The recoveries were higher than 80%, and the relative standard deviations of the intra- and inter-assays were lower than 10%. Measurement of GAZT in the incubation mixtures was performed according to a previously reported method39, with slight modification. BCRP. Further studies showed that ALF significantly increased the intestinal absorption of both rhodamine 123 and AZT without altering intestinal integrity, thus confirming an impairment of intestinal P-gp function. In conclusion, ALF significantly increases the oral plasma exposure of AZT in rats, a result partly attributed to the impaired function and expression of hepatic UGT2B7 and intestinal P-gp. have found a decreased expression of CYP3A, 2E1, and 1A2 in severe chronic liver disease and correspondingly attenuated activities of testosterone 6-hydroxylation (CYP3A), single-pass perfusion as previously described35. Briefly, rats were fasted overnight and anesthetized with 60 mg/kg pentobarbital (ip). After the isolation of 20 cm of jejunum, two cannulas were inserted into either end (input and output). The isolated jejunum was flushed with blank Krebs-Henseleit buffer (37 C) at a fast rate for 10 min, then with Krebs-Henseleit buffer containing 10 mol/L AZT or 0.5 mol/L rhodamine 123 at a 0.2 mL/min flow rate until equilibrium was reached. The total perfusion period was 120 min, and efflux samples were collected every 15 min from the output cannula. Rats were sacrificed at the end of the perfusion, and the perfused segments were removed for measurements of the absorption areas. The apparent effective permeability (is the entering intestinal perfusion flow rate, intestinal integrity was measured using a previously described method37. Rats received oral gavages of fluorescein isothiocyanate (FITC)-labeled 70-kDa dextran (FD-70) at 50 mg/kg body weight in phosphate-buffered saline (PBS, pH 7.4). After 1 and 4 h, 100 L of blood were collected from the postorbital venous plexus veins. Plasma was obtained by the addition of 10 g/mL heparin and centrifugation at 5000for 5 min at 4 C. Plasma (50 L) was mixed with an equal volume of PBS (pH 7.4) and added to a 96-well microplate. The concentration of fluorescein was determined by spectrophotometry (Varioskan Flash, Thermo Scientific, USA) with an excitation wavelength of 485 nm and an emission wavelength of 525 nm, using a calibration curve. Analytical procedures The concentrations of AZT and GAZT in plasma or urine were determined using liquid chromatography tandem mass spectrometry (LC-MS) according to a previously described method38. The linear ranges of AZT and GAZT in plasma were 0.05C10 g/mL and 0.025C2 g/mL, and the lowest limits of quantification of AZT and GAZT were 50 ng/mL and 25 ng/mL, respectively. The linear ranges of AZT and GAZT in urine were 0.05C10 g/mL and 0.025C5 g/mL, and the lowest limits of quantification of AZT and GAZT were 50 ng/mL and 25 ng/mL, respectively. The recoveries were higher than 80%, and the relative standard deviations of the intra- and inter-assays were lower than 10%. Measurement of GAZT in the incubation mixtures was performed according to a previously reported method39, with slight modification. Briefly, 10 L of terazosin (internal standard, 25 g/mL) Pyridoxal phosphate was added to 200 L of the microsomal incubation mixture, and the Rabbit polyclonal to AIP mixture was centrifuged at 20000 for 10 min. The supernatant was injected into an LC-MS system. The mobile phase consisted of acetonitrile and water (17:83, 442.00125.00 for GAZT and 388.15290.20 for the IS terazosin). The detection limit for GAZT was 0.02 mol/L, and the recoveries were greater than 75%. The intra-day and inter-day coefficients of variation for the assay were less than 10%. The concentrations of AZT and rhodamine 123 Pyridoxal phosphate in the intestinal eluate samples were determined using ultraviolet and fluorescence HPLC detectors, respectively, according to previously described methods40,41. The lowest limits of quantification of AZT and rhodamine 123 in the intestinal eluate samples were 0.5 and 0.02 mol/L, respectively. The linear ranges of AZT and rhodamine 123 in intestinal eluate samples were 0.5C40 mol/L and 0.02C1 mol/L, respectively. The recoveries of AZT and rhodamine Pyridoxal phosphate were higher than 85%, and the relative standard deviations of the intra-day and inter-day levels in the samples were lower than 10%. Statistical analysis The pharmacokinetic parameters were estimated using a noncompartmental analysis (Pheonix Winnonlin 6.4, Pharsight, St Louis, MO, USA). If the data fit a normal distribution, a standard analysis of variance was used. The significance of differences between two groups was determined with Student’s CON rats. 10.741.28 g h/mL in control rats). This result indicated that ALF may increase the plasma exposure of AZT after oral administration. Open in a separate window Figure 2 Plasma concentration-time profiles of zidovudine (AZT) and AZT CON rats. Table 2 Pharmacokinetic parameters of zidovudine (AZT) after oral administration of 20 mg/kg AZT or intravenous administration of 10 mg/kg AZT to acute liver failure (ALF) rats and.
