Identification and quantitative distribution of eight analogues of naturally occurring fecapentaenes in human feces by high-performance liquid chromatography

Abstract

Fecapentaenes, highly potent fecal mutagens originating from intestinal bacterial production, have been suggested to play an essential role in the initiation of colorectal cancer. Reviewing the data on fecapentaene occurrence in man, the applied methodologies for fecapentaene extraction and analysis appear to be very inconsistent. Therefore, we compared several methods and developed an optimal extraction and purification procedure for fecapentaene quantification in human feces. This method is based upon a dichloromethane extraction of freeze-dried material with application of a Potter homogenization instrument and subsequent HPLC analysis in combination with photodiode array detection. This system enables us to detect and quantify at least eight forms of fecapentaene-like substances generally occurring in human stool. We suggest that these peaks represent fecapentaene-12 (FP-12) and fecapentaene-14, both with a geometric isomer, as well as fecapentaene analogues that have never been reported before. Applying this methodology on feces of a group of young healthy persons, we were able to detect fecapentaene levels ranging from less than 5 micrograms to 6 mg/kg feces, and in 40% of the samples greater than 1.0 mg/kg feces. The newly identified fecapentaenes represent 21.7% of total fecapentaene concentration. It appears that some fecapentaenes are excreted in higher amounts by females as compared to males. Furthermore, we found that fecal mutagenicity to Salmonella tester strain TA100 appeared lower than hypothesized on the basis of overall fecapentaene contents, and that fecal extracts diminish the mutagenic effect of synthetic FP-12 dramatically. Apparently, optimal conditions for fecapentaene extraction result also in an increased level of co-extracted anti-mutagenic substances. Determination of fecal mutagenicity as an index for fecapentaene excretion or colorectal cancer risk is therefore not suitable. In order to assess the relevance of fecapentaenes in the etiology of colorectal cancer, we suggest that a distinction should be made between relative occurrence and degree of genotoxic effect in situ of the various fecapentaene analogues.