1 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD.
2 Department of Biomedical Research, Our Lady of Mercy Medical Center, Bronx, NY.
Received for publication November 27, 2001; accepted for publication September 6, 2002.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
antioxidants; ascorbic acid; prostatic neoplasms; vitamin A; vitamin E
Abbreviations: Abbreviation: PSA, prostate-specific antigen.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vitro studies suggest that antioxidant micronutrients have cancer chemopreventive properties, such as antioxidant effects; regulation of cellular signaling; enhancement of immunity; and modulation of gene expression, cell differentiation, cell proliferation, and apoptosis (6, 7). To date, the antioxidant micronutrients that have been the target of considerable research interest include vitamin A (a subgroup of retinoids that exhibit qualitatively the biologic activity of all-trans retinol (8)), carotenoids, vitamin C (ascorbic acid), and vitamin E (tocopherols). Studies of dietary intake have produced equivocal results with respect to the associations between antioxidant micronutrients and prostate cancer (9). Methodological issues such as inaccuracy and imprecision of dietary recall data collected by questionnaires may have hindered the interpretations and implications.
Epidemiologic evidence is limited in regard to the associations between prediagnostic concentrations of antioxidant micronutrients in serum and subsequent risk of developing prostate cancer (1017) (table 1). No previous studies have been known to examine the association between retinyl palmitate, a naturally occurring compound as well as a metabolite of retinol via esterification. Of the six studies that assessed serum concentrations of retinol, two (11, 14) reported statistically significant inverse associations, three (10, 12, 13) reported statistically nonsignificant inverse associations, and one (16) reported a positive association. No statistically significant association was observed between prostate cancer risk and serum ß-carotene (10, 1214), -carotene, ß-cryptoxanthin, lutein, or zeaxanthin (13, 16). Data on lycopene were equivocal; no association (13), a statistically nonsignificant inverse association (14), and a borderline significant inverse association (16) were reported. For
-tocopherol, inverse associations were found in some studies of smokers (12) or smokers who used vitamin E supplements (15), whereas no association was found in other studies (10, 13, 14, 16). Evidence on
-tocopherol (13, 16, 17) is inconsistent. Only one small study examined the association of prediagnostic serum concentrations of vitamin C with subsequent prostate cancer risk (12).
|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the summer and autumn of 1989, a similar campaign against cancer and heart disease, named CLUE II, was conducted to collect blood samples and data on age, gender, height, weight, weight at age 21 years, cigarette and cigar smoking, medication use, and vitamin supplement use within 48 hours prior to a blood donation. Participants filled out a modified 62-item Block food frequency questionnaire that contained questions about use of vitamin supplements (multivitamins, vitamin A, vitamin C, and vitamin E) over the past year. A total of 25,081 persons (10,456 men and 14,625 women), 30 percent of the Washington County residents, took part in this program. Participation was slightly higher among women, among those who had more years of education, and among those in the age group 4570 years. Blood samples were collected into Vacutainers containing heparin (Fisher Scientific, Pittsburgh, Pennsylvania). Samples were refrigerated at 4°C and were centrifuged within 26 hours. Plasma aliquots were stored at 70°C until assayed. For the ascorbic acid assays, an additional aliquot was collected and was mixed with an equal amount of 10 percent metaphosphoric acid solution.
In each cohort, incident cases of prostate cancer (International Classification of Diseases, Ninth Revision, code 185) were identified through linkage of the cohort participants with the Washington County Cancer Registry and, since 1992, with the Maryland State Cancer Registry. A comparison between the two registries for CLUE II cases diagnosed after 1992 showed that the Washington County Cancer Registry was more complete than the Maryland State Cancer Registry: 5 percent of the cases were identified solely from the Maryland State Cancer Registry, and 25 percent were identified solely from the Washington County Cancer Registry. Cases were confirmed histologically. The stage of prostate cancer was extracted from medical records at the Washington County Hospital and was determined by using the American Joint Committee on Cancer/Tumor, Node, and Metastasis system. Stage data from the Maryland State Cancer Registry, if available, were incorporated.
