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Circulating unmetabolized folic acid and 5-methyltetrahydrofolate and risk of breast cancer: a nested case-control study



Folates found in natural foods are thought to protect against cancer. However, folic acid (FA), a synthetic form of folate used in supplements and fortified foods, may increase breast cancer risk if present in unmetabolized form (UMFA) in the circulation. This study examined the associations of serum UMFA and 5-methyltetrahydrofolate (5-mTHF), the predominant form of circulating folate, with breast cancer risk.


We conducted a nested case-control study in a prospective cohort. In total, 553 cases of invasive breast cancer, diagnosed before mandatory FA fortification of grain in the US in 1998, were individually-matched to 1059 controls. Serum UMFA and 5-mTHF were measured using liquid chromatography-tandem mass spectrometry in stored serum samples, and 5-mTHF was corrected for storage degradation.


Serum UMFA was not associated with breast cancer risk: the percentage of women with detectable levels of UMFA was similar in cases and controls (18% and 20%, respectively; p = 0.46). Two tag-SNPs in the promoter region of the FA-metabolizing gene were also not associated with risk. There was a marginally significant inverse association of 5-mTHFcorrected with breast cancer risk (odds ratio for the highest vs. lowest quintile = 0.69, 95% CI = 0.49 to 0.97; ptrend = 0.08).


Circulating UMFA was not associated with breast cancer risk. These results apply to countries without mandatory FA food fortification. Studies are needed in countries with mandatory fortification, where levels of UMFA are much higher than in our study.

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This study was supported by research grants KG111502 from the Susan G. Komen Foundation, UM1 CA 182934 and P30 CA016087 from the US National Cancer Institute, and P30 ES000260 from the US National Institute of Environmental Health Sciences.

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KLK conceived of the study design, contributed to the analysis and interpretation of the data and to the writing of the manuscript. SS and AZJ conceived of the study design, acquired the data, contributed to the analysis and interpretation of the data and to the writing of the manuscript. YA analyzed the data and contributed to interpretation of the data. TVC selected the SNPs for analysis, assisted in the interpretation of the data and critically reviewed the manuscript. PMU performed the folate assays and critically reviewed the manuscript. All authors read and approved the final manuscript.

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Correspondence to Anne Zeleniuch-Jacquotte.

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Koenig, K.L., Scarmo, S., Afanasyeva, Y. et al. Circulating unmetabolized folic acid and 5-methyltetrahydrofolate and risk of breast cancer: a nested case-control study. Eur J Clin Nutr 74, 1306–1315 (2020).

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