Studies on the chromatographic fractionation of metabolites of benzo[a]pyrene in faeces and urine from germfree and conventional rats

Abstract

Rats, germfree and conventional, were dosed with 14C-labelled benzo[a]pyrene. Faeces and urine were collected. Metabolites in faeces were effectively extracted with a new method using a combination of solvents and solid sorbents. Metabolites in urine were extracted with octadecylsilane-bonded silica. The metabolites were fractionated into groups by chromatography on a cation exchanger (SP-LH-20 or SP-Sephadex C-25) and an anion exchanger (TEAP-LH-20). Some of the groups were further purified by column chromatography and analysed by HPLC and TLC. The analyses show a complex pattern of metabolism. A large part of the metabolites (9-24% depending on animal type and route of excretion) had amphoteric properties, e.g. like glutathione and cysteine conjugates. The abundance of conjugates sensitive to beta-glucuronidase and sulphatase was low. The relative amount of acidic conjugates in faeces was much higher in the germfree than in the conventional rats indicating the influence of the intestinal flora on the metabolism. The results support the view that the mercapturic acid pathway is a quantitatively important metabolic route for benzo[a]pyrene in rats. The methods of extraction and group fractionation were designed to be generally applicable to the analysis of lipophilic xenobiotics and their metabolites.

Fecal Metabolomics Reveals Distinct Profiles of Kidney Transplant Recipients and Healthy Controls

“…Fecal water was extracted by performing a successive two-solvent (ethanol and Diethyl ether)-based metabolite extraction protocol. It has been reported in a recent study that extraction efficiency was high for ethanol or methanol and isopropanol protocols [ 15 , 16 ]. As methanol is more carcinogenic than ethanol, we chose to use ethanol as an extraction solvent.…”

Characterization of Conjugated Metabolites of Benzo[a]pyrene in Germ-Free Rat Urine by Liquid Chromatography/Electrospray Tandem Mass Spectrometry

“…Sep-Pak C18 cartridges (Waters-Millipore, Milford, MA) were washed with methanol and water prior to use. SP-Sephadex C25 (Pharmacia, Uppsala, Sweden) and Lipidex-DEAP (Packard Instrument Co., Downers Grove, IL) were washed with aqueous ethanol and ethanol and converted to the H + and acetate forms, respectively, before use (31).…”

“…We have previously used lipophilic ion-exchange chromatography as the first step to separate metabolites of [ 14 C]BP given to germ-free and conventional animals and shown that the metabolite patterns of labeled metabolites in urine and feces are different as are the patterns in germ-free and conventional animals, indicating that the intestinal flora plays a role in the in vivo metabolism of this carcinogen (31). We have also carried out a thorough characterization of the neutral metabolites of BP in urine from germ-free rats by HPLC and GC/MS, which resulted in the identification of a number of novel unconjugated metabolites of BP (32).…”

High Throughput Analysis Reveals Changes in Gut Microbiota and Specific Fecal Metabolomic Signature in Hematopoietic Stem Cell Transplant Patients

“…Fecal water was extracted by performing a successive two solvent-based (ethanol and diethyl ether) metabolite extraction protocol. It was reported in a recent study that extraction efficiency was high for ethanol or methanol protocols [ 31 , 32 ]. The fecal samples were thawed, and 3 g of each stool specimen were mixed with pre-cooled ethanol (−20 °C) in the ratio of 3:20 (g: mL; feces: ethanol).…”