Fecal metabolites

Excretion of radioactivity in mice and rats was monitored for 48 hours following exposure to " C-labeled chloroform (Corley et al. 1990). In general, 92-99% of the total radioactivity was recovered in mice, and 58-98% was recovered in rats percentage of recovery decreased with increasing exposure. With increasing concentration, mice exhaled 80-85% of the total radioactivity recovered as " C-labeled carbon dioxide, 0.4-8% as " C-labeled chloroform, and 8-11 and 0.6-1.4% as urinary and fecal metabolites, respectively. Rats exhaled 48-85% of the total radioactivity as " C-labeled carbon dioxide, 2-42% as " C-labeled chloroform, and 8-11 and 0.1-0.6% in the urine and feces, respectively. A 4-fold increase in exposure concentration was followed by a 50- and 20-fold increase in the amount of exhaled, unmetabolized chloroform in mice and rats, respectively. 

The major fecal metabolites in male rats administered a single oral dose of "C-heptachlor are heptachlor epoxide, 1-exo-hydroxychlordene, 1-exo-hydroxy-2,3-exo-epoxychlordene, and 1,2-dihydroxydihydrochlordene, as well as two unidentified products (Figure 2-2) (Tashiro and Matsumura 1978). By day 3, 50% of the dose was excreted in the feces. About 72% of the radioactivity was eliminated in the feces in the form of metabolites and 26.2% as parent compound by day 10. The same metabolites were identified in the comparative in vitro study using rat and human microsomal preparations (Tashiro and Matsumura 1978). Heptachlor epoxide is metabolized one step further to a dehydrogenated derivative of 1-exo-hydroxy-2,3-exo-epoxychlordene. Less than 0.1 % of radiolabel was seen of this compound in an in vitro study using human liver microsomes (Tashiro and Matsumura 1978). 

Pigs dosed with radiolabeled avilamycin produced three unidentified fecal metabolites derived from the oligosaccharide and/or the eurekanate moieties. However, the primary metabolite in feces and liver was flambic acid. Mean total residues in muscle were all below 0.2 ppm, whereas residues in other edible tissues were all below 1 ppm. Most of tissue residues were derived from the oligosaccharide and/or the eurekanate portion of avilamycin, whereas very little was parent avilamycin. In fat, the avilamycin related residues was found to be due to radioactivity that had entered normal metabolic pathways and had been incorporated into the fatty acids. 

The data from animal studies demonstrate that DEHP and it metabolites are excreted in both the urine and the feces. Rats exposed to 50-300 mg/kg DEHP excrete 32-70% of the dose in the urine as metabolites (Astill 1989 Ikeda et al. 1980 Short et al. 1987). An additional 20-25% of the absorbed dose was excreted with the bile in the fecal matter. The remainder of the fecal excretion was unabsorbed DEHP and MEHP. In monkeys, approximately 30% of a 100 mg/kg dose and 4% of a 2,000 mg/kg dose were excreted in the urine (Astill 1989 Rhodes et al. 1986 Short et al. 1987 Sjoberg et al. 1985b). The remainder was in the feces. A portion of the fecal metabolites was contributed by the bile. The biliary excretory products represent approximately 15% of the absorbed DEHP. In rats and mice administered radiolabeled DEHP, 85-90% of the label was excreted in the first 24 hours (Astill 1989 Ikeda et al. 

At least four active metabolites have been identified, namely desethylene-ciprofloxacin, sulfo-ciprofloxacin, oxo-ciprofloxacin, and V-acetyl-ciprofloxacin. Oxo-ciprofloxacin appears to be the major urinary metabolite, and sulfo-ciprofloxacin the primary fecal metabolite [4, 11]. 

Figure 6.1 Common structures associated with ellagitannins include (A) free ellagic acid (hydrolytic product), (B) castalagin (oak wood), (C) Urolithin A (fecal metabolite), and (D) dimeric Sanguiin H-6 (Rubus species).

Propachlor is absorbed through the gastrointestinal tract, through intact skin, and through the respiratory system after inhalation of dust or spray mist. It is metabolized via the mercapturic acid pathway. The major fecal metabolite is a cysteine conjugate. Rats administered propachlor orally excreted 98.6% of the dose in the urine and feces within 48 h. Approximately 50% is excreted as metabolites through urine or feces within 24 h. 

Analysis of In Vivo Fecal Metabolites by LC/MS. Besides the analysis of urinary metabolites, animal metabolism studies of course require characterization of fecal metabolic products. We have found that rapid analysis of fecal extracts can also be accomplished by LC/MS, although more extensive cleanup is required. A general procedure for the extraction and LC/MS analysis of rat fecal metabolites is given in Figure 14. This procedure can easily be completed in an afternoon to provide a preliminary indication of metabolite structures. Based on this information the appropriate derivatives can be prepared for additional characterization, if necessary. 

