Erythrocyte Membrane Phospholipid Fatty Acid Concentrations and Risk of Colorectal Adenomas: A Case–Control Nested in the French E3N-EPIC Cohort Study

Colorectal cancer is the third common cancer worldwide and a leading cause of death (1). It is well accepted that a non-negligible proportion of colorectal cancers arise from colorectal adenomas, a very common lesion (2, 3). Thus, it has been hypothesized that preventing the growth of adenomas in the colon and rectum could prevent colorectal cancer (4).

However, prospective and case–control studies that assessed the ability of nutritional factors to prevent colorectal adenomas provided mixed findings (5, 6). Interventional trials did not provide convincing results (7–9). However, in the context of the long course of the adenoma–carcinoma sequence, the 3- or 4-year period assessed through clinical trials is very short.

The identification of bioactive nutrients that may reduce colorectal adenomas could help in primary prevention of cancer and have important public health implications. One such nutrient is represented by fatty acids. One study based on dietary food frequency questionnaires showed that high intakes of marine n-3 polyunsaturated fatty acids (PUFA) were associated with the decreased risk of adenoma in women (10), but other studies failed to show such inverse associations on risk or recurrence of colorectal adenomas (11, 12). Data derived from a cross-sectional study showed an increased risk of colorectal adenomas associated with a high consumption of trans fatty acids (13).

The assessment of lipid nutrients through food frequency questionnaires may be prone to measurement error (14). In addition, conversion of quantities of food items into their fatty acid content is exceptionally complex, as a consequence of the imprecision of qualitative and quantitative estimates of fat in foods. The fatty acid composition of a given food can vary according to cooking methods and industry supply, and food composition tables are incomplete. In contrast, biomarkers of dietary fatty acids offer objective measures of bioavailable amounts of these compounds, irrespective of the source and quality of food, particularly for fatty acids that are not endogenously synthesized (15, 16). However, few studies examined the association between biomarkers of fatty acids and colorectal adenomas. One study reported an inverse association between serum levels of n-3 PUFA and colorectal adenoma risk, whereas a positive association was found for n-6 PUFA (17). Another study found a positive association between serum levels of saturated and monounsaturated fatty acids and colorectal adenoma risk, whereas a negative association was found for some n-6 and n-3 PUFA (18). No previous study tested separately association with advanced and nonadvanced adenomas, which are two different histologic entities. Further prospective studies are needed to determine the association between the lipidome and colorectal adenoma risk.

In the present study, we investigated the association between erythrocyte membrane phospholipid fatty acid levels as biomarkers of past dietary fatty acids intake and fatty acid metabolism and risk of colorectal adenomas in a nested case–control study in the E3N- EPIC cohort.

The E3N Study (Etude Epidémiologique auprès des femmes de la Mutuelle Générale de l'Education Nationale) is an ongoing prospective study that was designed to examine associations between cancer risk and dietary, lifestyle, and reproductive factors in women (19). This cohort is the French component of the European Prospective Investigation into Cancer and Nutrition (EPIC; 20). The E3N cohort includes female members of a national health insurance scheme covering teachers in the French education system and their spouses. Overall, 98,995 female volunteers ages 40 to 65 years were enrolled between 1989 and 1991 after replying to a baseline questionnaire and giving their written informed consent. The study was approved by the French National Commission for Data Protection and Privacy. In the baseline questionnaire and subsequent self-administered questionnaires, participants provided information on anthropometric characteristics, reproductive history, health status, lifetime use of hormonal treatments, family history of breast cancer, and smoking status. Usual diet was assessed through a validated 208-item diet history questionnaire sent out between 1993 and 1995 (21). The response rate for the dietary questionnaire was 81% of the total cohort at baseline. Among E3N participants, 25,000 women volunteered for blood collection between 1995 and 1998. Following a common protocol, blood samples were collected, aliquoted into plasma, serum, lymphocytes, and erythrocytes, and stored in liquid nitrogen.

