1 Department of Health Promotion and Preventive Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
2 Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Japan.
3 Department of Public Health, Fujita Health University School of Health Sciences, Aichi, Japan.
4 Department of Public Health/Health Information Dynamics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
5 Department of Epidemiology for Community Health and Medicine, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan.
6 Department of Hygiene, Fujita Health University School of Medicine, Aichi, Japan.
7 Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan.
8 Department of Preventive Medicine/Biostatistics and Medical Decision Making, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Received for publication May 28, 2004; accepted for publication October 4, 2004.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
alpha-linolenic acid; chromatography; colorectal neoplasms; docosahexaenoic acids; eicosapentaenoic acid; fatty acids; prospective studies; serum
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Case ascertainment and control selection
Cases were defined as subjects who developed colorectal cancer (according to the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, codes C1820) during the follow-up period (mean, 7.1 years; standard deviation, 2.3), which ran until the end of 1997. We ascertained the incidence of cancer from population-based cancer registries, supplemented by a systematic review of death certificates (14). For each case, two or three controls with no previous history of cancer were selected from the population at risk. The controls were matched to each case by sex, age (±3 years, as close as possible), and participating institution. A total of 169 colorectal cancer cases (83 men and 86 women) and 481 controls (241 men and 240 women) were involved in the analysis. The numbers of colon cancer cases and matched controls were 119 (52 men and 67 women) and 336 (151 men and 185 women), respectively.
Serum fatty acid analysis
Serum was separated from the blood samples at local laboratories in or near the surveyed municipalities and was stored for 1114 years at 80°C. All samples were analyzed in November 2002 in one laboratory by trained staff blinded to case-control status. Samples were organized in batches of up to 50, which included two samples from a standard pool for quality control. Lipids in 0.2 ml of serum were extracted with Folchs solution under a nitrogen atmosphere (15). After methyl esterification by 0.4 M potassium methoxide and 14 weight percentage boron trifluoride methanol, fatty acids were measured by using a gas chromatograph (model GC17A; Shimazu, Kyoto, Japan) equipped with an Omegawax 250 capillary column (30-m x 0.25-mm inside diameter; 0.25-µm thickness; Supelco, Bellefonte, Pennsylvania). Peaks were determined by using a flame-ionization detector and were quantified with an electric integrator (model CR-7A; Shimazu) using pure standard mixtures (Sigma, St. Louis, Missouri).
A total of 24 fatty acids were identified from 12:0 to 24:1 -9. The serum level of each fatty acid was expressed as the composition, by weight percentage, of total lipids. The limit of detection for the assay was 0.02 weight percent. The respective repeatability and day-to-day variation of the standard sample coefficients of variation were as follows: 5.5 percent for both measurements for 16:0; 6.2 percent and 5.6 percent for 18:2; 4.9 percent and 6.5 percent for 20:3; and 2.9 percent and 4.5 percent for 24:1.
In particular, four -3 PUFAs and six
-6 PUFAs were measured:
-18:3
-3 (
-linolenic acid), 20:5
-3 (eicosapentaenoic acid), 22:5
-3 (docosapentaenoic acid), 22:6
-3 (docosahexaenoic acid), 18:2
-6 (linoleic acid),
-18:2
-6 (
-linolenic acid), 20:2
-6 (eicosadienoic acid), 20:3
-6 (dihomo-
-linolenic acid), 20:4
-6 (arachidonic acid), and 22:4
-6 (docosatetoraenoic acid). In addition, we calculated the content of total saturated fatty acids (12:0 + 14:0 + 16:0 + 18:0 + 20:0 + 22:0 + 24:0), monounsaturated fatty acids (MUFAs; 16:1
-7 + 18:1
-9 + 20:1
-9 + 22:1
-9 + 24:1
-9), and total
-3 and
-6 PUFAs. The ratio of
-6 to
-3 PUFAs was also determined.
Statistical methods
Background characteristics were compared between cases and controls by using the Cochran-Mantel-Haenszel test (16) and analysis of covariance (17), with adjustment for matching factors (age and area of enrollment) by sex. Spearmans correlation coefficients were calculated to determine the association between fatty acids. Conditional logistic regression models were used to calculate odds ratios for the incidence of colorectal cancer (18) for the serum level of each specific fatty acid. The odds ratios for colon cancer risk were also examined separately from those for rectal cancer. Cases and controls were divided into four groups according to the level of fatty acids in controls. Odds ratios were calculated for the second quartile ((Q)2), third quartile (Q3), and highest quartile (Q4) versus the lowest quartile (Q1). To test for linear trends in odds ratios over quartiles, we coded each quartile as 0, 1, 2, or 3 and incorporated these data into the logistic model as a single variable.
