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The study of the relationship between dietary intake of fatty acids and the risk of breast cancer has not yielded definite conclusions with respect to causality, possibly because of methodological issues inherent to nutritional epidemiology. To evaluate the hypothesis of possible protection of n-3 polyunsaturated fatty acids (PUFA) against breast cancer in women, we examined the fatty-acid composition of phospholipids in pre-diagnostic sera of 196 women who developed breast cancer, and of 388 controls matched for age at recruitment and duration of follow-up, in a prospective cohort study in Umeå, northern Sweden. Individual fatty acids were measured as a percentage of total fatty acids, using capillary gas chromatography. Conditional logistic-regression models showed no significant association between n-3 PUFA and breast-cancer risk. In contrast, women in the highest quartile of stearic acid had a relative risk of 0.49 (95% confidence interval, 0.22–1.08) compared with women in the lowest quartile (trend p = 0.047), suggesting a protective role of stearic acid in breast-cancer risk. Besides stearic acid, women in the highest quartile of the 18:0/18:1 n-9c ratio had a relative risk of 0.50 (95% confidence interval, 0.23–1.10) compared with women in the lowest quartile (trend p = 0.064), suggesting a decrease in breast-cancer risk in women with low activity of the enzyme delta 9-desaturase (stearoyl CoA desaturase), which may reflect an underlying metabolic profile characterized by insulin resistance and chronic hyper-insulinemia. Int. J. Cancer 83:585–590, 1999. © 1999 Wiley-Liss, Inc.
Data derived from animal experiments indicate that the tumor-promoting properties of high-fat diets may be more a function of differences in fatty-acid composition than of fat content per se or of total caloric intake. In several animal models, high-fat diets rich in n-6 polyunsaturated fatty acids (PUFA) generally stimulated mammary-tumor development and metastasis, whereas diets rich in n-3 PUFA appeared to inhibit tumor growth and metastasis (Fay et al.,1997). However, the promotion phase of chemically induced carcinogenesis has been shown to be significantly suppressed only when equal parts of high-fat diets rich in n-6 and n-3 long-chain PUFA were fed (Fay et al.,1997).
Epidemiological studies in which fat intake has been assessed in individuals by questionnaire methods have generally provided weak or no support for the hypothesis that dietary intake of n-3 PUFA might protect against breast cancer. Although some case-control studies (Ingram et al.,1991; Landa et al.,1994; Franceschi et al.,1995; Braga et al.,1997) showed inverse associations between breast-cancer risk and consumption of fish rich in long-chain n-3 PUFA, these findings were not confirmed by several prospective cohort studies (Vatten et al.,1990; Toniolo et al.,1994). Pooled analyses of multiple case-control (Howe et al.,1990) or cohort studies (Hunter et al.,1996) showed no association between intake of PUFA and the risk of breast cancer. In none of these studies, however, was any distinction made between n-6 and n-3 PUFA. Furthermore, conclusive evidence for a role of individual fatty acids in breast-cancer risk may be precluded by the many methodological limitations in measurements of dietary intake of fatty acids.
Beyond food-questionnaire methodology, objective data points on consumption of fish can be obtained from long-chain n-3 PUFA analysis of serum or plasma phospholipids, which have been shown to accurately reflect recent dietary intake of long-chain fatty acids from fish (Bjerve et al.,1993; Ma et al.,1995). Therefore, measurement of serum phospholipid fatty acids may be appropriate for examining whether the type of PUFA is related to breast-cancer risk.
To test the hypothesis that n-3 PUFA may have a protective role against breast cancer, we conducted a prospective cohort study in Umeå, northern Sweden, in which we compared the fatty-acid composition of serum phospholipids of women who developed breast cancer and that of a sub-set of cohort members who did not. Main findings were an absence of association between breast-cancer risk and phospholipid levels of n-3 PUFA of marine origin. A less expected finding, however, was a negative association of risk with level of stearic acid (18:0) and with the ratio 18:0/18:1n-9c, and a positive association with level of palmitic acid (16:0).
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- MATERIAL AND METHODS
In this study, we examined whether n-3 PUFA of marine origin may protect against the risk of breast cancer. For this purpose, n-3 PUFA (20:5 n-3, 22:6 n-3) levels of serum phospholipids were used as a biomarker of dietary intake of n-3 PUFA, to investigate the relation between exposure to n-3 fatty acids and breast cancer. We compared the fatty-acid composition of phospholipids in pre-diagnostic sera of 196 women who developed breast cancer and of 388 controls matched for age at recruitment and duration of follow-up, in a prospective cohort study in Umeå, northern Sweden. Contrary to our starting hypothesis, no significant association between n-3 PUFA and breast-cancer risk was found. In contrast, we found that individual saturated fatty acids were associated with risk. Women in the highest quartile for palmitic acid (16:0) had an increased risk compared with women in the lowest quartile, with an OR of 2.09, while the OR for breast cancer among women in the highest quartile of stearic acid (18:0) compared with the lowest quartile was 0.49, suggesting a protective role of stearic acid. Moreover, women in the highest quartile of the 18:0/18:1 n-9c ratio had a relative risk of 0.50 compared with women in the lowest quartile, suggesting a decrease in breast-cancer risk in women with low activity of the enzyme delta 9-desaturase (stearoyl CoA desaturase).