Repeated mails were sent to participants who reported intestinal polyps in the questionnaires and to their physicians, requesting pathologic and colonoscopy reports. The polyp database has been previously described (22). The histologic features, size, number, and precise location of the tumors were coded. Advanced lesions were defined as adenomas more than 1 cm in diameter, with high-grade dysplasia (severe or in situ adenocarcinoma), or with a villous component; women simultaneously diagnosed with advanced and nonadvanced adenomas were classified in the “advanced adenoma” category.

From the initial 74,531 E3N-EPIC women who completed the baseline dietary questionnaire, a case–control study nested within the E3N cohort was designed among women who underwent at least one colonoscopy, and provided blood samples at baseline (n = 19,934). We excluded 4,654 women with prevalent cancer, 810 lost to follow-up after the baseline questionnaire, and 1,364 with extreme values of energy intake. In the remaining 13,106 women who underwent a colonoscopy during follow-up, we further excluded 193 women with inflammatory bowel disease, 9 with colectomy, 1 with familial adenomatous polyposis, 1,929 with a colorectal adenoma or unspecified polyp diagnosed before baseline, 783 with a hyperplastic polyp as the first diagnosed polyp, 115 whose removed polyp was not analyzed, and 421 with no available histologic report. During the follow-up period (1993–2002, date of the seventh questionnaire mailing), 328 cases of incident colorectal adenomas were identified in the remaining cohort, 143 of them were classified as advanced adenomas. Controls (n = 619) were randomly selected among women with polyp-free colonoscopies. Two controls per case were matched to 291 cases, and one control per case was matched to 37 cases, on age (± 1 year) at blood collection, collection center, and date of blood collection (same year).

Fatty acid concentrations were determined according to a methodology described previously (23). Samples were divided into batches of blinded tubes corresponding to samples from cases and their matched controls in random order and quality controls. Total lipids were extracted from erythrocytes' membrane phospholipids (250 μL) with chloroform–methanol 2:1 (v/v) containing antioxidant butylated hydroxytoluene and L-A-phosphatidylcholine-dimyristoyl-d₅₄ (Cambridge Isotope Laboratories, Inc.) as an internal standard. Phospholipid fraction was purified on SPE tubes (Supelco). Fatty acid methyl esters (FAME) were formed by methylation with Methyl-Perp II (Alltech) and extracted in hexane. Analyses were carried out on a 6890N gas chromatograph (Agilent). A BP×70 capillary column was used for separation of FAME (SGE). The relative amount of each fatty acid, expressed as a percentage of total fatty acids, was quantified by integrating the area under the peak and dividing the result by the total area. Fatty acids were also expressed as absolute concentrations (μmol/L) based on the quantity of the methyl-deuterated internal standard.

For quality control, we added one or two aliquots of the same serum sample to each batch of samples analyzed, in random order. Coefficients of variation varied from 0.75% for major peaks to 10.5% for minor peaks.

Baseline characteristics of controls and cases were compared using the χ² test for categorical variables and Student t test or non-parametric test (Wilcoxon) for continuous variables according to the normality assumption.

Individual saturated, cis- and trans- monounsaturated, n-6 and n-3 PUFA were considered in the analysis. In addition, the desaturation indexes (DI), as the ratio of oleic to stearic acid (DI_n-9) and the ratio of cis-palmitoleic to palmitic acid (DI_n-7) were determined. The DI is an indicator of activity of the rate-limiting enzyme stearoyl-CoA desaturase-1 (SCD-1), which transforms palmitic and stearic acids into monounsaturated palmitoleic and oleic acids, respectively. For analyses, fatty acids and DIs were divided into tertiles based on the distribution among controls.

OR and 95% confidence intervals (CI) were estimated using conditional logistic regression matched on the triplets (n = 291) or pairs (n = 37).