We adjusted for the following factors by including them in the logistic models (19): age at completing final education (18 years or
19 years); history of colorectal cancer in parents or siblings (yes or no); body mass index (weight (kg)/height (m)2; <20.0 kg/m2, 20.024.9 kg/m2, or
25.0 kg/m2) calculated from reported height and weight at baseline; smoking status (never, former, or current); daily alcohol consumption (never, former, or current); frequency of intake of green leafy vegetables (almost every day or
34 days per week), and time spent exercising (<3 hours per week or
3 hours per week) (20). The results were not significantly affected by adjustment for potential confounding factors; therefore, only the multivariate-adjusted odds ratios are presented in the tables in this paper. To eliminate the influence of undiagnosed colorectal cancers at baseline, the analyses were repeated by excluding men and women who developed colorectal cancer within the first 2 or 5 years of follow-up, respectively, along with their matched controls.
All analyses were performed by using SAS software, release 8.2 (SAS Institute, Inc., Cary, North Carolina). In the conditional logistic regression analysis, missing values for each categorical covariate were treated as an additional category of the variable and were included in the model. Two-tailed probability (p) values of <0.1 were considered marginally significant, and p values of <0.05 were considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Table 2 shows the associations between the serum levels of each group of fatty acids and the risk of colorectal cancer by sex. For men, total saturated fatty acids and -6 PUFAs failed to show significant associations with colorectal cancer risk. A marginally significant positive trend was observed between serum level of total MUFAs and colorectal cancer risk (p for linear trend = 0.06). Total
-3 PUFAs were inversely associated with colorectal cancer risk, showing a 76 percent risk reduction when Q4 and Q1 were compared (odds ratio = 0.24, 95 percent confidence interval (CI): 0.08, 0.76; p for linear trend = 0.08). For the
-6/
-3 ratio, Q2 showed a marginally significant association, with a 2.36-fold increased risk of colorectal cancer relative to Q1 (95 percent CI: 0.99, 5.66), although the dose-response relation was unclear. In the analysis of colon cancer risk alone (data not shown), there was no statistically significant association with the serum levels of any of the fatty acids. However, the directions of the nonsignificant trends were similar to those observed in the combined analyses. The odds ratios for Q4 versus Q1 of each group of fatty acids for the risk of colon cancer in men were as follows: 1.00 for saturated fatty acids (95 percent CI: 0.28, 3.52; p for linear trend = 0.84), 1.19 for MUFAs (95 percent CI: 0.34, 4.22; p for linear trend = 0.60), 0.40 for
-3 PUFAs (95 percent CI: 0.10, 1.55; p for linear trend = 0.43), and 1.04 for
-6 PUFAs (95 percent CI: 0.34, 3.16; p for linear trend = 0.86).
|
To exclude the influence of undiagnosed colorectal cancer at baseline, the analyses were repeated by excluding men and women who developed colorectal cancer during the first 2 or 5 years of follow-up, respectively, along with their matched controls (table 3). For men, excluding those who developed colorectal cancer within the first 5 years strengthened both the positive association of total MUFAs and the inverse association of total -3 PUFAs with colorectal cancer risk. For women, excluding those who developed colorectal cancer within the first 2 years revealed a significant association for
-3 PUFAs, with a 70 percent decreased risk of colorectal cancer at the Q2 compared with the Q1 level (odds ratio = 0.30, 95 percent CI: 0.11, 0.79).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our study design is similar to that of the nested case-control study based on data from the Multiple Risk Factor Intervention Trial (MRFIT) (21). Simon et al. examined 108 cancer cases and 215 controls and found no association between any serum fatty acid component and the risk of fatal cancer. Unfortunately, because of the limited number of subjects, they were unable to estimate risk by organ site. This prospective study is the first known to report an association between colorectal cancer risk and specific serum fatty acids.
