Journal: Drug Metabolism and Disposition

Article Title: Characterization of fecal deglucuronidation activity in healthy subjects and in patients treated with fecal microbiota transplantation

doi: 10.1016/j.dmd.2025.100205

Figure Lengend Snippet: Structures of the GUS reporter substrates, drug glucuronides, and steroid glucuronides investigated in this study (A). Deglucuronidation of 200 μ M 4-MUG, 500 μ M PNPG, and 100 μ M drug and steroid glucuronides in the presence of fecal lysates produced from fecal samples of 12 healthy donors (HD01–HD12) (B). After the initiation of the reaction, 4-MU and p -nitrophenol formation in the presence of 10 μ g fecal lysates was monitored continuously at 1-minute intervals. The deglucuronidation rates of 4-MUG and PNPG, defined as the amount of formed aglycone per time per amount of total protein in the reaction, were determined with linear regression. The drug and steroid glucuronides were incubated with 5 μ g of the fecal lysates for a predetermined time, after which the formed aglycones were measured with ultraperformance liquid chromatography. The incubation times were 1 minute and 3 minutes in all reactions for indomethacin-G and telmisartan-G, respectively. For estradiol-17G, the incubation time was 3 minutes, except for sample HD04 (6 minutes) and samples HD02 and HD07 (1 minute). Similarly, the incubation time was 3 minutes for testosterone-G, except for samples HD02 and HD07 (1 minute). The concentration of formed estradiol for sample HD04 was below the limit of quantification (BLQ). The deglucuronidation data were collected from 3 independent experiments (2 for estradiol-17G and HD04) shown as individual data points representing the deglucuronidation rate of the substrate with the bar representing the mean. Heatmap of the relative deglucuronidation activities of lysates from donors HD01–HD12 (C). Here, the deglucuronidation rate of each substrate was normalized to between 0 and 1 according to the lowest and highest deglucuronidation rates. Spearman correlation matrix of the deglucuronidation rates of the investigated GUS substrates (D). In panels C and D, yellow corresponds to high values and blue to low values. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001. Presented are the unadjusted P values; see for adjusted (Benjamini-Hochberg) values. Estradiol-17G, β -estradiol 17- β - d -glucuronide; GUS, β -glucuronidase; indomethacin-G, indomethacin acyl- β - d -glucuronide; 4-MU, 4-methylumbelliferone; 4-MUG, 4-methylumbelliferyl- β - d -glucuronide; PNP, p -nitrophenol; PNPG, p -nitrophenyl- β - d -glucuronide; telmisartan-G, telmisartan acyl- β - d -glucuronide; testosterone-G, testosterone β - d -glucuronide.

Article Snippet: The GUS reporter substrates 4-methylumbelliferyl- β - d -glucuronide dihydrate (4-MUG) and p -nitrophenyl- β - d -glucuronide (PNPG) were obtained from Santa Cruz Biotechnology and Sigma-Aldrich, respectively, whereas the aglycones 4-methylumbelliferone (4-MU) and p -nitrophenol (PNP) were both from Sigma-Aldrich. ( R,S )-Ezetimibe phenoxy- β - d -glucuronide (ezetimibe-G), indomethacin acyl- β - d -glucuronide (indomethacin-G), and telmisartan acyl- β - d -glucuronide (telmisartan-G) were purchased from Synthose.

Techniques: Produced, Incubation, Liquid Chromatography, Concentration Assay

Journal: Drug Metabolism and Disposition

Article Title: Characterization of fecal deglucuronidation activity in healthy subjects and in patients treated with fecal microbiota transplantation

doi: 10.1016/j.dmd.2025.100205

Figure Lengend Snippet: Deglucuronidation of 4-MUG by cultured bacterial strains (A). Lysates (1 μ g total protein per reaction) produced from 19 bacterial isolates were incubated with 200 μ M 4-MUG for 24 hours at +37 °C with 250 rpm shaking, after which the released 4-MU aglycone was measured using fluorescence detection. The data are presented as the fraction of 4-MUG converted to 4-MU in percentage. The bars represent the mean of 2 individual experiments (shown as points) conducted on 2 separately prepared lysate batches. Deglucuronidation of 250 μ M 4-MUG, 200 μ M indomethacin-G, and 200 μ M telmisartan-G (B) in the presence of Bacteroidales lysates (10 μ g total protein for 4-MUG and 5 μ g for indomethacin-G and telmisartan-G). For 4-MUG, aglycone formation was monitored continuously at 1-minute intervals. The deglucuronidation rate, defined as the amount of formed aglycone per time per amount of total protein in the reaction, was determined with linear regression. Indomethacin-G and telmisartan-G were incubated in the presence of the bacterial lysates for a predetermined time, after which the formed aglycone was detected with ultraperformance liquid chromatography. For Bacteroides uniformis and Bacteroides thetaiotaomicron , the incubation times were 15 minutes, whereas Phocaeicola vulgatus was incubated with indomethacin-G for 5 minutes and telmisartan-G for 7.5 minutes. Here, the bars represent the mean of 3 technical replicates (shown as points) originating from a single experiment. Indomethacin-G, indomethacin acyl- β - d -glucuronide; 4-MU, 4-methylumbelliferone; 4-MUG, 4-methylumbelliferyl- β - d -glucuronide; telmisartan-G, telmisartan acyl- β - d -glucuronide.