Models were adjusted for age (as a continuous variable), body mass index [weight (kg)/height² (m²), as a continuous variable], physical activity (metabolic equivalents/week, as a continuous variable), total energy intake (kcal/day, as a continuous variable), alcohol consumption (g/day, as a continuous variable), smoking status (in 3 categories: nonsmoker, past smoker, current smoker), educational level (in 3 categories: <12,12–14, ≥15 years), menopausal status (yes vs. no), menopausal hormone use (yes vs. no), and family history of colorectal cancer (yes vs. no). Tests for linear trend across tertiles of fatty acids were carried out using the median level in each tertile. Comparisons of ORs between advanced and nonadvanced adenomas were carried out using the Wald χ² test (P_{heterogeneity}).

All tests were two-sided, and statistical significance (P) was set at the 0.05 level. All analyses were conducted by using SAS, version 9.3, software (SAS Institute, Inc.).

Compared with controls, cases tended to have more often a family history of colorectal cancer and reported statistically significant higher alcohol consumption than controls (Table 1). Body mass index, menopausal status, ever use of menopausal hormones, physical activity, smoking status, and years of education did not differ significantly between cases and controls.

Table 2 shows OR for colorectal adenoma by tertiles of erythrocyte membrane phospholipid fatty acids, expressed as a percentage of total fatty acids. Among saturated fatty acids, pentadecanoate (15:0) plus heptadecanoate (17:0), fatty acids with an odd number of carbons, tended to be inversely associated with colorectal adenoma risk [for the highest tertile compared with the lowest, OR_T3vsT1 = 0.68 (95%CI 0.45–1.01); P_trend = 0.055]. On the opposite, the association between adenoma risk and monounsaturated oleic acid is near the significance, a higher concentration of C18:1n-9cis increased the risk of colorectal adenoma [OR_T3vsT1 = 1.54 (1.00–2.37); P_trend = 0.052]. Trans-monounsaturated fatty acids were not associated with colorectal adenoma risk. No association was found with n-6 PUFAs, except di-homo-γ-linolenate positively associated with colorectal adenoma risk [OR_T3vsT1 = 1.43 (0.99–2.07) P_trend = 0.045]. Finally, no significant association was found with long-chain n-3 PUFA, except a trend for an inverse association between colorectal adenoma risk and α-linolenate, the essential fatty acid of the n-3 family [OR_T3vsT1 = 0.71 (0.49–1.03); P_trend = 0.078]. No association was found between the ratio of α-linolenate/linoleate or the ratio of long chain n-3 PUFA to n-6 PUFA and colorectal adenoma risk.

The associations between erythrocyte membrane phospholipid fatty acids concentrations and colorectal adenoma risk were tested separately for advanced and nonadvanced adenomas (Table 3). Although some fatty acids showed their influence on the risk of advanced adenomas, no significant effect was highlighted on the risk of nonadvanced adenomas. High level of pentadecanoate plus heptadecanoate was associated with a decreased risk of advanced adenomas [OR_T3vsT1 = 0.40 (0.20–0.79) P_trend = 0.009] but not of nonadvanced adenomas (P_{heterogeneity} = 0.042). Oleic acid was associated with an increased risk of advanced adenomas [OR_T3vsT1 = 2.32 (1.16–4.64); P_trend = 0.018]. Among n-6 PUFA, di-homo-γ-linolenate doubled the risk of advanced adenomas [OR_T3vsT1 = 2.07 (1.15–3.72); P_trend = 0.013] but was not significantly associated with risk of nonadvanced adenomas (P_{heterogeneity} = 0.191). A protective effect of long-chain n-3 PUFA was found regarding advanced adenoma risk but not for nonadvanced adenoma risk. Eicosapentaenoic and docosahexaenoic acids had a clear protective effect on advanced adenoma risk [OR_T3vsT1 = 0.50 (0.27–0.93); P_trend = 0.044 and OR_T3vsT1 = 0.50 (0.26–0.96) P_trend = 0.028, respectively] but not on nonadvanced adenoma risk (P_{heterogeneity} = 0.044 and P = 0.045, respectively). The strongest inverse association was found between the ratio of long-chain n-3 PUFA to long chain n-6 PUFA and advanced adenoma risk [OR_T3vsT1 = 0.40 (0.21–0.74) P_trend = 0.004]. Association between advanced adenoma risk and α-linolenic acid concentration did not reach the significance level [OR_T3vsT1 = 0.66 (0.38–1.14) P_trend = 0.138].