Several experimental findings on stearic acid or on the ratio 18:0/18:1n-9c and breast cancer fit well with our observation. Decreased level of stearic acid in red-cell membranes, as well as increased level of oleic acid, have been reported for patients with cancers at different sites (Wood et al.,1985; Kelly et al.,1990; Persad et al.,1990; Pandey et al.,1995). In addition, a prospective study of survival of breast-cancer patients showed a strong negative association between risk of metastases and stearic-acid level in membrane phosphatidylcholine in the primary tumor (Bougnoux et al.,1992).
The inverse association of breast-cancer risk with stearic acid and with the ratio 18:0/18:1 n-9c is not known. A possible interpretation is that stearic acid is directly involved in the inhibition of tumor development. Stearic acid had already been found to inhibit in vitro proliferation of various human cancer cell lines (Fermor et al.,1992), including mammary tumor cells, and additional observations suggest that stearic acid may inhibit epidermal-growth-factor(EGF)-induced breast-cancer cell growth (Wickramasinghe et al.,1996). Furthermore, parenteral administration of stearic acid in a chemically induced mammary-tumor model in rats delayed mammary-tumor development (Habib et al.,1987). However, the direct effects of dietary stearic acid on breast cancer warrant further experimental studies.
If we assume that a low stearic-acid level in plasma lipids or cellular membranes was indeed a direct cause of breast cancer in our cohort, the next question is which factors might have caused the decrease in stearic-acid level. Although dietary fatty acids are known to influence serum phospholipid fatty-acid levels, no association, or only weak associations, have been reported between levels of saturated fatty acids in plasma phospholipids and dietary intake levels (Ma et al.,1995). In fact, except for the essential fatty acids, linoleic acid and alpha-linolenic acid, which must be obtained from diet, all other fatty acids in serum lipids may come either from diet or from de novo endogenous synthesis. Furthermore, all fatty acids can undergo modifications via desaturation, elongation, retroconversion and/or oxidation. Most stearic acid comes either directly from diet or from elongation of palmitic acid, which is also synthesized de novo in considerable amounts. In this regard, the contribution of dietary stearic acid to its level in serum phospholipid is difficult to evaluate. We have no information on estimated dietary intake of stearic acid in our population. Therefore, the possibility that a low level of stearic acid reflects reduced dietary intake of stearic acid warrants further studies.
Lowered stearate in blood cells of patients with malignancies was proposed as being the consequence of increased delta-9 desaturation to oleic acid (Wood et al.,1985), but the reason for an increase in the enzyme activity is not known. The delta-9 desaturase enzyme or stearoyl-CoA desaturase enzyme is encoded by the SCD-gene family. Various fatty acids have been shown to regulate, in vitro and in vivo, expression of the SCD-1 gene in liver (Ntambi, 1995). Administration of sterculic acid, an uncommon fatty acid from plants which inhibits delta-9 desaturase, caused a decrease in the ratio of oleic acid to stearic acid in peripheral red cells, serum and liver of rats bearing NMU-induced mammary tumors, and inhibited tumor growth (Khoo et al.,1991). These results suggest that delta 9-desaturase activity may be directly involved in cancer development; but it is also possible that the inhibition of enzyme activity is merely a correlated phenomenon, without any mechanistic relation to tumor development.
Besides fatty acids, insulin is a well-documented regulator of stearoyl-CoA desaturase activity (Ntambi, 1995). Direct evidence for a regulatory effect of insulin comes from studies showing insulin-stimulation of SDC-1-gene expression in liver of diabetic mice (Waters and Ntambi, 1996), in mammary glands of normal mice (Kaput et al.,1994), in cultured hepatocytes (Legrand et al.,1994) and adipocytes (Weiner et al.,1991). These various observations suggest that decreased serum phospholipid levels of stearic acid, increased levels of oleic acid and decreased 18:0/18:1n-9 ratio, associated with increased risk of breast cancer in our cohort, might reflect an underlying metabolic profile characterized by chronic hyper-insulinemia.
Contrary to our starting hypothesis, we found no significant association of breast-cancer risk either with total n-3 polyunsaturated fatty acids or with long-chain n-3 fatty acids of marine origin. The relatively wide ranges of variation in both EPA (0.47–12.12%, controls and cases combined) and DHA (2.33–9.85%) appear to rule out the possibility that inter-individual differences in n-3 PUFA intake were too small to allow a decrease in risk to appear. Our results are similar to those of another prospective cohort study, in Norway, which also reported no association of breast-cancer risk with n-3 PUFA levels in serum phospholipids (Vatten et al.,1993). The use of adipose-tissue fatty-acid composition as a biomarker of dietary fatty acids showed an inverse association between the ratio of long-chain n-3 PUFA to n-6 PUFA and breast-cancer risk, in an ecological study conducted in 5 European countries, reinforcing the hypothesis that the degree of inhibition of fatty acids of the n-3 on breast cancer may depend on levels of n-6 PUFA (Simonsen et al.,1998). However, in our population, we found no significant association between the ratio 20:5 n-3/20:4 n-6 or the ratio n-3 PUFA/n-6 PUFA and breast-cancer risk (data not shown).
In conclusion, this study showed no association between n-3 PUFA in serum phospholipids and risk of breast cancer, and thus does not support the hypothesis that n-3 PUFA may be protective. In contrast, we found a decreased risk of breast cancer among women in the highest quartile of stearic acid, as compared with women in the lowest quartile, suggesting a protective effect of stearic acid. Further epidemiological and experimental data are needed to precisely identify the role of stearic acid in breast cancer.