Combined chromatographic—isotopic dilution analysis of fecapentaenes in human feces

Abstract

Fecapentaene-12 (FP-12) and fecapentaene-14 (FP-14) are genotoxic unsaturated ether lipids produced by colonic bacteria in man. We have developed and applied to feces collections from normal volunteers direct isotopic dilution procedures using tritium-labeled (at C 5) FP-12 and FP-14 for measuring these compounds. FPs were recovered from feces by solvent extraction, silica cartridge clean-up, and analytical liquid chromatography. Low levels of FP-12 and FP-14 (<0.1 to 2.4 μg/g of freeze-dried feces) were observed. Identity of chromatographic peaks was established by co-elution and by ultraviolet absorption spectra obtained via photodiode array scanning. Two unknown peaks were tentatively identified from absorption spectra as closely related compounds with increased (hexaene?) or decreased (tetraene?) number of double bonds. Levels of FPs increased after incubation of feces at 37°C for 96 h under anaerobic conditions and pre-FP-12 and pre-FP-14 peaks were observed, which showed identical spectra with authentic FPs. These were interpreted to be isomeric forms of the all- trans-[ 3H]FPs used for the isotopic dilution analysis. Total FPs (including pre-FP) yielded a range of 0.3–80 μg FP-12 and 2.8–44 μg FP-14 per g of freeze-dried feces from the study group.