The selectivity of electron capture ionization for fluorinated compounds provides a convenient means to locate and identify fecal metabolites of dithiopyr. 

The PPINICI experiment again proved to be very useful in the analysis of fecal metabolites, by providing complementary information from both the positive and negative... 

Figure 15. Total ion current trace from negative ion analysis of dithiopyr fecal metabolites.

The lower panel displays the negative ion trace from the PPINICI analysis and contains essentially only the peaks associated with fecal metabolites, whereas the positive ion trace (upper panel) contains numerous peaks associated with normal fecal constituents. The two monoacids 2 and 2 were the only pyridine-derived metabolites which could be identified from the positive ion chromatogram. The positive ion spectra were useful nonetheless, because they provided confirmation of molecular weights, and in some cases provided molecular weight information for peptide conjugates which underwent substantial fragmentation after electron capture. 

Figure 16. PPINICI chromatograms from LC/MS analysis of dithiopyr rat fecal metabolites.

No studies were located regarding possible fecal metabolites of 2,4-DNP. 

Metabolites were isolated and identified in the urine, feces, liver and kidneys of animals dministered two consecutive daily intravenous doses of 2.2 mg u-flunixin active/kg body weight. Approximately 90X of the total administered dose was recovered in the urine and feces within 24 hours after the second dose. Radioactivity was excreted in approximately equal percentages in the urine and feces. Urine and feces were extracted with methanol and the extracts analyzed by HPLC and TLC. Urinary and fecal metabolites were identified by co-chromatography with known synthetic metabolite standards. The major radioactive component in both cow and steer urine had similar chromatographic retention characteristics to unchanged flunixin. Two minor metabolites, with retention times corresponding to the 2 -methylhydroxy-flunixin and 5-hydroxyflunixin metabolite standards, were also detected. The only radioactive component present in both cow and steer feces had similar elution characteristics to 5-hydroxyflunixin. 

A study in the rat reinvestigated the nature of the major fecal metabolite of dieldrin. Its structure is thought to be that of dieldrin with an hydroxyl group at position-9. In mice, the toxicity of the metabolite was at least five times less than that of dieldrin. 

Figure 16.6 compares six technologies for high-sensitivity metabolic chromatography using " C molecular labels. Data plots for HPLC—ARC of human fecal metabolites (Colizza et al., 2007), HPLC—LSC and HPLC—Top-Count of human liver microsome metabolites (Zhu et al., 2005), UPLC RAM... 

For several other retinoids, actual spectral data have not been published, but characteristic fragmentation patterns have been reported. These compounds include three urinary metabolites of retinoic acid in which the chain is shortened and the cyclohexenyl ring is oxidized (Hanni et al., 1976 see Fig. 7b, Chapter 11), three fecal metabolites of retinoic acid (4-oxoretinoic acid and two hydrox-ylated metabolites, see Fig. 6, Chapter 11 Hanni and Bigler, 1977), several metabolites (Hanni et al., 1972 see Fig. 14, Chapter 11) and isomers (Englert et al., 1978) of etretinate, 3-hydroxyanhydroretinol, a metabolite of 3-hy-droxyretinol (Barua et al., 1979), and a 4-oxo-Ci9 aldehyde metabolite of retinoic acid (Rockley et al., 1980 see Fig, 8, Chapter 11). With the advent of HPLC and an increase in the sensitivity of mass spectrometry, this technique should continue to be of great usefulness for the identification of retinoid metabolites. 

Measurement of urinary metabolites for assessment of vitamin A status was suggested by Varma and Beaton (1972) on the basis of an observed correlation in rats between liver stores and the quantitative excretion of total urinary and fecal metabolites of vitamin A. These studies were done on rats with high liver stores. Rietz et al. (1974) were unable to demonstrate in rats a sufficiently high correlation between liver stores over a range of values and total urinary metabolites for them to recommend this approach for measuring vitamin A status. 

The majority of the fecal metabolites probably originate from the bile. As is also the case with urinary and biliary metabolites, the amount of the administered dose found in the feces varies depending on the position of the label, the mode of administration, and on the amount administered. Values from 0.3% of a 10- x.g dose in 24 h (Sundaresan and Sundaresan, 1975) to 26% of a 2-p,g dose in 5 days (Roberts and DeLuca, 1%7) have been reported. There appears to be an initial delay in the fecal excretion of metabolites after injection of retinol (Olson, 1968 Varma and Beaton, 1972). Again, however, little has been done to obtain structural data on these compounds. 

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