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American Journal of Clinical Nutrition, Vol. 87, No. 3, 734-743, March 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Plasma folate, vitamin B-6, vitamin B-12, and risk of breast cancer in women1,2,3

Jennifer Lin, I-Min Lee, Nancy R Cook, Jacob Selhub, JoAnn E Manson, Julie E Buring and Shumin M Zhang

1 From the Division of Preventive Medicine, Department of Medicine (JL, IML, NRC, JEM, JEB, and SMZ), Channing Laboratory (JEM), and Department of Ambulatory Care and Prevention (JEB), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; the Department of Epidemiology, Harvard School of Public Health, Boston, MA (IML, NRC, JEM, JEB, and SMZ); the Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA (JS)

2 Supported by grants CA47988, CA104871, and CA112529 from the National Cancer Institute and by grant HL43851 from the National Heart, Lung, and Blood Institute.

3 Address reprint requests to J Lin, Division of Preventive Medicine, Brigham and Women's Hospital, Harvard Medical School, 900 Commonwealth Avenue East, Boston, MA 02215. E-mail: jhlin{at}rics.bwh.harvard.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: B vitamins such as folate, vitamin B-6, and vitamin B-12 are coenzymes that are important for DNA integrity and stability. Deficiency in these B vitamins may promote tumor carcinogenesis.

Objective: We prospectively evaluated plasma concentrations of folate, pyridoxal 5-phosphate (PLP; the principal active form of vitamin B-6), and vitamin B-12 in relation to breast cancer risk.

Design: We included 848 incident cases of invasive breast cancer identified as of 31 March 2004, and 848 individually matched control subjects from 28 345 women in the Women's Health Study aged ≥45 y who provided blood samples and had no history of cancer and cardiovascular disease at baseline in 1993. Logistic regression controlling for matching factors and other risk factors for breast cancer was used to estimate relative risks (RRs) and 95% CIs. All statistical tests were 2 sided.

Results: Plasma concentrations of folate, PLP, and vitamin B-12 were not associated with overall risk of breast cancer. Women in the highest quintile group relative to those in the lowest quintile had multivariate RRs of 1.42 (95% CI: 1.00, 2.02) for plasma folate (P for trend = 0.21), 0.91 (95% CI: 0.63, 1.30) for plasma PLP (P for trend = 0.48), and 1.29 (95% CI: 0.92, 1.82) for plasma vitamin B-12 (P for trend = 0.18). However, higher plasma folate concentrations were moderately associated with an increased risk of developing premenopausal breast cancer (P for trend = 0.04) and for developing estrogen receptor (ER)–positive or progesterone receptor (PR)–positive breast tumors (P for trend ≤ 0.06). Conversely, an inverse association was seen between plasma PLP and postmenopausal breast cancer (P for trend = 0.04).

Conclusions: Data from this study suggest that B vitamins, including folate, vitamin B-6, and vitamin B-12, may confer little or no reduction in overall risk of developing breast cancer. The observed positive associations of folate status with risk of developing premenopausal breast cancer and ER-positive or PR-positive tumors are unexpected. Additional research is needed to elucidate the role of folate in breast cancer development.

Key Words: Folate • vitamin B-6 • vitamin B-12 • breast cancer • cohort study • plasma marker


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
B vitamins such as folate, vitamin B-6, and vitamin B-12 function as coenzymes in one-carbon metabolism, which is critical for the synthesis and methylation of DNA (1-4). Folate is involved in the conversion of homocysteine into methionine and eventually into S-adenosylmethionine, the primary methyl group supplier for most methylation reactions (1-5). Folate also assists in the synthesis of purines and thymidylate for DNA synthesis (1-4, 6). Vitamin B-6 is a coenzyme that is involved in homocysteine catabolism and the conversion of tetrahydrofolate to 5,10-methelene-tetrahydrofolate for DNA synthesis (2, 7). Vitamin B-12 facilitates the methyl transfer to homocysteine to form methionine (5). Deficiency in these B vitamins may interfere with DNA methylation and synthesis, leading to aberrant gene expression and DNA instability and eventually the development of cancer or birth defects (8, 9).