Article Snippet: The GUS reporter substrates 4-methylumbelliferyl- β - d -glucuronide dihydrate (4-MUG) and p -nitrophenyl- β - d -glucuronide (PNPG) were obtained from Santa Cruz Biotechnology and Sigma-Aldrich, respectively, whereas the aglycones 4-methylumbelliferone (4-MU) and p -nitrophenol (PNP) were both from Sigma-Aldrich. ( R,S )-Ezetimibe phenoxy- β - d -glucuronide (ezetimibe-G), indomethacin acyl- β - d -glucuronide (indomethacin-G), and telmisartan acyl- β - d -glucuronide (telmisartan-G) were purchased from Synthose.

Techniques: Cell Culture, Produced, Incubation, Fluorescence, Liquid Chromatography

Journal: Drug Metabolism and Disposition

Article Title: Characterization of fecal deglucuronidation activity in healthy subjects and in patients treated with fecal microbiota transplantation

doi: 10.1016/j.dmd.2025.100205

Figure Lengend Snippet: Phylum-level compositions (A) and the amplicon sequence variant (ASV)–level alpha diversities (B) of the fecal microbiotas of healthy donors HD01–HD12. Principal coordinate analysis of the microbiota compositions of HD01–HD12 at the ASV level and genus level (C) calculated with the Bray-Curtis dissimilarity index. Donors with high and low deglucuronidation activity are colored in yellow and purple, respectively, in the alpha and beta diversity plots. Spearman correlation of the relative abundance of the most abundant genera and deglucuronidation activity (D). In panel D, yellow corresponds to high ρ values and blue to low values. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001. Presented are the unadjusted P values; see for adjusted (Benjamini-Hochberg) values. Estradiol-17G, β -estradiol 17- β - d -glucuronide; indomethacin-G, indomethacin acyl- β - d -glucuronide; 4-MUG, 4-methylumbelliferyl- β - d -glucuronide; PNPG, p -nitrophenyl- β - d -glucuronide; telmisartan-G, telmisartan acyl- β - d -glucuronide; testosterone-G, testosterone β - d -glucuronide.

Article Snippet: The GUS reporter substrates 4-methylumbelliferyl- β - d -glucuronide dihydrate (4-MUG) and p -nitrophenyl- β - d -glucuronide (PNPG) were obtained from Santa Cruz Biotechnology and Sigma-Aldrich, respectively, whereas the aglycones 4-methylumbelliferone (4-MU) and p -nitrophenol (PNP) were both from Sigma-Aldrich. ( R,S )-Ezetimibe phenoxy- β - d -glucuronide (ezetimibe-G), indomethacin acyl- β - d -glucuronide (indomethacin-G), and telmisartan acyl- β - d -glucuronide (telmisartan-G) were purchased from Synthose.

Techniques: Amplification, Sequencing, Variant Assay, Activity Assay

Journal: Drug Metabolism and Disposition

Article Title: Characterization of fecal deglucuronidation activity in healthy subjects and in patients treated with fecal microbiota transplantation

doi: 10.1016/j.dmd.2025.100205

Figure Lengend Snippet: Deglucuronidation rate of 4-MUG grouped by sex of the healthy donors HD01–HD12 (A). Statistical significance of the difference in 4-MUG deglucuronidation rates between sexes was tested with the Mann-Whitney U test. Phylum-level microbiota compositions of HD01–HD12 averaged by sex (B). Alpha diversities of the microbiotas of donors HD01–HD12 grouped by sex (C). Statistical significance in the difference of alpha diversity indices between sexes was tested with the Kolmogorov-Smirnov test. Relative abundances of Alistipes , Bacteroides , Faecalibacterium , and Roseburia (D) in the fecal microbiotas of donors HD01–HD12 grouped by sex. The Mann-Whitney U test was used for the assessment of statistical significance in the difference in relative abundance between the sexes. Presented are the unadjusted P values; see for the adjusted (Benjamini-Hochberg) P values for the differential abundance analysis. The boxes extend from the 25th to the 75th percentile, with the line representing the median, the cross representing the mean, and the whiskers extending from the minimum to the maximum value. 4-MU, 4-methylumbelliferone; 4-MUG, 4-methylumbelliferyl- β - d -glucuronide.

