6 g, K2HPO4 1.85 g, 1% (v/v) reducing solution (30 g/l L-aminothiopropionic acid and NVP-HSP990 supplier 30 g/l sodium hyposulfite, dissolved in PBS), and 1 g NH4Cl. Medium C was the same as medium B except the absence of any nitrogen source. Culture was conducted as follows: 0.3 g of defatted flaxseeds was added into each of tubes containing either medium A, B or C (3 ml), which were then sealed with liquid paraffin and autoclaved at 121°C for 15 min. Into the medium, 0.3 g of fresh human feces was added and incubated at 37°C for 72
h. Supernatant of the cultures was then inspected for the appearance of END. Collection and processing of fecal samples Initially, fresh fecal specimens (ca. 4.0 g each), obtained from 28 healthy young subjects (fourteen females and fourteen males, 22-33 years old), were suspended in 20 ml sterile phosphate buffer saline (PBS, 2.6 g l-1 KH2PO4, 1.85 g l-1 K2HPO4, PH 7.4) and 2 ml such fecal suspension was transferred to 20 ml medium, followed by incubation at 37°C for 36 h. During the fecal collection and culture preparation, no strictly anaerobic techniques or instruments were used. The fecal specimen that we used for END production was from a 33 years old female. High-performance liquid chromatography
(HPLC) The HPLC system consisted of Agilent 1200 series HPLC Thiazovivin research buy apparatus (Agilent Technologies, USA), including high-pressure binary-gradient solvent-delivery pump, DAD detector, autosampler, thermostat column compartment and chemstation (9.01 edition). 6-phosphogluconolactonase Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm) was used to analyze all of the samples. Mobile phase consisted of
water (A) and acetonitrile (B) in a linear gradient change from 100% A to 50% A and 50% B in 30 min. Detection wavelength was 280 nm, and the temperature of the column oven was 25°C with a flow rate of 1.0 ml min-1. Calibration of the END and SECO curves The stock solutions of END standard (1.98 mg ml-1) and SECO standard (175.5 μg ml-1) were prepared by accurately weighing and transferring each of them into a volumetric flask (1 ml) and dissolving it in methanol. Solutions for END calibration (0.0198 ~ 1.98 mg ml-1) and SECO calibration (175.5 ~ 2.74 μg ml-1) were prepared by dilution of the stock solutions with methanol, with six dilution series being analyzed (1.98, 0.99, 0.396, 0.198, 0.099, 0.0198 mg ml-1) for END calibration and seven dilution series being analyzed (175.5, 87.75, 43.86, 21.94, 10.97, 5.48, 2.74 μg ml-1) for SECO calibration. For each calibration curve, independent 4EGI-1 chemical structure dilutions were analyzed. The calibration equation of END was obtained by plotting HPLC peak areas (Y) versus the concentration of calibrators (X, mg ml-1), which was as follows: Y = 4433.46 X + 63.86 (R2 = 0.9999), with a good linearity over the range from 0.0198 mg ml-1 to 1.