In this nested case–control study based on the French prospective E3N-EPIC cohort, we found evidence that some fatty acids showed their influence on the risk of advanced colorectal adenomas, whereas no significant effect was highlighted on the risk of nonadvanced adenomas. Increasing levels of oleic acid and di-homo-γ-linolenic acid were associated with increased advanced colorectal adenoma risk. In contrast, increasing levels of pentadecanoic plus heptadecanoic acids and long-chain n-3 PUFA were associated with decreased risk of advanced adenoma.

Among saturates, fatty acids containing an odd number of carbon atoms cannot be synthesized de novo and only derive from ruminant products (24). Accordingly, we already reported a positive association of serum phospholipid pentadecanoate (15:0) plus heptadecanoate (17:0) levels with dietary dairy products (25). In our study, increasing levels of pentadecanoate plus heptadecanoate were associated with decreased risk of advanced colorectal adenomas, suggesting that high dietary intakes of dairy products may be associated with decreased risk of advanced colorectal adenomas. This finding strengthens results from the E3N-EPIC cohort that showed the association between high dietary intake of dairy products, calcium and phosphorus, and risk of colorectal tumors (adenomas and cancer; 26). Similarly, a review of epidemiologic studies concluded that dairy products had a potential protective effect on adenoma occurrence (27). The main hypothesis underlying a protective effect of dairy products on colorectal cancer has been related to their calcium content, and to a lesser extent to vitamin D, conjugated linoleic acid, sphingolipids, butyric acid, and fermentation products (28). Another hypothesis might be related to the effect of specific saturated fatty acids pentadecanoate and heptadecanoate on insulin concentrations. Epidemiologic studies indicated a link between chronic hyperinsulinaemia and increased risk of colorectal adenoma and cancer (29), and findings derived from animal studies suggested that insulin is an important growth factor of colonic epithelial cells and a mitogen of tumor cell proliferation in vitro (30). In a prospective study, serum phospholipid pentadecanoate and heptadecanoate levels were inversely associated with serum levels of insulin and also plasminogen activator inhibitor-1 and leptin (31). Further experimental studies are needed to investigate the effects of these specific fatty acids on colorectal carcinogenesis and insulin responses to these fatty acids.