We found a marginally significant inverse association between serum level of -3 PUFAs and the risk of colorectal cancer in men. The odds ratios for the highest versus lowest quartiles of all
-3 PUFAs examined were less than 1.0 and were statistically significant (p < 0.05), except for eicosapentaenoic acid (p = 0.13). These findings support the potential preventive effects of fish oil supplements rich in
-3 PUFAs against colorectal cancer (22, 23), which have been suggested by a number of clinical studies (35, 24, 25). The chemopreventive activity of nonsteroidal antiinflammatory drugs on colorectal tumors has been well documented in a number of experimental studies (2629). Suppression of cyclooxygenase and inhibition of prostaglandin E2 synthesis by nonsteroidal antiinflammatory drugs is thought to be the main mechanism for this activity.
-3 PUFAs are thought to influence the carcinogenic process via their effects on the synthesis of prostaglandins and thromboxanes (1) through a mechanism similar to that of nonsteroidal antiinflammatory drugs. Increased intake of eicosapentaenoic and docosahexaenoic acids might also promote apoptosis in cells of the normal human colonic mucosa (30).
We found no significant association between -3 PUFAs and the risk of colorectal cancer in women.
-linolenic and linoleic acid were associated with colorectal cancer incidence in the opposite direction for men and women. MUFAs showed a marginally significant positive association for men only. On the basis of the available data, we cannot suggest a plausible explanation for the gender difference in the association between fatty acids and colorectal cancer risk. Genetic and hormonal factors, nutritional status, and disease are all thought to influence fatty acid metabolism (31). In addition, it has been suggested that female sex hormones play a role in the etiology of colorectal cancer (32, 33). Interestingly, the odds ratios for the highest versus lowest quartiles were less than 1.0 for all of the
-3 PUFAs examined in women when those women who developed colorectal cancer within the first 5 years of follow-up were excluded. It is possible that physical disorders or medications not evaluated in the present analyses might have influenced the results. Unfortunately, we did not collect data on use of nonsteroidal antiinflammatory drugs and other medications that might have interfered with the association between
-3 PUFAs and colorectal cancer risk. Further investigation of diet and metabolism will therefore be necessary to clarify these gender interactions. In addition, we did not observe an obvious dose response between serum levels of
-3 PUFAs and colorectal cancer risk. Additional studies should thus examine whether an optimal level of
-3 PUFAs is associated with colorectal cancer prevention.
The risks of colorectal cancer are reported to vary by subsite (34, 35). The available data showed no obvious differences between the separate risk of colon cancer and the combined risk of colorectal cancer regarding the association with fatty acids. Although we were unable to estimate the independent risks by subsite in our study because of the limited number of cases, they should be confirmed in future studies with larger sample sizes.
Some limitations that affected interpretation of our results must be noted. First, our subjects were selected from among the participants of a large cohort study. As Kato et al. (36) discussed previously, subjects in cohort studies tend to be homogeneous and health conscious, which might reduce the between-person variation in food consumption and other health-related factors and make detection of associations between individual fatty acids and disease risk more difficult. Moreover, the subjects in the present study were limited to those who donated blood samples: only 36.6 percent of the total cohort. In fact, those who did not donate blood samples were more likely to be highly educated, to consume alcohol daily, and to exercise less compared with those who donated blood samples, regardless of gender. In addition, compared with nonparticipants, male participants tended to be older and female participants tended to be younger. The differences in the background characteristics of the subjects should be considered to generalize our findings. Second, because this was a multicenter study (12), the procedures used to collect blood were not uniform. However, we confirmed that no area had a greatly different distribution of fatty acid levels. In addition, we matched cases and controls by participating institution; therefore, any bias due to differences between areas should have been accounted for.
Third, we used serum samples that were stored at 80°C for 1114 years to evaluate the levels of fatty acids. Iso et al. (37) examined 31 serum samples taken from subjects in the present cohort in 1990 and again in 1998. They reported an increase in the composition of saturated fatty acids (29.2 percent vs. 30.3 percent) and 20:3 (dihomo- linolenic acid) (0.85 percent vs. 0.98 percent), a decline in the composition of MUFAs (22.9 percent vs. 22.4 percent), and no changes in the other fatty acids over this 8-year time interval. Zeleniuch-Jacquotte et al. (11) reported that storage for up to 12 years at 80°C effectively protected PUFAs from oxidation. However, the long-term effects of storage for up to 14 years have not been confirmed. Fourth, although we used the fatty acid composition of serum total lipids as a biomarker, several alternative methods are available for biologic assessment of fat intake. Adipose tissue and the erythrocyte membrane reflect long- and medium-term fatty acid intake, respectively, whereas serum reflects only short-term (weeks to months) intake (10). Although measuring these alternative biomarkers is more expensive and invasive, they might be a better index for use in predicting colorectal cancer. Therefore, our results should be confirmed by using these media. Fifth and finally, we evaluated the fatty acids and background characteristics of the subjects only once, at baseline. These measurements might not accurately reflect the long-term habits of the subjects. Thus, repeated measurements should be considered to reduce measurement errors (11).
