Folate is also potentially involved in early molecular changes that take place in colon carcinogenesis (2). Understanding this relationship is important because colorectal cancer is one of the leading causes of death by cancer in the United States, with 129,400 new cases and 56,600 deaths expected in 1999 (3). A genetic predisposition has been implicated, but less than 10% of colorectal cancers are attributable to inherited mutations. Varying incidence rates throughout the world suggest there may also be a causal relationship with dietary intake (4).

Folate and Colorectal Adenomas

Most cases of colorectal cancer develop from pre-cancerous colorectal adenomas (5). Several studies have found a link between lower levels of folate intake and a higher incidence of these adenomas (6-8), which suggests that folate may play a protective role early in the carcinogenic process. Theoretically, if folate has a protective association against precancerous colorectal adenomas, then folate would be expected to also have a protective association against colorectal cancer.

In a case-control study of diet and colorectal adenoma risk, Benito and colleagues (6) found that subjects with folate intakes of greater than 222 mcg /day were approximately one fourth as likely to have adenomas when compared to subjects with intakes below 141 mcg /day. Unfortunately, the only dietary factor the data were adjusted for was total calorie intake. Other dietary factors including fat, fiber and alcohol intake may also play a role in colon carcinogenesis.

Tseng and colleagues (7) pursued this possible cancer/folate connection by conducting a similar case-control study to evaluate the relationship between micronutrients and colorectal adenoma risk. Although their results did not achieve statistical significance, they did observe a gender specific trend. After adjusting for other dietary factors, their results showed a 60% decrease in adenoma risk for women in the highest quartile of folate intake (319.9-2877.7 mcg/day) compared with the lowest quartile (44.4-172.3 mcg/day). The cause of the observed sex specificity is unclear, but it has been suggested there may be other physiological factors involved that change risk patterns between men and women.

A prospective study of similar subjects from two large cohorts, the Nurses' Health Study and the Health Professionals Follow-up Study, was conducted by Giovannucci and colleagues (8). They were interested in the association with folate because they saw a mechanism by which low folate levels might contribute to the development of adenomas by decreasing the availability of methyl groups. DNA with insufficient methyl groups has been observed in both colon cancer and colorectal adenomas (9,10). The decrease in risk for all adenomas in the highest quartile of intake (median value 711 mcg/day - women, 847 mcg/day - men) compared with the lowest quartile (median value 166 mcg/day - women, 241 mcg/day - men) was 29%. The data were adjusted for saturated fat, dietary fiber and alcohol intake. An important finding of this study was that an association existed even though relatively few subjects had folate intakes of less than 200 mcg/day. This suggests that subjects do not need to be folate deficient in order to obtain the potential benefits from increased folate intake.

Folate and Colorectal Cancer

Studies that have looked at associations between folate and the risk of colorectal cancer itself (11-19) rather than at the development of adenomas have had varying results. However, at least some of the discrepancy in their findings may be accounted for by the different methods used to conduct the studies. Some of these studies measured plasma folate, some looked at food folate intake and others included supplemental folic acid.

Plasma Folate. The studies which looked at plasma folate levels observed no significant association with colorectal cancer (11,12). Subjects with plasma folate indicating clinical deficiency (<3 ng/ml) were compared with subjects having adequate levels (³ 3 ng/ml). Both were case-control studies involving only male subjects. Plasma folate levels are limited to measuring folate status at one point in time. Low levels of folate may indicate folate depletion, but may also be a marker of early negative folate balance. In measuring colorectal cancer risk, dietary intake which measures folate status over longer periods of time may be more accurate.

Dietary Folate. Several studies utilized folate intake from only dietary sources (13-16). One study limited the measurement of dietary folate to plant foods and found no association with colon cancer (13). Due to the exclusion of folate from other than plant sources, approximately one fourth of the total dietary folate intake was missing. The three studies that found an association between increased dietary folate and decreased risk of colon cancer were all case-control studies from various geographical locations in the United States and Europe (14-16). The results in these studies did not support a strong association. After further adjustment of the data by nutrients which had a high correlation with folate, the association disappeared or was greatly reduced.

In the Western New York Diet Study, Freudenheim and colleagues (14) observed a 70% decrease in male rectal cancer for calorie-adjusted folate intakes >385 mcg/day compared with <250 mcg/day. Vitamin C, carotenoids and vegetable fiber had a high correlation with folate. After further adjustment of the data for these nutrients, the protective trend only remained with vitamin C in the model. This indicates that the protective association observed with folate may instead be due to the protective role of carotenoids and vegetable fiber.

Similar results were observed by Ferraroni and colleagues (15) in a study conducted in Milan, Italy. Subjects with dietary folate exceeding 261.49 mcg/day were one half as likely to have colon, rectal or colorectal cancer than subjects with intakes below 162.63 mcg/day. This trend disappeared after adjustment for vitamin E, beta-carotene and vitamin C.

Meyer and White (16) observed a decreased risk for colon cancer from increased dietary folate intake, but this association was limited to women. Women with intakes above 294 mcg/day were approximately one half as likely to have colon cancer as those with intakes below 156 mcg/day. When the data were further adjusted for fiber intake, the protective association was reduced by half.