In our study, an increased risk of advanced colorectal adenoma was found with increasing levels of erythrocyte membrane phospholipid oleic acid. Similarly, a case–control study in Japan reported the same association with the overall risk of adenoma (18). Oleic acid is not an essential fatty acid and can be either diet derived or formed endogenously by the desaturation of stearic acid into oleic acid via microsomal enzyme SCD-1 (32). Data derived from a cross-sectional study within the EPIC cohort showed that dietary oleic acid originates mainly from olive oil in south Europe and from meat in north-central Europe (33). However, the weak correlations calculated at the individual level between plasma phospholipid oleic acid and olive oil or meat intake suggested that dietary contributors of plasma phospholipid oleic acid concentration may not be strong determinants compared with endogenous hepatic synthesis (16). Thus, increased risk of advanced colorectal adenoma associated with high level of oleic acid, as previously reported in relation to gastric (34) and to breast cancer risks (23), may be the consequence of increased desaturation of stearic acid into oleic acid rather than to a diet rich in oleic acid. However, in our study, increasing ratio of oleic acid to stearic acid, as a biomarker of hepatic SCD-1 expression/activity (32), was not significantly associated with increased risk of advanced colorectal adenoma. Diet may also have an important effect on SCD-1 activity. In EPIC studies, a high consumption of alcohol was associated with increased ratio of oleic to stearic acid, whereas a high consumption of olive oil counteracted this effect (16, 25). High blood level of phospholipid oleic acid, presumably reflecting both a specific complex dietary pattern and increased stearic acid desaturation via SCD-1, is related to increased risk of advanced colorectal adenomas. The potential pathway underlying the relation between prediagnostic blood phospholipid oleic acid and advanced colorectal tumors is not known. Despite the overactivation of enzymes that synthesize saturated fatty acids (35), abundant amounts of monounsaturated fatty acid (oleate) are found in numerous cancer cells (36, 37). Monounsaturates can serve as mediators of signal transduction and cellular differentiation, and unbalanced levels of these mediators have also been implicated in carcinogenesis (38). The expression of SCD-1, the main SCD isoform in humans, is increased in several human cancers and chemically induced tumors (37, 39, 40). The suppression of SCD-1 expression reduces cancer cell proliferation and in vitro invasiveness, and dramatically impairs tumor formation and growth (41, 42). These data may suggest that endogenously synthesized oleic acid, rather than exogenous dietary oleic acid, act as regulators of cancer cell growth (15).

In the present study, higher concentration of di-homo-γ-linolenic acid was associated with increased risk of advanced colorectal adenoma. The main dietary sources of this n-6 are oil seeds (such as borage, evening primrose and blackcurrant), but its blood concentration is likely mostly due to the metabolism of linoleic acid via delta-6 desaturation and elongation (16). N-6 PUFA leads to production of prostaglandins and thromboxanes, which are critical regulators of platelet aggregation, T-cell development, inflammation, and cancer (43). COX-derived prostaglandin E (PGE2) is the predominant prostanoid found in most colorectal cancers and is known to stimulate colon carcinoma growth and invasion (44). In addition, PGE2 has been shown to transactivate PPAR-δ through PI3K/Akt signaling, which promotes cell survival and adenoma formation (44).

In our study, erythrocyte membrane phospholipid long-chain marine n-3 PUFA levels originating from marine sources were inversely associated with advanced colorectal adenoma risk. Two case–control studies reported an inverse (but nonsignificant) association between serum n-3 long-chain PUFA levels and colorectal adenoma risk (17, 18). Two prospective studies based on food frequency questionnaires did not find any association between n-3 PUFA intakes and risk of colorectal adenomas (11, 12), but association was not tested separately on advanced and nonadvanced colorectal adenoma risk. Mechanistic studies suggest that n-3 PUFA may decrease tumor cell proliferation or growth by decreasing prostaglandin formation from n-6 PUFA, suppressing COX-2 induction, and proliferation in the colorectal mucosa (45). This will lead to an increase in eicosanoid synthesis from long-chain n-3 PUFA, resulting in a shift in production of 2-series to 3-series prostaglandins (46). Our observation of a strong inverse association between the ratio of long-chain n-3PUFA to long-chain n-6 PUFA and advanced colorectal adenoma risk supported the hypothesis of a competition between long-chain omega-6 and long-chain omega-3 PUFA for eicosanoid production as an underlying mechanism. In addition, in a sensitivity analysis, we estimated the association between long-chain n-3 PUFA and risk of advanced colorectal adenoma after adjusting for long-chain n-6 PUFA. The inverse association between long-chain n-3 PUFA and risk of advanced colorectal adenoma was no longer significant [OR_T3vsT1 = 0.57, 95% CI = 0.28–1.16, P_trend = 0.11, data not shown), further supporting the hypothesis that the effect of long chain n-3 PUFA on the risk of advanced colorectal adenoma is dependent on background levels of long-chain n-6 PUFA.