Completeness of case ascertainment was assessed by comparing the number of identified cases with the number based on the American Cancer Society estimates (101.4 percent of estimated cases). On the basis of a survey of 4 percent of the CLUE I households, the annual emigration rate was 1.4 percent from 1974 to 1985. Among the 8,396 persons who participated in both CLUE I and CLUE II, 750 were lost to follow-up from 1974 to 1998. Hence, the annual rate of loss to follow-up was less than 1 percent in this group. Until September 1996, 182 and 142 incident prostate cancer cases in the CLUE I cohort and CLUE II cohort, respectively, were included in the present studies. Serum specimens from the majority of the CLUE I cases diagnosed during the period 19751985 were used for other studies (14) and thus were not available for the present study.
Each case was matched with two controls on age, race, gender, and date of blood donation. Controls were selected from the cohort participants who were not known to have died or to have been diagnosed with cancer (except for basal or squamous cell skin cancer) at the time that a case was diagnosed. Henceforth in this paper, the terms "CLUE I" and "CLUE II" are used to refer to the nested case-control studies conducted among the cohorts established in 1974 and 1989, respectively.
We previously reported an interaction of selenium, -tocopherol, and
-tocopherol with respect to prostate cancer risk in a subset (117 matched sets) of the CLUE II participants who provided toenail samples along with blood samples (17). For completeness, we have included the results of blood concentrations of tocopherols for the total CLUE II study (142 matched sets).
Laboratory assay
Frozen samples were thawed in ice water under yellow, dim light. The aliquots for assays were refrozen and were shipped to the laboratory with dry ice in Styrofoam boxes (The Dow Chemical Company, Midland, Michigan). The samples were then thawed at room temperature and were protected from light (20 minutes), mixed by vortex (15 seconds), and centrifuged (at 2,500 g, 5 minutes, 4°C). The fibrin-free upper fraction was used to measure retinol, carotenoids, and tocopherols by reversed-phase high-pressure liquid chromatography with multiwavelength detection (18). Ascorbic acid assays were performed on additional CLUE II samples. These samples were thawed at room temperature (
20 minutes), mixed by vortex (15 seconds), and centrifuged (10,000 g, 5 minutes, 4°C); the clear supernatant was used to measure ascorbic acid with 2,4-dinitrophenylhydrazine as chromogen (19). Repeated freezing to 70°C and thawing for up to six cycles had no appreciable effect on the plasma or serum concentrations of micronutrients (20). In CLUE I, serum samples were thawed a maximum of two times prior to the aliquots for prostate cancer study, for a total of three thaws. Only 15 percent of the samples were thawed three times. None of the CLUE II samples in this study had been thawed previously. Serum concentrations of retinol, ß-carotene, and
-tocopherol were stable for at least 15 years of storage at 70°C (21).
Laboratory technicians were instructed to analyze multiples of triplet sets (including one case and two controls) on the same day by using the same solvents and reagents. Reproducibility of the laboratory measurements was assessed in 20 sets of serum specimens for the CLUE I study and in 15 sets of plasma specimens for the CLUE II study. For the CLUE I study, the intra-assay coefficients of variation were 11.6 percent for -carotene; 11 percent for ß-cryptoxanthin; 18.8 percent for retinyl palmitate; and 26 percent for ß-carotene, total carotene, lutein, lycopene, retinol,
-tocopherol, and
-tocopherol. For CLUE II, the intra-assay coefficients of variation were 16.0 percent for
-carotene; 13.6 percent for retinyl palmitate; and 38 percent for ß-carotene, total carotene, ß-cryptoxanthin, lutein, lycopene, retinol,
-tocopherol,
-tocopherol, and ascorbic acid.
Statistical analysis
Baseline characteristics of cases and controls were summarized and compared by using paired t tests for continuous variables and conditional logistic regression models for categorical variables. Mean micronutrient concentrations for the two controls in a matched set were calculated, and data from cases and the mean values of their respective controls were compared by using the Wilcoxon signed-rank test. Micronutrient levels were further categorized into fifths according to the distributions among controls. The odds ratios and 95 percent confidence intervals for developing prostate cancer for the upper fifths compared with the lowest fifth of each micronutrient were estimated by using conditional logistic regression models. Adjustments for total lipid levels (continuous variable) in the blood, hours since last meal (<3, 37, 8), and education (years of schooling) were performed. Body mass index at age 21 years (continuous variable) was also included in the adjustment analyses for CLUE II. Tests for trend were performed by entering into a conditional logistic regression model a categorical variable defined by the median in each fifth of micronutrient concentration. Stratification analyses were conducted according to age at diagnosis (by median), number of years since blood was drawn, disease stage at diagnosis, history of cigarette smoking, and body mass index at age 21 years. To retain adequate sample size, thirds of micronutrient concentrations in controls were used to define the categories for the odds ratio estimates in these stratification analyses.
