Colorectal cancer, one of the most common types of cancer in many Western countries, has a high mortality rate (Weisburger 1991). Distinct differences in the incidence of colon cancer among inhabitants of different countries and the disappearance of these differences among immigrants and natives point to a close association with environmental factors (Boyle et al. 1985). Epidemiological studies indicate that differences in nutrition, in particular, contribute to this variability (Miller et al. 1994, Statland 1992). In these studies, a high consumption of fat and meat and a low intake of vegetables were associated with a higher risk of developing colorectal cancer. However, it is not yet known which nutritional factors are responsible for these observations. On the one hand, the composition of the diet may influence the supply of toxic and protective nutrients to the body. On the other hand, the composition of the feces, which is influenced by dietary factors, has potentially injurious local effects on the colon mucosa. In addition to some components of feces such as bile acids, reactive oxygen species are also thought to have a promoting effect on colorectal cancer (Babbs 1990). Until now, little information has been available about the level of these species in feces (Graf and Eaton 1993). Reactive oxygen species can evolve when bacteria produce superoxide, which is converted to the highly reactive hydroxyl radical under the catalytic effect of iron. Hydroxyl radicals are capable of converting nonmutagenic to mutagenic substances.

The objective of the present study was to investigate the effect of two diets markedly different in fat and dietary fiber content on the in vitro production of reactive oxygen species in human feces. To obtain information on the influence of these diets on the antioxidant supply to the body, the plasma concentrations of vitamin C, carotenoids and vitamin E were measured. In addition, the plasma malondialdehyde concentration was determined as a potential indicator of oxidative stress in the body.

SUBJECTS AND METHODS

Table 1. Subjects participating in the study [View Table]

Table 2. Composition of the two diets [View Table]

Table 3. Examples of the food components of four daily meals for each diet period [View Table]

At the end of each study period, feces were collected in fractions in plastic bags on the morning of d 13, after the subjects had drunk 2-3 L physiological saline solution. Four samples of feces were taken from each subject. The third sample contained only small amounts of feces and the fourth sample from each subject consisted of cloudy purging water only. To determine the in vitro hydroxyl radical production, the first feces portion was mixed and an aliquot of 1 g used immediately for the hydroxyl radical production. A second aliquot of 1 g was lyophilized to determine the dry weight of this sample. The remainder of the combined three samples of feces was frozen at 80°C, lyophilized and homogenized in a mortar to measure the dry matter and iron concentrations of all samples.

At the beginning of the study and after each dietary period, 10 mL of blood were collected after overnight fasting. Plasma was prepared and stored at 80°C until the cholesterol, antioxidative vitamins and malondialdehyde concentrations were determined.

The study was approved by the Ethical Committee of the Robert-Bosch Hospital (Stuttgart, Germany) and written permission obtained from each subject.

All samples were measured in triplicate. For validation, a feces sample was divided into 10 equal portions and each portion was frozen at 80°C. Each of the portions was then analyzed in the same manner as the fecal samples of each subject.

To determine the concentration of iron in the feces, the method of Fries (1971) with ferrozine as a color reagent was used after wet digestion of the feces with H₂SO₄/HNO₃.

RESULTS

Table 4. Dry weight of feces, production of hydroxyl radicals and fecal iron concentration in human subjects after consuming the fat rich, low fiber diet 1 and the low fat, fiber rich diet 22 [View Table]

The plasma concentrations of -carotene and vitamin C were significantly lower after consumption of diet 1 than after diet 2 [57 and 27%, respectively (Table 5)]. The concentration of malondialdehyde was significantly higher (+ 47%) after the consumption of diet 1 than after diet 2 (Table 5).

Table 5. Plasma concentration of cholesterol, vitamins with antioxidant capacity and malondialdehyde in human subjects before and after the periods during which the rich fat, low fiber diet 1 and the low fat, fiber rich diet 2 were consumed1 [View Table]

DISCUSSION

Ten years ago the first paper was published offering the hypothesis that free radicals could arise in feces and may be involved in the pathogenesis of colorectal cancer (Graf and Eaton 1985). Five years later, Babbs (1990) extended this hypothesis to a comprehensive theory, which has repeatedly been cited but never verified (Graf and Eaton 1993, Weinberg 1994). Babbs postulated that in the oxygen-containing area near the mucosa of the colon, bacteria release superoxides, which are converted to the highly reactive hydroxyl radical through the catalytic activity of Fe²⁺. Because these hydroxyl radicals react at once with the contents of the feces, they have no direct damaging effect. They can, however, convert nonmutagenic to mutagenic substances in the feces, which then possibly damage the stem cells in the colon. Bile acids and pigments are believed to promote this process, because they can solubilize peroxidizable fatty acids and iron, whereas phytate may inhibit this process through the binding of iron. A diet rich in meat and fat and poor in dietary fiber is therefore expected to promote the formation of these reactive oxygen species. Epidemiological findings support the hypothesis of Babbs (1990) and Graf and Eaton (1993). The following examples concerning colon cancer formation were reported: iron-rich meat possibly has a promoting effect (Giovannucci and Willett 1994) and phytate rich dietary fiber from cereals may have a protective effect (Trock et al. 1990). However, another explanation for the latter epidemiological finding could be a direct effect of phytate on the proliferation and differentiation of colon cells (Shamsuddin 1995).

To our knowledge, the present study is the first in which the formation of hydroxyl radicals in human feces was tested under different dietary conditions. In accordance with the hypothesis of Graf and Babbs, we found a distinct increase in the in vitro free radical production in feces after consumption of a diet with a high fat and meat and a low fiber content (diet 1). This coincided with a significantly higher concentration of iron in the feces, which was probably the result of the higher meat consumption. However, it is unclear which factors were responsible for the higher hydroxyl radical formation in feces of subjects after they consumed diet 1. Explanations may include the higher iron concentration in the feces or the diminished binding of iron to phytates, which increases the free iron in feces. A highly effective binding capacity of phytate for iron was shown by Graf et al. (1987).

Because it was not clear whether and to what extent hydroxyl radical formation may be influenced by dietary factors, the study was designed in such a manner that a maximum effect of differences in the feces could be expected. Additional studies are required to further clarify the role of specific factors suspected of having a promoting or inhibiting effect on the formation of hydroxyl radicals in feces.

In addition to the hydroxyl radical formation in feces, we also measured some plasma components that may influence the antioxidant system of the body and therefore possibly play a role in colon cancer formation. After subjects consumed a diet rich in vegetables and wholemeal products and poor in fat for 12 d, greater concentrations of the antioxidants vitamin C and -carotene were found in the plasma. These findings are in accordance with other studies (Jackson 1994). In addition, a lower malondialdehyde concentration, a potential marker for lipidperoxidation, was observed in subjects after they consumed diet 2 than after they consumed the fat rich diet 1. However, in a recent study undertaken to examine the effects of certain nutrients on the formation of polyps (Greenberg et al. 1994), there was no preventive effect of vitamin C, -carotene or vitamin E on the development of colorectal polyps (which are regarded as precursors of colorectal cancer). This suggests that, in general, other factors associated with a diet rich in vitamin C and -carotene may play a more important role than the vitamins themselves (Byers and Guerrero 1995). The inhibition of hydroxyl radical production in feces may be such a factor.