The association between folate, vitamin B-6, and vitamin B-12 and breast cancer risk has been extensively evaluated in recent studies (10-31). Many case-control studies have reported an inverse association between dietary intakes of folate or other B vitamins and breast cancer risk (10-16). Several prospective cohort studies observed an inverse association between folate intake and breast cancer risk among women consuming alcohol (20-22, 24, 31) and among current smokers (19), although most of the studies have not found an overall association between intakes of folate or vitamins B-6 and B-12 and breast cancer risk (19-24, 31). Data relating circulating concentrations of folate, pyridoxal 5-phosphate (PLP; the principal active form of vitamin B-6), or vitamin B-12 to breast cancer risk are limited (25-28). Three studies (26-28) but not another (25) suggested an inverse association between circulating folate concentrations and breast cancer risk.

In this prospective study with a large sample size relative to the previous studies of circulating B vitamins, we examined plasma concentrations of folate, PLP, and vitamin B-12 in relation to the risk of invasive breast cancer. To gain a better understanding of the role of these B vitamins in breast cancer risk, we investigated whether the association between plasma folate, PLP, and vitamin B-12 and breast cancer risk was modified by potential risk factors for breast cancer, including menopausal status (32), alcohol intake (33), cigarette smoke (34, 35), and supplement use (21, 36). Because estrogen receptor (ER)–negative and perhaps progesterone receptor (PR)–negative breast tumors, resulting from the hypermethylation of the promoter region of ER and PR (37-39), were linked to the low intake of folate and potentially other B vitamins (40-42), we also examined the association according to hormone receptor status.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study population
The Women's Health Study is a completed randomized trial that evaluated low-dose aspirin and vitamin E for the primary prevention of cancer and cardiovascular disease (43-45). Beginning in 1993, 39 876 US female health professionals aged ≥ 45 y and free of cancer and cardiovascular disease participated in the study and completed a baseline questionnaire about their medical history and potential risk factors for breast cancer. Blood samples were collected in both EDTA- and citrate-containing tubes from 28 345 women (71% among the total) before randomization and were stored in liquid nitrogen freezers at –170 °C. Baseline characteristics of women who gave blood samples were largely similar to those who did not (46). Briefly, both groups were not different in mean age (54.7 and 54.4 y), proportions of women with body mass index (BMI; in kg/m2) of ≥25 (48.4% compared with 51.1%), alcohol intake (1.1 compared with 0.9 g/d), physical activity (median expenditure per week: 599 compared with 526 kcal/wk), and current use of multivitamin supplements (29.4% compared with 28.6%) (46).

Dietary assessment
At baseline, participants were also asked to fill out a 131-item food-frequency questionnaire (FFQ), which asked the average intake of food and beverages during the past year. Participants chose from 9 possible answers, ranging from "never or less than once per month" to "6 or more times per day." The response for each food item was then converted into an average daily intake of the food item in servings per day. Nutrient values in foods were computed by multiplying the frequency of responses by the nutrient content of specified portion sizes according to the US Department of Agriculture food composition data (47) and were supplemented by food manufacturers. Total alcohol intake per day was estimated by the sum of the alcohol content contributed from beer, wine, and liquor, assuming 12.8 g of ethanol for 360 mL (12 oz) of beer, 11 g for 120 mL (4 oz) of wine, and 14 g for 45 mL (1.5 oz) of liquor. Information on type and brand of multivitamin supplement use was also asked from the FFQ, and a database relating the folate, vitamin B-6, and vitamin B-12 content of the multivitamin was developed.

The validity and reproducibility of the FFQ was assessed in the Nurses' Health Study. Pearson's correlation coefficient between vitamin B-6 intake from the FFQ and that from four 1-wk dietary records spaced over a year was 0.56 (48). The correlation coefficient between total folate intake and blood concentrations of folate ranged from 0.49 to 0.55 (27, 49). The correlation coefficients between other B vitamins and their blood concentrations were 0.52 for vitamin B-6 and 0.25 for vitamin B-12 (27).

Identification of case and control subjects
Every 6 mo during the first year of follow-up and then annually thereafter, participants were asked whether they had been newly diagnosed with breast cancer. We then confirmed women who reported a diagnosis of breast cancer and those who were deceased through the review of medical records and extracted detailed information on the diagnosis of these women. As of March 31, 2004, we documented 889 incident cases of invasive breast cancer among women with blood samples. Each breast cancer case was individually matched to one control with no diagnosis of cancer on age (up to 5 y of difference), ethnicity, menopausal status (premenopausal, postmenopausal, or uncertain or unknown), fasting status (≥8 h or fewer), month and year of blood return (4-mo interval), postmenopausal hormone use (never, past, or current), and trial randomization date (12-mo difference). On the basis of these matching criteria, a total of 850 invasive breast cancer cases were individually matched to 850 controls.