Article Snippet: The GUS reporter substrates 4-methylumbelliferyl- β - d -glucuronide dihydrate (4-MUG) and p -nitrophenyl- β - d -glucuronide (PNPG) were obtained from Santa Cruz Biotechnology and Sigma-Aldrich, respectively, whereas the aglycones 4-methylumbelliferone (4-MU) and p -nitrophenol (PNP) were both from Sigma-Aldrich. ( R,S )-Ezetimibe phenoxy- β - d -glucuronide (ezetimibe-G), indomethacin acyl- β - d -glucuronide (indomethacin-G), and telmisartan acyl- β - d -glucuronide (telmisartan-G) were purchased from Synthose.

Techniques: MANN-WHITNEY

Journal: Drug Metabolism and Disposition

Article Title: Characterization of fecal deglucuronidation activity in healthy subjects and in patients treated with fecal microbiota transplantation

doi: 10.1016/j.dmd.2025.100205

Figure Lengend Snippet: Deglucuronidation of 200 μ M 4-MUG (A) and 100 μ M estradiol-17G (B) in the presence of fecal lysates produced from the sequential fecal samples of 3 FMT donors FD01–FD03 (A). After the initiation of the reaction, 4-MU formation was followed at 1-minute intervals. The 4-MUG deglucuronidation rate, defined as the amount of formed aglycone per time per amount of total protein in the reaction, was determined with linear regression. The formed estradiol was measured with ultraperformance liquid chromatography after a 5-minute incubation. The 4-MUG data were collected from 3 individual experiments shown as individual data points. The estradiol data are from a single experiment conducted in triplicate samples shown as individual data points. For both substrates, the line connects the mean deglucuronidation rates of the different sample collection time points. The highlighted area represents the mean ± 50% of the deglucuronidation rates of the different fecal sample collection time points. The dagger symbol (†) denotes fecal sample collection time points where the deglucuronidation rate deviates from the mean deglucuronidation rate of all time points by >50%. Time points where donor FD02 underwent oral antibiotic treatment (amoxicillin 500 mg 3 times daily on days 188–194 and cefalexin 500 mg twice daily on days 239–246, after which they quit as a FMT donor) are shown as arrows. estradiol-17G, β -estradiol 17- β - d -glucuronide; FMT, fecal microbiota transplantation; 4-MU, 4-methylumbelliferone; 4-MUG, 4-methylumbelliferyl- β - d -glucuronide.

Article Snippet: The GUS reporter substrates 4-methylumbelliferyl- β - d -glucuronide dihydrate (4-MUG) and p -nitrophenyl- β - d -glucuronide (PNPG) were obtained from Santa Cruz Biotechnology and Sigma-Aldrich, respectively, whereas the aglycones 4-methylumbelliferone (4-MU) and p -nitrophenol (PNP) were both from Sigma-Aldrich. ( R,S )-Ezetimibe phenoxy- β - d -glucuronide (ezetimibe-G), indomethacin acyl- β - d -glucuronide (indomethacin-G), and telmisartan acyl- β - d -glucuronide (telmisartan-G) were purchased from Synthose.

Techniques: Produced, Liquid Chromatography, Incubation, Transplantation Assay

Journal: Drug Metabolism and Disposition

Article Title: Characterization of fecal deglucuronidation activity in healthy subjects and in patients treated with fecal microbiota transplantation

doi: 10.1016/j.dmd.2025.100205

Figure Lengend Snippet: Deglucuronidation of 200 μ M 4-MUG by fecal lysates produced from the fecal samples of patients P01–P07 with recurrent Clostridioides difficile infection before FMT, 1 month after FMT, and 1 year after FMT. After the initiation of the reaction, 4-MU formation was followed at 1-minute intervals. The 4-MUG deglucuronidation rate, defined as the amount of formed aglycone per time per amount of total protein in the reaction, was determined with linear regression. The data were collected from 3 independent experiments, the means of which for each patient are shown as individual data points. The bars represent the mean deglucuronidation rates of all lysates at the different fecal sample collection time points. The lines connect the deglucuronidation rates of the individual patients. Statistical significance of the difference between the means was tested with the Friedman test with Dunn post hoc test for multiple comparisons. FMT, fecal microbiota transplantation; 4-MU, 4-methylumbelliferone; 4-MUG, 4-methylumbelliferyl- β - d -glucuronide.

Article Snippet: The GUS reporter substrates 4-methylumbelliferyl- β - d -glucuronide dihydrate (4-MUG) and p -nitrophenyl- β - d -glucuronide (PNPG) were obtained from Santa Cruz Biotechnology and Sigma-Aldrich, respectively, whereas the aglycones 4-methylumbelliferone (4-MU) and p -nitrophenol (PNP) were both from Sigma-Aldrich. ( R,S )-Ezetimibe phenoxy- β - d -glucuronide (ezetimibe-G), indomethacin acyl- β - d -glucuronide (indomethacin-G), and telmisartan acyl- β - d -glucuronide (telmisartan-G) were purchased from Synthose.

Techniques: Produced, Infection, Transplantation Assay