We observed 70% lower risk of colon cancer among men with high (>41.6 pmol/ml) compared with low (<=15.9 pmol/ml) serum vitamin B6 concentrations. Hypothesized protective associations between higher serum folate concentrations and risk of colorectal cancer were not observed, and serum vitamin B12, riboflavin, and homocysteine were also unrelated to risk. Further, combinations of the five one-carbon serum biomarkers did not provide convincing evidence that these serologic phenotypes collectively influenced colorectal cancer risk in this population.
The colon cancer - serum vitamin B6 association appeared limited to the highest quintile and was stronger for the distal colon, whereas rectal cancer was unrelated to B6. Our finding is another among studies suggesting that the risk factors for colon and rectal cancer may differ (3
). One other study found significantly lower risk of colon (but not rectal) cancer with higher plasma vitamin B6 concentrations, with significant dose-response trends (21
). Mean vitamin B6 concentrations in controls were higher (53.2 pmol/ml) than in our study (39.5 pmol/ml), with corresponding high quantile medians of 131.2 pmol/ml and 65.4 pmol/ml. The threshold association we observed for the highest quintile could be a consequence of the lower than adequate serum vitamin B6 levels in our population. With the exception of one study (13
), the prospective epidemiologic evidence for vitamin B6 dietary intake and supplementation appears to be fairly consistent in suggesting an inverse association with colorectal cancer (21
), a conclusion drawn by a recent meta-analysis (35
). Interestingly, we observed a fairly strong correlation between serum and dietary vitamin B6 (r=0.46) in the present study, relative to the weaker correlations with the other nutrients examined. The food sources of vitamin B6 in the ATBC cohort included potatoes, rye and wheat products, fish, pork, sausages, milk, fruits, and vegetables, accounting for 76% of the total vitamin B6 intake. Less than 13% of all subjects in this nested study reported intake of supplemental vitamin B6.
Vitamin B6 is a cofactor for an enzyme in the one-carbon remethylation pathway and for two enzymes in the transsulfuration pathway, which metabolizes homocysteine (6
). Because there are no risk associations or interactions with the other one-carbon biomarkers, the protective association with serum vitamin B6 may be working through the transsulfuration pathway in which homocysteine is degraded to cysteine, a limiting factor in the synthesis of glutathione. Glutathione has numerous functions such as antioxidant defense, detoxification, and reduction of oxidative stress (36
). Alternatively, high vitamin B6 concentrations may protect against carcinogenesis directly through hypothesized roles of inhibiting cell proliferation, nitric oxide production, oxidative stress, and angiogenesis, and improving immune function (37
). Finally, we can not rule out confounding by another highly correlated factor to explain the protective effect of serum vitamin B6.
We found no evidence of a protective association between serum folate and colon or rectal cancer risk – our strongest a priori
hypothesis. In fact, increased risk of colon cancer for men in the middle quintile was suggested, similar to a pattern observed in a prospective study in Sweden (15
). Our study enrolled only men aged 50−69 years at baseline who smoked at least five cigarettes daily at study entry, and research suggests that smokers have reduced biochemical status (tissue and blood) of folate, vitamin B6, and vitamin B12 compared with non-smokers (40
), possibly due to lower intakes (especially of folate) or the effects of oxidizing compounds in tobacco smoke which inactivate folate and vitamin B12 cofactors (41
). Smokers may therefore require higher folate status for colorectal cancer protection; however, the folate concentrations and overall findings we observed were very similar to those in the Swedish cohort study where only 20% of the subjects were active smokers and where younger subjects were included at baseline (15
), and we found no evidence that the serum biomarker –colorectal cancer risk associations differed by age or by smoking dose or duration.
Although folate intake and colorectal cancer risk has been extensively examined, the number of prospective studies of folate status are few, and their results have been inconsistent. We previously found no significant association between baseline serum folate and colon and rectal cancer risk in the ATBC Study cohort, based on 144 cases and approximately 8 years of follow-up (14
). (The present investigation includes all cases from this previous analysis, but uses only new and expanded biomarker data). Two other studies were null (18
), two reported inverse associations (16
), and one (discussed above) observed elevated risk of colorectal cancer in the third and fourth quintiles of plasma folate (15
). The two studies finding protective relationships reported higher mean/median serum folate concentrations (approximately 15 to 22 nmol/L) (16
), compared with the other studies (14
) and the present analysis (approximately 8 to 10 nmol/L). It is possible that the folate levels in the null studies were not high enough to provide protection against colorectal carcinogenesis. However, a recently published study with higher median plasma folate concentrations (15.4 nmol/L) was also null (19
), and a recent trial reported that supplementation with 1 mg/day folic acid did not reduce but may have increased the recurrence of multiple and advanced colorectal adenomas (43
). A dual role for folate in carcinogenesis has been suggested to explain these divergent observations, whereby folate may prevent colorectal cancer by reducing DNA damage, but may also promote the growth of preexisting adenomas (44
). It has been conjectured by some that folic acid fortification in the U.S. and Canada has led to increased rates of colorectal cancer in these countries (46
). The lack of a protective effect of folate in the current study could be explained if the subjects had already accumulated colorectal mutations by the time they were recruited into the study.