Laboratory analyses
All assays were conduced at the Jean Mayer US Department of Agriculture Human Nutrition Research Center on Aging at Tufts University. Plasma concentrations of folate and vitamin B-12 were measured with the use of the IMMULITE 1000 immunoassay system (Diagnostic Products Corporation, Los Angeles, CA). Plasma PLP, the major active form of vitamin B-6, was measured by an enzymatic procedure based on the radioactive tyrosine and apo-enzyme tyrosine decarboxylase as described by Shin-Buehring et al (50).

Blood samples for the 850 cases and 850 controls were handled identically and together and shipped in the same batch. Each pair of the samples was assayed together, and all pairs were analyzed in one run. All laboratory personnel were blinded to case or control status. To assess the laboratory precision, 29 triplets of replicate plasma samples were randomly interspersed and labeled to preclude their identification. The mean CVs for these triplets were 9.0% for folate, 11.2% for PLP, and 10.8% for vitamin B-12.

Statistical analyses
A total of 848 case-control pairs were included in the analysis after excluding 2 pairs who had missing plasma information in one member of the pair. Because plasma concentrations of folate, PLP, and vitamin B-12 were skewed from normality, plasma nutrients were first loge-transformed. We then categorized plasma nutrients into quintiles on the basis of the distribution in the controls. Differences between case-control pairs in mean concentrations of plasma nutrients and other continuous covariates were tested with the use of a paired t test. McNemar's test was used to compare the difference between case-control pairs in proportions of covariates as categorical variables.

Conditional logistic regression was used to estimate relative risks (RRs) and 95% CIs for invasive breast cancer. Analyzed models, taking into account the matching factors, were adjusted for age (in y), random treatment assignment (aspirin compared with placebo, vitamin E compared with placebo), and additionally for risk factors for breast cancer assessed at baseline, including BMI (<25, 25 to <30, ≥30), physical activity (energy expenditure in kcal/wk, in quartiles), family history of breast cancer in a first-degree relative (yes, no), history of benign breast disease (yes, no), age at menarche (≤11, 12, 13, ≥14 y), parity (0, 1–2, 3–4, ≥5 children), age at first birth (≤19, 20–24, 25–29, ≥30 y), smoking status (never, past, current), alcohol consumption (none, >0 to <5, 5 to <15, ≥15 g/d), and age at menopause (<45, 45 to <50, 50 to <52, ≥52 y). When we further adjusted for screening mammogram (yes, no) obtained during the first 12-mo follow-up questionnaire, we excluded cases confirmed during the first year of follow-up.

We also evaluated whether the overall associations were modified by menopausal status (premenopause, postmenopause), alcohol intake (<10, ≥10 g/d), and smoking status (current, past, never) among all women and by hormone therapy (never, current) among postmenopausal women. Beginning in 1996 and becoming effective in 1998, the Food and Drug Administration issued a regulation that required enriched grain products to be fortified with folic acid (140 µg/100 g) (51). We, therefore, tested whether the association between plasma nutrients and breast cancer risk was different before and after this regulation by evaluating the association according to the calendar year of folate fortification (follow-up period before, after 1998). Finally, we examined whether the association between plasma nutrients and breast cancer risk was different by hormone receptor status (ER positive, ER negative, PR positive, PR negative). All stratified analyses were performed by unconditional logistic regression controlling for matching factors, including year of age, ethnicity, month of blood collection, fasting status, postmenopausal hormone use, menopause status, and randomization date, as well as risk factors for breast cancer. Tests for multiplicative interactions between nutrient concentrations and modifiers were performed by entering each product term into the multivariate model with a Wald statistic. Tests for difference in the estimates of nutrient concentrations for ER breast tumors (ER positive, ER negative) and for PR breast tumors (PR positive, PR negative) were conducted with the squared T statistic, which has a chi-square test distribution with 1 df. We used SAS statistical software (version 8.2; SAS Institute, Cary, NC) for all analyses. Tests for trend were performed by fitting the median of the plasma nutrient for each quintile as a continuous variable in the models. All P values were 2 sided.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We first compared the baseline characteristics of patients with breast cancer patients and control subjects (Table 1Go). Cancer patients were more likely than were control subjects to have a previous history of benign breast disease and to report as current smokers. Cancer patients were also more likely to be older at first child birth and when experiencing menopause and to have fewer childbirths. Dietary intakes of folate, vitamin B-6, and vitamin B-12 were not different between cancer patients and control subjects. There was also no difference between cancer patients and control subjects in plasma concentrations of these 3 vitamins.