There is increasing concern that consumption of industrial trans fatty acids may contribute to disease risk such as heart disease (47) and type II diabetes (48). We previously showed that plasma phospholipid trans elaidic acid level was correlated to dietary intake of highly processed foods in a cross-sectional study within the EPIC cohort (15). Using serum phospholipid trans elaidic and palmitelaidic acid levels as biomarkers of industrial foods, we found an increased risk of breast cancer associated with increasing levels of these specific trans fatty acids (23). In our present study, we did not find evidence of an association between risk of colorectal adenomas and levels of erythrocyte membrane phospholipid trans elaidic and palmitelaidic acid. Two cohort studies failed to find a positive association between trans fatty acid intake estimated by food frequency questionnaires and colorectal cancer risk (49, 50). A cross-sectional study showed that high dietary intake of trans fatty acids increased the risk of colorectal adenomas (13) and distal colorectal cancer (51). The biologic plausibility underlying such association may involve increased oxidative stress and inflammation in the colonic mucosa (52). The present study will be further extended by investigating the association between both erythrocyte membrane and plasma phospholipid fatty acid levels and risk of colorectal cancer in the French E3N-EPIC study.

This study has important strengths. Each participant underwent complete colonoscopy, limitating misclassification of case–control status. Individual fatty acids were measured on prediagnostic blood samples, several years before potential diagnosis of adenoma. There are also some limitations to the present study. Cases and controls came from a selected population of highly educated women who volunteered to participate in both the dietary survey and the blood collection. However, although this population was not representative of the general population, it is not clear how selection could have seriously affected the associations estimated in this study. As in other epidemiologic studies, we cannot rule out the possibility that the associations we observed resulted from confounding bias, although we adjusted for known risk factors available in the questionnaire. Then, dietary changes or changes in dietary food composition may have occurred after baseline blood collection and beforethe onset of the disease. Because of the lack of repeated blood sample collection, we could not correct for such measurement errors, which could have led to an underestimation of the associations. Finally, because of a lack of information on aspirin and nonsteroidal anti-inflammatory drugs, which could influence COX-2 and thus, the associations between n-6 and n-3 PUFA and risk of colorectal adenoma, we cannot adjust for these potential confounding factors in our regression model.

In summary, findings from this study suggest that a prediagnostic erythrocyte membrane phospholipid fatty acid profile, presumably reflecting both a complex dietary pattern and impaired metabolism, is associated with increased risk of advanced colorectal adenomas, whereas no significant association was found with nonadvanced adenomas.

No potential conflicts of interest were disclosed.

The study sponsors had no role in the design of the study, the analysis or interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication.

Conception and design: V. Cottet, F. Clavel-Chapelon, V. Chajès

Development of methodology: F. Clavel-Chapelon, V. Chajès

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Collin, A.-S. Gross, S. Morois, F. Clavel-Chapelon, V. Chajès

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): V. Cottet, M. Collin, A.-S. Gross, F. Clavel-Chapelon, V. Chajès

Writing, review, and/or revision of the manuscript: V. Cottet, M. Collin, A.-S. Gross, M.C. Boutron-Ruault, S. Morois, F. Clavel-Chapelon, V. Chajès

Study supervision: F. Clavel-Chapelon, V. Chajès

The authors thank practitioners for providing pathology reports. The authors also thank all members of the E3N-EPIC study group and M. Niravong, L. Hoang, R. Chait, G. Guillas, and M. Fangon for technical assistance.

This study was based on data from the E3N cohort and was supported by grants from the French League Against Cancer (LNCC), the Fondation de France (FDF), the Association for Research on Cancer (ARC), and Lipidomic Core Facilities TA2012. The E3N Study is being carried out with the financial support of the LNCC, the European Community, the Mutuelle Générale de l'Education Nationale (MGEN), the French Institute of Health and Medical Research (INSERM), and the Gustave Roussy Institute (IGR). V. Cottet was partly supported by a French Government grant managed by the French National Research Agency under the program “Investissements d'Avenir” with reference ANR-11-LABX-0021.

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