Alcohol intake can have a negative impact on folate status and metabolism (47
), and higher risk of colorectal cancer has been observed among subjects with high alcohol intake and low serum/dietary folate, although not all risk estimates were elevated or statistically significant (12
). We observed no interaction between alcohol, folate status, and colon or rectal cancer, and additional consideration of methionine or protein intake showed no striking interaction. In our earlier analysis, there was no significant elevation in risk of colon cancer for the high alcohol-low serum folate-low protein intake category, while the same analysis using folate intake showed a significantly increased risk in the high risk group (OR=4.8) (14
), Only two other studies examined the interaction with folate status rather than intake, and neither reported significant interactions (17
). In regard to serum vitamin B6 and alcohol, one study observed the most benefit of vitamin B6 intake when alcohol intake was high (26
), but two others, in addition to ours, observed no interactions (21
Serum homocysteine, vitamin B12, and riboflavin were unrelated to risk of colon or rectal cancer. There are very few other prospective studies of these circulating factors and colorectal cancer. One study reported a non-significant elevation in risk with higher serum homocysteine (17
), a second found reduced risk with low homocysteine among subjects with the MTHFR valine/valine genotype (20
), while a third found no association (15
). Circulating vitamin B12, in conjunction with MTHFR and MTR genotypes, was not associated with colorectal cancer risk in one study (20
) but was associated with reduced risk of rectal, but not colon, cancer in another (22
). In terms of dietary intake, there are also few prospective studies. Vitamin B12 intake was not materially associated with risk of colorectal cancer in three studies (13
). One study found a reduced risk of colorectal cancer with higher riboflavin intake, primarily among subjects with the MTHFR TT genotype (25
A major strength of the present study is the simultaneous consideration of several key biomarkers of one-carbon metabolism in addition to serum folate, which provided a thorough examination of the hypothesis. Based on these multiple measurements, a priori
high and low risk combinations of the serum biomarker levels were investigated but provided no clear evidence to support a collective influence on colorectal cancer risk. We utilized a prospective design which minimizes an effect of cancer on the serum biomarker concentrations, and our study encompassed a long follow-up period (17 years). Finally, while folic acid fortification of enriched grain products was mandated in the United States in 1998 (51
), there is no such national fortification program in Finland, so our results are free from this perturbation of folate intake. Limitations resulting from the parent study include our investigation of only smokers, although results are similar to those of other studies which were not limited to smokers (15
), and only men. We measured serum vitamin B12, rather than methylmalonic acid, a functional marker of vitamin B12 status. In addition, we had no baseline data on use of aspirin or non-steroidal anti-inflammatory medications. Our use of quintile cuts for the main exposures of interest has the advantage of enabling comparisons between quantiles that are more widely spread apart, thus allowing us to explore potential threshold effects, however risk estimates using quintiles are a bit less stable than using quartiles or tertiles. Finally, our sample size limited our ability to detect interactions among the one-carbon biomarkers and with other factors. Analyses regarding genetic variants in the one-carbon pathway are planned in order to evaluate direct effects of and effect modification by genotypes.
In summary, we observed a significantly reduced risk of colon cancer among men with higher serum concentrations of vitamin B6, and similar evidence for protective associations with vitamin B6 has been accumulating. In addition to other serological studies, investigation of vitamin B6-related genetic polymorphisms may provide additional useful information in this regard. By contrast, the stronger a priori
hypothesis favoring an inverse folate-colorectal cancer relationship was not observed for the serum biomarker, even in the context of and simultaneous adjustment for other key one-carbon factors. In fact, we observed a suggested increased risk of colon cancer for men in the middle quintile. Thus, although an extensive literature exists regarding a possible benefit for higher folate intake, our study adds to the few studies of serum or plasma folate that have been inconsistent. There were also no associations for other serum one-carbon factors - vitamin B12, riboflavin, and homocysteine - either singly or jointly with folate or vitamin B6. Coupled with the recent trial of folic acid and colorectal adenoma showing no effect or potential harm from supplementation (43
), additional careful study of folate and one-carbon biomarker relationships with colorectal cancer, including consideration of genetic variants in the one-carbon pathway and possible isolated associations among genetic subgroups, is warranted.