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  		TABLE 1. Baseline characteristics of invasive breast cancer cases and their matched controls in the Women's Health Study1

 
We next evaluated the partial correlations of loge-transformed plasma folate, PLP, and vitamin B-12 and loge-transformed intakes of these 3 nutrients in the control subjects (Table 2Go). As expected, plasma concentrations of folate, PLP, and vitamin B-12 were mutually positively correlated with each other (r = 0.35–0.50) and were inversely correlated with homocysteine concentrations (partial correlation coefficients with adjustment for matching factors: –0.33 for plasma folate, –0.39 for plasma PLP, and –0.35 for plasma vitamin B-12). Plasma concentrations of these 3 nutrients were also moderately positively correlated with dietary intakes; the correlations were stronger with dietary nutrient intakes from all sources than with intakes from food only, suggesting the influence of multivitamin supplements on these nutrient concentrations. It is notable that intake of vitamin B-12 from food only was not correlated with any of these 3 plasma nutrients (Table 2Go). Total alcohol intake and tobacco use were also not related to these plasma nutrients (Table 2Go).


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  		TABLE 2. Partial correlation coefficients between dietary intakes and plasma concentrations of folate pyridoxal 5'-phosphate (PLP) and vitamin B-12 among control subjects (n = 848) adjusted for matching factors1

 
Plasma concentrations of folate, PLP, and vitamin B-12 were not significantly associated with overall risk of breast cancer in the analysis controlling for matching factors and age and randomized treatment assignment (Table 3Go). The results were not appreciably changed after further adjustment for other potential risk factors for breast cancer; women in the highest quintile relative to those in the lowest quintile had multivariate RRs of 1.42 (95% CI: 1.00, 2.02; P for trend = 0.21) for plasma folate, 0.91 (95% CI: 0.63, 1.30; P for trend = 0.48) for plasma PLP, and 1.29 (95% CI: 0.92, 1.82; P for trend = 0.18) for plasma vitamin B-12. Additional adjustment for mammogram screening test also did not change the results (data not shown). In addition, no significant interaction was observed among these 3 plasma nutrients with breast cancer risk (P for interactions > 0.42). When we modeled these 3 plasma nutrients simultaneously in the multivariate model, we also observed no appreciable change of the results (data not shown).


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  		TABLE 3. Relative risks (RRs) and 95% CIs of invasive breast cancer according to quintiles of plasma concentrations and nutrient intakes of folate, pyridoxal 5'-phosphate (PLP), or vitamin B-6 and vitamin B-12 in the Women's Health Study1

 
A linear trend of association was not observed between total intakes of folate and vitamin B-6 from the baseline FFQ and overall risk of breast cancer. A possible J-shaped pattern in RRs was seen in these 2 total intakes (Table 3Go). However, total vitamin B-12 intake was positively associated with an increased risk of breast cancer with a multivariate RR of 1.44 (95% CI: 1.02, 2.04) in the highest quintile group relative to the lowest quintile group (P for trend = 0.03). Intakes of folate, vitamin B-6, and vitamin B-12 from food only were not significantly associated with breast cancer risk (Table 3Go). Intake of methionine, another methyl-related nutrient, was not associated with breast cancer risk (data not shown).

When analyzing the association of these 3 plasma nutrients by menopausal status, we observed an inverse association between plasma PLP and breast cancer risk among postmenopausal women (P for interaction = 0.11); women in the highest quintile group had a multivariate RR of 0.64 (95% CI: 0.42, 0.99; P for trend = 0.04). The inverse association was more prominent among never users of hormone therapy (P for interaction < 0.01; Table 4Go). We, however, saw an unexpected positive association between plasma folate and premenopausal breast cancer (P for interaction = 0.05). Folate concentrations were not significantly associated with breast cancer risk among postmenopausal women, although the association was modified by hormone use (P for interaction = 0.02; Table 4Go). No significant association was observed between vitamin B-12 concentrations and breast cancer risk by menopausal status (P for interaction = 0.82) and hormone use (P for interaction = 0.32). In addition, there was no evidence of an interaction between these plasma nutrients and alcohol consumption (P for interaction ≥ 0.14) with breast cancer risk. The association between these 3 plasma nutrients and breast cancer risk was also not modified according to supplement use and smoking (P for interaction ≥ 0.53). However, we observed a marginally positive association between plasma folate concentrations and breast cancer risk during follow-up subsequent to mandatory fortification with folic acid (P for trend = 0.06; Table 4Go).


