Retigabine N-glucuronidation and its potential role in enterohepatic circulation.
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The metabolism of retigabine in humans and dogs is dominated by N-glucuronidation (), whereas in rats, a multitude of metabolites of this new anticonvulsant is observed (). The comparison of the in vivo and in vitro kinetics of retigabine N-glucuronidation in these species identified a constant ratio between retigabine and retigabine N-glucuronide in vivo in humans and dog. An enterohepatic circulation of retigabine in these species is likely to be the result of reversible glucuronidation-deglucuronidation reactions. Rats did not show such a phenomenon, indicating that enterohepatic circulation of retigabine via retigabine N-glucuronide does not occur in this species. In the rat, 90% of retigabine N-glucuronidation is catalyzed by UDP-glucuronosyltransferase (UGT)1A1 and UGT1A2, whereas family 2 UGT enzymes contribute also. Of ten recombinant human UGTs, only UGTs 1A1, 1A3, 1A4, and 1A9 catalyzed the N-glucuronidation of retigabine. From the known substrate specificities of UGT1A4 toward lamotrigine and bilirubin and our activity and inhibition data, we conclude that UGT1A4 is a major retigabine N-glucuronosyl transferase in vivo and significantly contributes to the enterohepatic cycling of the drug. (+info)
Quantitative evaluation of alternative mechanisms of blood and testes disposition of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in rats.
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Di(2-ethylhexyl) phthalate (DEHP), a commercially important plasticizer, induces testicular toxicity in laboratory animals at high doses. After oral exposure, most of the DEHP is rapidly metabolized in the gut to mono(2-ethylhexyl) phthalate (MEHP), which is the active metabolite for induction of testicular toxicity. To quantify the testes dose of MEHP with various routes of exposure and dose levels, we developed a physiologically based pharmacokinetic (PBPK) model for DEHP and MEHP in rats. Tissue:blood partition coefficients for DEHP were estimated from the n-octanol: water partition coefficient, while partition coefficients for MEHP were determined experimentally using a vial equilibration technique. All other parameters were either found in the literature or estimated from blood or tissue levels following oral or intravenous exposure to DEHP or MEHP. A flow-limited model failed to adequately simulate the available data. Alternative plausible mechanisms were explored, including diffusion-limited membrane transport, enterohepatic circulation, and MEHP ionization (pH-trapping model). In the pH-trapping model, only nonionized MEHP is free to become partitioned into the tissues, where it is equilibrated and trapped as ionized MEHP until it is deionized and released. All three alternative models significantly improved predictions of DEHP and MEHP blood concentrations over the flow-limited model predictions. The pH-trapping model gave the best predictions with the largest value of the log likelihood function. Predicted MEHP blood and testes concentrations were compared to measured concentrations in juvenile rats to validate the pH-trapping model. Thus, MEHP ionization may be an important mechanism of MEHP blood and testes disposition in rats. (+info)
Phenotypic characterization of lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice. Pathophysiology Of biliary lipid secretion.
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The inbred C57L strain but not the AKR strain of mice carry Lith genes that determine cholesterol gallstone susceptibility. When C57L mice are fed a lithogenic diet containing 15% fat, 1% cholesterol, and 0.5% cholic acid, gallbladder bile displays rapid cholesterol supersaturation, mucin gel accumulation, increases in hydrophobic bile salts, and rapid phase separation of solid and liquid crystals, all of which contribute to the high cholesterol gallstone prevalence rates (D. Q-H. Wang, B. Paigen, and M. C. Carey. J. Lipid Res. 1997. 38: 1395;-1411). We have now determined the hepatic secretion rates of biliary lipids in fasting male and female C57L and AKR mice and the intercross (C57L x AKR)F(1) before and at frequent intervals during feeding the lithogenic diet for 56 days. Bile flow and biliary lipid secretion rates were measured in the first hour of an acute bile fistula and circulating bile salt pool sizes were determined by the "washout" technique after cholecystectomy. Compared with AKR mice, we found that i) C57L and F(1) mice on chow displayed significantly higher secretion rates of all biliary lipids, and larger bile salt pool sizes, as well as higher bile salt-dependent and bile salt-independent flow rates; ii) the lithogenic diet further increased biliary cholesterol and lecithin outputs, but bile salt outputs remained constant. Biliary coupling of cholesterol to lecithin increased approximately 30%, setting the biophysical conditions necessary for cholesterol phase separation in the gallbladder; and iii) no gender differences in lipid secretion rates were noted but male mice exhibited significantly more hydrophobic bile salt pools than females. We conclude that in gallstone-susceptible mice, Lith genes determine increased outputs of all biliary lipids but promote cholesterol hypersecretion disproportionately to lecithin and bile salt outputs thereby inducing lithogenic bile formation. (+info)
The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis.
