, gender and serum cholesterol but this was no longer significant when other potential confounders were controlled for. A nested-case control study also combining six individual carotenoids, reported significant inverse associations with upper aerodigestive tract cancer.7
A possible protective effect of serum ß-cryptoxanthin and lutein on mortality as observed in our study is supported by three other observational studies. Howard et al.6 found significantly lower plasma levels of ß-cryptoxanthin in a population living in an area with a high CHD incidence (Belfast) than in a population from a low CHD incidence area (Toulouse). In a cross-sectional study5 significantly lower serum concentrations of ß-cryptoxanthin and lutein plus zeaxanthin were observed in cases with asymptomatic atherosclerosis (90th percentile of carotid intima media thickness) than in controls (below 75th percentile of carotid intima media thickness). For men with upper aerodigestive tract cancer lower levels of ß-cryptoxanthin were reported than for matched controls.7 Although a mechanism is not specified yet, the suggestion of Howard et al.6 that the oxygenated carotenoids ß-cryptoxanthin, lutein and zeaxanthin may be of special interest in preventing CHD is carefully supported by the results of the study of Irribarren et al.5 and by our study. We observed an inverse trend (P = 0.006) between the sum of the oxygenated carotenoids (including ß-cryptoxanthin, lutein and zeaxanthin) and total mortality, which was higher than for the specific carotenoids. If oxygenated carotenoids are especially important in CHD the strong association with total mortality in our study might be due to the large contribution of CHD to total mortality in Dutch elderly.
We studied all-cause mortality to cover the assumed associations of the different carotenoids on both CHD and cancer as main causes of total mortality. The possible protective effects of the carotenoids on different diseases may be due to common mechanisms like their antioxidant activity in vitro,2123 although the effect in vivo is debated.24 Besides a common protective mechanism of the individual carotenoids on a range of diseases, their differences in structure, metabolism, transport and tissue distribution25,26 may explain differential effects on different diseases as hypothesized for lycopene on prostate cancer,3 lutein and zeaxanthin on degenerative macular eye disease, ß-carotene and -carotene on specific cancers,2,7 and the oxygenated carotenoids ß-cryptoxanthin, lutein and zeaxanthin on CVD.5,6 Unfortunately, we were not able to further explore cause-specific disease and mortality as no such data were available.
In our study ß-cryptoxanthin (26%), ß-carotene (31%) and lutein (25%) were the three main contributors to total carotenoids. In a study comparing individual carotenoids among elderly from cities in the US and 10 European countries, it was seen that median serum concentrations of ß-carotene, lutein and ß-cryptoxanthin were mostly of similar magnitude to our study.27 Serum lycopene concentrations varied considerably among study populations. In our population lycopene concentrations were fourfold less than in the elderly populations from the US, France, Ireland and Italy.6,27 In these populations lycopene was the main contributor to serum total carotenoids. This resulted in higher absolute total carotenoid values than in our study. As lycopene is mainly derived from the intake of tomato and tomato products, differences in diet between these countries are probably responsible.
For cholesterol-adjusted serum vitamin E we found lower concentrations in deceased men than in survivors. No significant increased risk or inverse association with total mortality was found after adjustment for major confounders. In line with other observational studies serum vitamin E in normal physiological ranges is in general not strongly associated with mortality risk. The inverse associations found are usually due to intake of high-dosed vitamin supplements.28 Our study was not influenced by vitamin E supplement use as only six subjects used vitamin E supplements and none of the elderly reported the use of specific carotenoid-containing supplements. The antioxidant index results in our study give no support to the suggested interaction among carotenoids themselves and -tocopherol.22,29
The carotenoids we studied are mainly derived from fruit and vegetables the intake of which is consistently inversely associated with CVD and cancer.30,31 It can, of course, not be ruled out that the observed associations may be linked to other protective constituents or factors related to fruit and vegetable intake.32 Associations of specific carotenoids with specific diseases would be more suggestive of a possible real association with the carotenoids.
Methodological considerations
Serum concentrations were measured only once in 1991/1992 and possible changes over 6 years follow-up were not registered. We assumed in our analyses that the serum vitamin concentrations of 1991/1992 were indicative for serum concentrations during follow-up time. This is supported by the findings that mean plasma concentrations of -tocopherol, ß-carotene,
-carotene, lycopene, and lutein measured in a subgroup of 77 subjects33 in 1994 were similar to values in 1991/1992. Moreover, tertile classification of serum vitamins between the two time periods was concordant in 5472% of the cases and misclassification by more than one tertile occurred for less than 10% of the cases.
Misclassification by a conscious shift in diet, altering serum concentrations, after diagnosis of risk factors or chronic diseases prior to baseline measurements may have occurred but is not thought to have highly influenced our results as excluding the first year of follow-up from analyses did not markedly change the observed associations (data not shown).
Finally, loss of carotenoids or -tocopherol by storage for 6 years at a temperature of -80°C is not likely.34,35 Furthermore, laboratory personnel were unaware of the subject' status, and sampling, storage and further handling of the sera was carried out identically, so bias in comparing deceased and censored subjects was excluded.
In conclusion, this study provides information on the six major serum carotenoids and combinations of them in relation to all-cause mortality risk and showed that the inverse association is especially profound for the sum of the oxygenated carotenoids (ß-cryptoxanthin, lutein and zeaxanthin). Research into the possible protective constituents of fruit and vegetable intake should consider individual carotenoids and/or their combinations instead of focusing primarily on ß-carotene.
Acknowledgments
We thank Dr CEJ van den Hombergh for her work and responsibilities in collecting the baseline data in 1991/1992 of this study population. T Uneken and T Hoekstra for their contribution to completing the follow-up data and JG Kosmeijer-Schuil for the analyses of the carotenoids and tocopherol in serum.
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