![]() |
ACKNOWLEDGMENTS |
---|
The authors express their appreciation to Dr. Kunio Aoki, Professor Emeritus, Nagoya University School of Medicine and the former chairman of the JACC Study Group; and to Dr. Haruo Sugano, the former director of the Cancer Institute of the Japanese Foundation for Cancer Research, who greatly contributed to initiating the study.
The present members of the JACC Study and their affiliations are as follows: Dr. Akiko Tamakoshi (present chairman of the study group), Nagoya University Graduate School of Medicine; Dr. Mitsuru Mori, Sapporo Medical University School of Medicine; Dr. Yutaka Motohashi, Akita University School of Medicine; Dr. Ichiro Tsuji, Tohoku University Graduate School of Medicine; Dr. Yosikazu Nakamura, Jichi Medical School; Dr. Hiroyasu Iso, Institute of Community Medicine, University of Tsukuba; Dr. Haruo Mikami, Chiba Cancer Center; Dr. Yutaka Inaba, Juntendo University School of Medicine; Dr. Yoshiharu Hoshiyama, University of Human Arts and Sciences Graduate School; Dr. Hiroshi Suzuki, Niigata University Graduate School of Medical and Dental Sciences; Dr. Hiroyuki Shimizu, Gifu University School of Medicine; Dr. Hideaki Toyoshima, Nagoya University Graduate School of Medicine; Dr. Shinkan Tokudome, Nagoya City University Graduate School of Medical Science; Dr. Yoshinori Ito, Fujita Health University School of Health Sciences; Dr. Shuji Hashimoto, Fujita Health University School of Medicine; Dr. Shogo Kikuchi, Aichi Medical University School of Medicine; Dr. Akio Koizumi, Graduate School of Medicine and Faculty of Medicine, Kyoto University; Dr. Takashi Kawamura, Kyoto University Center for Student Health; Dr. Yoshiyuki Watanabe and Dr. Tsuneharu Miki, Kyoto Prefectural University of Medicine Graduate School of Medical Science; Dr. Chigusa Date, Faculty of Human Environmental Sciences, Mukogawa Womens University; Dr. Kiyomi Sakata, Wakayama Medical University; Dr. Takayuki Nose, Tottori University Faculty of Medicine; Dr. Norihiko Hayakawa, Research Institute for Radiation Biology and Medicine, Hiroshima University; Dr. Takesumi Yoshimura, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan; Dr. Naoyuki Okamoto, Kanagawa Cancer Center; Dr. Hideo Shio, Moriyama Municipal Hospital; Dr. Yoshiyuki Ohno (former chairman of the study group), Asahi Rosai Hospital; Dr. Tomoyuki Kitagawa, Cancer Institute of the Japanese Foundation for Cancer Research; Dr. Toshio Kuroki, Gifu University; and Dr. Kazuo Tajima, Aichi Cancer Center Research Institute.
The past members of the JACC Study, other than the following eight, are listed in reference 9 (affiliations are those at the time of participation in the JACC Study): Dr. Takashi Shimamoto, Institute of Community Medicine, University of Tsukuba; Dr. Heizo Tanaka, Medical Research Institute, Tokyo Medical and Dental University; Dr. Shigeru Hisamichi, Tohoku University Graduate School of Medicine; Dr. Masahiro Nakao, Kyoto Prefectural University of Medicine; Dr. Takaichiro Suzuki, Research Institute, Osaka Medical Center for Cancer and Cardiovascular Diseases; Dr. Tsutomu Hashimoto, Wakayama Medical University; Dr. Teruo Ishibashi, Asama General Hospital; and Dr. Katsuhiro Fukuda, Kurume University School of Medicine.
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|