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  		TABLE 4. Relative risks (RRs) and 95% CIs of invasive breast cancer according to quintiles of plasma nutrient concentrations by categories of covariates in the Women's Health Study1

 
Finally, plasma concentrations of these 3 plasma nutrients were not significantly inversely associated with risk of developing ER-negative breast tumors (Table 5Go). However, plasma folate was marginally positively associated with risk of developing ER-positive or PR-positive breast tumors (P for trend ≤ 0.06). These 3 plasma nutrients were not related to PR-negative breast tumors (Table 5Go).


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  		TABLE 5. Relative risks (RRs) and 95% CIs of invasive breast cancer according to quintiles of plasma nutrient concentrations by hormone receptor status in the Women's Health Study1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this large prospective case-control study nested in a female cohort, plasma concentrations of folate, PLP, and vitamin B-12 were not associated with overall risk of breast cancer. There was also no conclusive effect modification of the association by alcohol consumption, supplement use, and smoking. However, plasma PLP was inversely associated with the risk of developing postmenopausal breast cancer. By contrast, higher plasma folate was positively associated with the risk of developing premenopausal breast cancer as well as ER-positive or PR-positive breast tumors.

Many (10-16) except a few (29, 30) case-control studies have reported an inverse association between folate intake and breast cancer risk. However, most prospective cohort studies have not found an overall association with breast cancer risk (20-22, 24, 31), although many of these studies have reported an inverse association between folate intake and breast cancer risk among women consuming alcohol (19-24, 31). Three studies assessing blood concentrations of folate and breast cancer risk have reported a suggestive inverse association between blood folate concentrations and breast cancer risk (26-28). We and one other study from the Washington County Study cohort (25), however, did not observe the association. Different distributions of folate concentrations between our study cohort and the others may have contributed to the different findings. Although plasma folate concentrations in our study were similar to those in the Washington County Study (25) (median value: 8 ng/mL), our plasma folate concentrations (highest quintile range: ≥16 ng/mL) were substantially higher than those in the 2 Australian studies (highest quartile ranges: ≥6 and ≥9 ng/mL, respectively), which observed an inverse association between folate status and breast cancer risk (26, 28). Two recent meta-analysis studies further concluded no clear association between folate intake and folate concentrations and breast cancer risk (52, 53), suggesting that high folate status may be associated with little or no risk reduction of breast cancer in the entire population.

Despite its central function in maintaining DNA integrity and stability, the role of folate in cancer prevention was recently re-scrutinized. Experimental studies in animals suggest that folate may have dual effects on breast cancer development, depending on its timing (9). Folate administration can prevent tumor development before the existence of preneoplastic lesions and increasing tumorigenesis once preneoplastic lesions are established. Three animal studies in rats reported that folate supplementation may promote, whereas folate deficiency suppressed, mammary tumors (54-56). Rapidly proliferating tissues, such as tumors, require nucleotide synthesis, and folate is efficient in fulfilling these needs (57). In addition, several antifolate agents such as methotrexate and 5-fluorouracil are considered effective chemotherapy to interfere with DNA synthesis in breast tumors (58, 59). Folic acid may, therefore, contribute to breast carcinogenesis at specific stages of the neoplastic process.

Since 1998, the US population has been potentially exposed to an increase in folate intake. It was estimated that the mandatory fortification with folic acid has resulted in a mean increase in folate intake by approximately twice as much as previously expected in the general population (60). Our finding of a marginally positive association between baseline plasma concentrations of folate and breast cancer risk during follow-up after mandatory fortification with folic acid raises the question of appropriate folate dosage in relation to cancer risk. In a randomized trial of folic acid supplementation during pregnancy, women who received folate supplements (0.2 or 5 mg/d) had a nonsignificantly increased risk of breast cancer mortality compared with those in the placebo group (61). Notably, women who received the higher dose of 5 mg daily had the highest risk of breast cancer mortality (RR = 2.02; 95% CI: 0.88, 4.02), although the association was not statistically significant (61). Nevertheless, we found no difference in breast cancer risk between folate supplement users and nonsupplement users. More studies are necessary to resolve this issue.