Supplemental Folate. The decrease in colon cancer risk with increasing folate intake was more apparent when supplemental sources of folate were included. This may be due to the high level and bioavailability of folate in supplements. Most supplements contain 400 mcg of folate in the form of folic acid or pteroylmonoglutamic acid (17). The folate in food is primarily present as pteroylpolyglutamates, which must be hydrolyzed to pteroylmonoglutamic acid to be absorbed. Many of the foods which are good sources of folate contain conjugase inhibitors that prevent the hydrolysis of polyglutamate forms to the monoglutamate form (20).

In their prospective study of a large cohort, the Nurses' Health Study, Giovannucci and colleagues (17) found total folate intake appeared to be protective against colon cancer. They observed a 30% decrease in the risk of colon cancer for women with total folate intake exceeding 400 mcg/day when compared to women with an intake of (200 mcg/day. The total folate intake in the two highest quartiles included supplemental sources. In this cohort more than four out of five women in the highest quartile of intake used multivitamins. In the second highest quartile of intake, 9.1% of the women were multivitamin users. The amount of daily folate intake from multivitamins was ³ 400 mcg for 69% of users. The large use and amount of folate in the multivitamins suggest that supplemental folate intake may have a causal relationship with decreased risk for colon cancer.

This finding is supported by White and colleagues (18) in their population based case-control study of vitamin supplement use and colon cancer. They found a 50% decrease in colon cancer risk for subjects with a supplemental intake of folate ³ 400 mcg/day compared with subjects having no intake. This association was also noted for subjects using multivitamins once a day compared with those never using multivitamins.

In a prospective (19) and a case-control study (12) using only male subjects, there were no significant associations between total folate intake and colon cancer risk. Giovannucci and colleagues (19) also found no significant association with multivitamin use and colon cancer risk. These findings may be due to the overall lower percentage of men using multivitamins (21).

Folate is rarely taken as an individual supplement. It may be difficult to determine whether folate or the multivitamin is responsible for the decreased colon cancer risk. It may be that multivitamin users have more healthful lifestyles in general (22). So while these findings show that supplemental folate is associated with a decreased risk for colon cancer, they do not conclusively demonstrate that folate is the factor responsible for reducing that risk.

Folate and Colon Cancer in Rats

Animal studies provide a controlled environment in which researchers can observe specific intakes of folate and their effect on colorectal cancer. Rats treated with dimethylhydrazine provide a model that in many ways imitates human colorectal carcinogenesis (23). Researchers have used this model to study the effect of varying doses of folate on colorectal cancer development (23, 24).

Cravo and colleagues (23) found that rats fed a diet including 8 mg/kg folic acid compared to those fed a diet with no folic acid supplementation had a lower incidence of dysplasia and carcinoma. Another important finding in their study was the lack of neoplastic changes in rats fed the folate deficient diet with no dimethylhydrazine treatment. These results provide evidence that the lack of folate is not carcinogenic on its own. In this study, the diet containing no folic acid supplementation created a moderate deficiency of folate that was not severe enough to result in growth retardation or anemia. This observation may have clinical significance for humans, as moderate folate deficiency is more common than severe deficiency in people.

The 8 mg/kg diet used in the Cravo study was four times the accepted basal folate requirement for rats (25). Kim and colleagues (24) obtained results that suggest supplementation above 8 mg/kg does not add any additional benefit. With folate supplementation up to 8 mg/kg, there was a decrease both in the percentage of rats with macroscopic tumors and in the average number of tumors per rat. Supplementation at 40 mg/kg did not result in any further decrease. A possible explanation for this occurrence is that the 8 mg/kg diet causes the mucosal cells to become saturated with folate. It is apparent that folate supplementation above basal dietary requirements may have a protective role against colorectal cancer in dimethylhydrazine treated rats. The protective benefit may be limited to doses up to four times the basal requirement. Results from human clinical studies will be needed for clarification of any human implications that may exist from these studies.

Conclusion and Clinical Applications

Folate deficiency is one of the most common vitamin deficiencies in the United States (23). The results from epidemiological studies suggest that diets low in folate are associated with an increased risk of colorectal adenomas and cancer (6,8,14-18). This may be important for the many Americans who have a low folate intake.

The potential benefit of adequate folate intake was more observable for women than men (5,6,11,12,16,17,19). This may be due to gender specific physiological factors having an influence on colorectal cancer risk, but women are also more likely to take multivitamins, which usually contain 400 mcg of folate in a highly bioavailable form (17). The inclusion of supplemental folate strengthened the protective association against colorectal cancer (14-18). The subjects taking multivitamins had folate intake levels that exceeded the accepted daily requirements, so it may be that intakes greater than the minimum DRI will increase the potential protective benefit of folate.

Animal studies demonstrated a decreased risk of colorectal cancer with folate intake levels up to four times the basal requirement for rats. No additional benefit was observed with diets containing higher levels. Results from human clinical studies will be needed to determine whether or not this also applies to humans. Until these results are available, it would be prudent for all people to maintain folate intake levels of at least 400 mcg/day. This level of intake may be difficult to obtain through diet alone, so inclusion of a multivitamin containing folate may be a good option.

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Jeanette Kennedy is an MS candidate in Food & Nutrition and
Suzanne Neubauer is a Professor of Food & Nutrition at Framingham State College in Framingham, MA.