P nitrophenyl sulfate

Acenaphthenol, benzyloxyresorufin, bicinchoninic acid (BCA) assay kit, cytochrome c, NADH, NADPH, NADP⁺, 7-ethoxyresorufine, menadione, 7-methoxyresorufine, β-naphthoflavone, p-nitrophenyl sulfate, resorufin, R-sulforaphane, 3′-phosphoadenosin-5′-phosphate, and UDP-glucuronic acid were purchased from Sigma-Aldrich (Prague, Czech Republic). Midazolam was obtained from Toronto Research Chemicals (North York, ON, Canada). All other chemicals used were of HPLC or analytical grade. For real-time Polymerase Chain Reaction (PCR) analyses, ProtoScript^® II Reverse Transcriptase was purchased from NEB (Ipswich, UK), TriReagent from Biotech (Prague, Czech Republic), and the qPCRCore kit for SYBR Green I from Eurogentec (Seraing, Belgium).

UGT1a1 and SULT1a1 Supersomes were purchased from Corning (Netherlands). Sulfuric acid (96%), hydrogen peroxide (30%), sodium phosphate dibasic heptahydrate (98%), sodium phosphate monobasic monohydrate (98%), ethanol (95%), sodium hydroxide (98%), glutaraldehyde (25%), (3-aminopropyl)trimethoxysilane (97%), UDP-glucuronic acid (UDP-GA) (98%), 3′-phosphoadenosine-5′-phosphosulfate (PAPS, 60%), resorufin (95%), resorufin β-d-glucuronide (90%), p-nitrophenol (99%) and p-nitrophenyl sulfate (98%), LC-MS grade methanol, LC-MS grade water and triosephosphate isomerase, trifluoroacetic acid (TFA, 99%) were all obtained from Sigma-Aldrich (UK).

For the activity assay of fecal β-glucosidase, β-glucuronidase, and sulfatase, the reaction mixture (total volume of 0.2 mL), which contained 20 μL of 2.5 mmol/L p-nitrophenyl-β-D-glucopyranoside (Sigma-Aldrich) for β-glucosidase, 2.5 mmol/L p-nitrophenyl-β-D-glucuronide (Sigma-Aldrich) for β-glucuronidase, 2.5 mmol/L p-nitrophenyl palmitate (Sigma-Aldrich) for lipase, or 2.5 mmol/L p-nitrophenyl sulfate (Sigma-Aldrich) for sulfatase, 75 μL of 0.05 mol/L phosphate buffer (pH 7.0), and 20 μL of the fecalase, was incubated at 37 °C for 20 min and added 0.2 mL of 0.1 N NaOH, as previously reported [23 (link)]. Enzyme activities were indicated as the amount required to catalyze the formation of 1.0 nmole of p-nitrophenol per hour. Specific activity was defined in terms of μmol/h/g wet feces.