In our study, folate concentrations were positively associated with the risk of developing premenopausal breast cancer. Although 3 other cohort studies observed no significant association between folate intake and risk of developing premenopausal breast cancer (19, 21, 22), the reported overall risk estimates were >1. Folate plays an important role in DNA synthesis, repair, and methylation, all of which are essential processes during cell mitoses. Because the proliferation rate in breast epithelial cells was suggested to be higher in premenopausal than in postmenopausal women (62), time available for DNA repair may be reduced in premenopausal women. More studies are necessary to confirm our findings.

We also observed a positive association of plasma folate with the risk of developing ER-positive breast tumors. Previous data on the relation between folate and estrogen in cancer development are limited. Two in vitro studies in reproductive cancer cells have shown that the expression of folate receptor-{alpha}, through which folate acts within the cells, is repressed in the presence of estradiol but derepressed by tamoxifen in an ER-{alpha}–dependent manner, suggesting a direct regulation of ER on folate receptors (63, 64). Two other in vivo studies have, however, found that the expression of folate receptor-{alpha} was either elevated (65) or unaffected in ER-positive breast tumors (66). These experimental data suggest that the interaction between folate and estrogen is complex. At least 2 other isoforms of folate receptor were identified, each of which exhibits a distinct tumor or tissue specificity (67, 68). It is possible that folate exerts different physiologic function in breast cancer development through the interaction between the 3 isoforms of folate receptor and ER-{alpha}. In this study, we also observed a moderately positive association between plasma folate and PR-positive breast tumors. The interaction between folate or folate receptor and PR in relation to breast cancer risk remains unknown.

Consistent with the findings from the Nurses' Health Study (27), we also observed an inverse association between plasma PLP and postmenopausal breast cancer. However, the other study did not observe the association in either premenopausal or postmenopausal women (25). Recent in vitro studies have shown that administration of vitamin B-6 inhibits cell proliferation in the mammary cancer cells (69, 70), although it remains unclear whether vitamin B-6 interacts with sex steroid hormones such as estrogens in relation to growth inhibition of breast cancer cells (70, 71). In our data, the risk reduction in postmenopausal women was stronger among women who had never used hormone therapy, suggesting an effect of vitamin B-6 on breast cancer development in the absence of estrogens. However, our data are exploratory and need to be interpreted with caution.

The strengths of this study include a large sample size, long-term duration of follow-up, and the measurement of biomarkers of these 3 B vitamins. However, there are also several limitations present in our study. We used only a single measurement for circulating concentrations of B vitamins, which may not reflect the true status of B vitamins during the study period as a result of changes in diet or fortification. We also have no data on one-carbon metabolizing genes to test potential gene-nutrient interaction in relation to breast cancer development. Because many subgroup analyses were performed in the study, our findings may be subject to chance. We also cannot exclude the possibility of confounding by uncontrolled variables.

In conclusion, our data provide little evidence that higher circulating concentrations of folate, vitamin B-6, and vitamin B-12 are associated with overall risk reduction of breast cancer. The unexpectedly positive associations seen in plasma folate and the risk of developing premenopausal breast cancer and ER-positive or PR-positive breast tumors suggest a possible complex role of this nutrient in breast cancer development, and more studies are needed to elucidate the true relation of folate and other B vitamins with breast cancer.


   ACKNOWLEDGMENTS
 
We thank the entire staff of the Women's Health Study under the leadership of David Gordon, as well as Mary Breen, Susan Burt, Marilyn Chown, Georgina Friedenberg, Inge Judge, Jean Mac-Fadyean, Geneva McNair, David Potter, Claire Ridge, and Harriet Samuelson. We also thank the Endpoints Committee of the WHS (Dr Wendy Y Chen and Jim Taylor), Eduardo Pereira, and Natalya Gometskaya for technical assistance with the manuscript.

The author's responsibilities were as follows—JL and SMZ: contributed to study design and data analysis; JL: was responsible for writing and revising the manuscript; SMZ, NRC, JEM, IML, JS, and JEB: provided substantial editorial comments on manuscript drafts. None of the authors had any personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication April 11, 2007. Accepted for publication October 5, 2007.




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