All analyses were performed separately for each cohort because of the differences in storage (serum vs. plasma) and the time between blood donation and diagnosis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the CLUE I and CLUE II studies, information was available on vitamin supplement use within the 48 hours prior to blood draw. Regular users were likely to be captured by this approach, but duration of use was not determined. In CLUE II, additional information on supplement use in the past year was available from the food frequency questionnaire administered at baseline. Restriction of analyses to nonvitamin users identified by either one or both approaches did not alter the observed associations.
Our findings for -carotene, ß-carotene, total carotene, lutein, ß-cryptoxanthin, lycopene, and ascorbic acid are consistent with previous evidence (10, 1214, 16) that suggests no association with prostate cancer risk. Lycopene has recently received considerable attention regarding a possible protective association against prostate cancer. Of the three previous reports (table 1), only one found a statistically borderline significant decreased risk, which was limited to aggressive cases of prostate cancer (16). A previous study of the first 103 cases of prostate cancer in the CLUE I cohort (14) found a statistically nonsignificant lower risk of prostate cancer in men whose serum lycopene concentrations were in the highest fourth level. In the present study, the cutoff points for the fifths of lycopene concentrations in controls were similar to those in the study by Gann et al. (16), but we did not observe an association of circulating lycopene concentrations with prostate cancer risk. Lycopene is thought to be important primarily because of the higher rate constant, as compared with several other carotenoids, in quenching singlet oxygen in in vitro photosensitization reactions (22). It is unknown whether significant numbers of singlet oxygen molecules occur in biologic systems, particularly in prostate tissue. The fate and the impact of the lycopene molecules attached with singlet oxygen are also unknown. Whether and how the chemopreventive effects of lycopene would emerge only at late stages of prostate cancer (16) is particularly intriguing.
Retinyl esters (such as retinyl palmitate, retinyl stearate, and retinyl oleate) are the primary storage forms of vitamin A (2325). Retinyl palmitate is formed and stored mostly in the liver (23), and it accounts for 8090 percent of stored vitamin A (25). In serum or plasma, retinyl palmitate was found to be the predominant retinyl ester, followed by retinyl stearate (18, 26). Approximately 4 hours after using vitamin A supplements, circulating levels of retinyl palmitate and retinyl stearate increase transiently (26). On the other hand, long-term use of vitamin A supplements (>5,000 IU/day) leads to a consistent elevation in circulating retinyl palmitate (27, 28). Retinyl esters have been linked to functionality in the vision cycle and may protect the retina from night blindness (23, 29). Functional dependence on vitamin A also occurs in the tracheobronchial tract (30). It is unknown whether retinyl palmitate alone or following conversion to retinol or retinol metabolites (retinoic acid or others) has functional importance in the prostate tissue.
Previous studies on retinol and prostate cancer have produced inconsistent results (10, 14, 16), which may be in part due to small sample sizes in some studies. The study based on the first 103 cases in the CLUE I cohort suggested a protective association between circulating retinol concentrations and prostate cancer (14). However, we did not replicate the finding in this larger study with cases diagnosed 10 or more years following blood donation (CLUE I) or cases diagnosed within 7 years of blood donation (CLUE II).
We did not observe an overall protective association against prostate cancer with higher -tocopherol concentrations, a result consistent with some previous studies (10, 13, 14, 16). Unlike some other studies suggesting that serum
-tocopherol level is particularly important for smokers (12) and for cases with aggressive prostate cancer (16), we did not observe such associations in the subgroups. These findings may be due to the generally low intake of
-tocopherol in the US diet (typically 79 mg/day in adults);
-tocopherol is the major or sole ingredient in vitamin E supplements, and levels are high in certain types of vegetable oil, for example, wheat germ oil, sunflower oil, and cottonseed oil (31). Previous studies have suggested that supplementation with vitamin E in doses higher than those consumed from food may confer benefits on reducing the risk of prostate cancer. In the Alpha-Tocopherol Beta-Carotene (ATBC) trial of Finnish heavy smokers, a 32 percent reduction in prostate cancer incidence and a 41 percent reduction in prostate cancer mortality were observed in the group that used
-tocopherol supplements of 50 mg/day for 58 years compared with the placebo group (32); a statistically nonsignificant protective association was observed between serum
-tocopherol levels at baseline and subsequent prostate cancer risk, but only in the group that received
-tocopherol supplementation (15). Vitamin E supplementation was also associated with a lower prostate cancer risk in a case-control study (33) and a prospective study in which the association for metastatic or fatal prostate cancer was limited to current smokers or recent quitters (34). We previously reported possible interactive associations of
-tocopherol,
-tocopherol, and selenium with respect to prostate cancer risk (17). It appears that benefits, yet to be confirmed, of
-tocopherol on prostate cancer may be acquired from supplements of doses higher than those from diet or from simultaneously adequate levels of other nutrients such as
-tocopherol and/or selenium.
This expanded analysis of the role of -tocopherol in prostate cancer showed differences between the CLUE I and CLUE II cohorts, with stronger associations observed in CLUE II. In 1989, 4 percent of the cases and 8 percent of the controls ever used vitamin E supplements regularly. Although
-tocopherol supplement use decreases circulating levels of
-tocopherol, because use was less common in cases than in controls, it is unlikely that the lower
-tocopherol concentration in cases was a consequence of
-tocopherol supplement use. We know of only two previous prospective studies that have examined the association of serum
-tocopherol with prostate cancer risk. A study of 142 sets of Japanese-American men (13), during a study period similar to CLUE I, reported a nonsignificant inverse association, a result similar to our findings from CLUE I. In contrast, a study among US physicians observed no association (16). The potential role of
-tocopherol in prostate cancer prevention was supported by several in vitro studies showing
-tocopherol to be more effective than
-tocopherol in inhibiting prostate cancer cells (35), reducing oxidative DNA damage as measured by sister chromatid exchange (36), and scavenging electrophilic mutagens such as peroxinitrite, a potent oxidizing nitrating compound (37).
Tests of prostate-specific antigen (PSA) have been increasingly used in prostate cancer screening and diagnosis since the early 1990s. Frequent use of screening may result in detection of disease in the early stages. In the present study, we found no evidence of overdiagnoses of early-stage prostate cancer by using PSA tests and digital rectal examinations. In CLUE I, we observed no important changes in the association between micronutrients and prostate cancer after excluding cases diagnosed before 1990. In CLUE II (19901996), 92 percent of the cases and 64 percent of the controls who responded to a survey questionnaire in 1996 reported ever having a PSA test; 94 percent of the cases and 82 percent of the controls reported ever having a digital rectal examination. Among the prostate cancer cases who ever underwent a PSA test, 14 percent, 54 percent, and 31 percent were diagnosed to have stage 0 or 1, stage 2, and stage 3 or 4 disease, respectivelythe same frequency distribution applied to digital rectal examination. No statistically significant difference was found in disease stage by PSA testing (p = 0.77).
An interesting aspect of this study is the difference in time from blood draw to clinical diagnosis for CLUE I and CLUE II participants. Duration of preclinical asymptomatic prostate cancer in Caucasians was estimated to be at least 1112 years (38). Whether inconsistencies in the results of -tocopherol and retinyl palmitate between the two cohorts can be attributed to chance or other factors such as secular changes in diet over time need to be determined by additional studies. With respect to potential changes in diet over time, changes in fat intake from the 1970s to the late 1980s among the study participants could have accounted for inconsistencies in the
-tocopherol results. For example, polyunsaturated fat, including corn oil and soybean oil, both of which contain the highest amount of
-tocopherol compared with other types of oil, has been increasing in the US diet since 1970 (39). In the present study, circulating levels of
-tocopherol were indeed higher in 1989 than in 1974, particularly among controls. Replication of these findings in other studies with prolonged follow-up is needed to improve our understanding of potentially modifiable factors in the prevention of prostate cancer.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|