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A new stable isotope procedure has been developed and validated in rats, applying [1-(13)C]acetate infusion to quantify the production of bile salts from de novo synthesized cholesterol making use of the mass isotopomer distribution analysis (MIDA) principle. Ions (m/z) 458-461, 370-373 and 285-288 were monitored by GC/MS (EI-mode) for the methyl trimethylsilylether derivatives of cholate, chenodeoxycholate and beta-muricholate, respectively. Rats with intact exteriorized enterohepatic circulation and rats with chronic bile diversion were infused with [1-(13)C]acetate for up to 14 h. After 10 h of infusion the enterohepatic circulation of the intact group was interrupted to deplete the existing bile salt pool (acute bile diversion). The fractions of biliary cholesterol and individual bile salts derived from newly synthesized cholesterol were determined by MIDA at t=14 h. In rats with acute bile diversion, these fractions were 20, 25, 27 and 23% for biliary cholesterol, cholate, chenodeoxycholate and beta-muricholate, respectively. After bile diversion for 8 days to induce hepatic cholesterol and bile salt synthesis, these fractions increased significantly to 32, 47, 41 and 47%, respectively. Calculated enrichments of the acetyl-CoA precursor pools were similar for all bile salts and biliary cholesterol within the two rat groups. However, chronic enterohepatic interruption decreased the acetyl-CoA pool size almost two-fold. We conclude that MIDA is a validated new stable isotope technique for studying the synthetic pathway from acetyl-CoA to bile salts. This technique provides an important new tool for studying bile salt metabolism in humans using stable isotopes. (+info)
Pharmacokinetics, biliary excretion, and tissue distribution of novel anti-HIV agents, cosalane and dihydrocosalane, in Sprague-Dawley rats.
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Cosalane and dihydrocosalane are potent inhibitors of HIV replication with a broad range of activity. The purpose of this study was to investigate: 1) the pharmacokinetic disposition of both cosalane and dihydrocosalane in male Sprague-Dawley rats, and 2) biliary excretion, enterohepatic circulation, and tissue distribution of cosalane after i.v. and/or oral administration. Animals were administered i.v. (10 mg/kg) cosalane or dihydrocosalane through a jugular vein to obtain plasma profiles. Dose dependence of cosalane was studied over a dose range of 1.0 to 10 mg/kg. The extent of enterohepatic recycling, biliary excretion, and tissue distribution were studied after i.v. administration. Both cosalane and dihydrocosalane exhibited a biexponential disposition with very long half-lives of 749 +/- 216 and 1016 +/- 407 min, along with very large volumes of distribution 23.1 +/- 4.4 and 24.4 +/- 2. 5 liter/kg, respectively. Both cosalane (nondetectable) and dihydrocosalane (<1%) showed very poor oral bioavailability. The biliary and renal excretions of cosalane were found to be negligible with no detectable metabolites either in urine or bile. After oral administration, more than 87% of the cosalane dose was excreted in the feces as the parent compound. Also, cosalane was sequestered significantly in liver with quantifiable levels in all tissues tested, even 48 h after the dose was administered. Therefore it was concluded that the poor oral bioavailability of cosalane may be due to its poor enterocytic transport coupled with sequestration in liver parenchymal cell membrane layers. (+info)
Synthesis and receptor sites of endothelin-1 in the rat liver vasculature.
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Immunocytochemical localization of big endothelin-1 (big ET-1), ET-1, and ET receptor A and B (ET(A) and ET(B)), and gene expression of prepro ET-1 mRNA were examined on the rat liver vasculature. Immunoreactivities for big ET-1 and ET-1 were preferentially seen along the endothelium of interlobular veins (IV) and artery (IA), although the staining intensity was more pronounced in IV. Expression of preproET-1 mRNA was detected in both vascular endothelia and the signal intensity was more prevalent in IV. Immunoelectron microscopy showed that rough endoplasmic cisterns were immunoreactive for big ET-1, while Weibel-Palade (WP) bodies, a storage site for ET-1, were immunoreactive for ET-1 in endothelial cells of IV. These results indicate that endothelial cells of IV are the major site of synthesis of ET-1, which is extracellularly secreted by degranulation and/or exocytosis of WP bodies. Hepatic stellate cells (HSCs), especially of the plasma membrane of perisinusoidal and interhepatocellular processes, were immunoreactive for both ET(A) and ET(B) receptor antibodies. These findings suggest that ET-1 receptor-mediated HSC contraction is involved in the regulation of hepatic sinusoidal blood flow as previously cited in mammalian liver cirrhosis. We also showed that sarcolemma and caveoles in the smooth muscle cells of the media of IV, and its branches before reaching the hepatic sinusoids, were immunoreactive for ET(A) receptor antibody. The results suggest that such vessels, which contains a large amount of hepatic blood inflow, participate in pump mechanism toward hepatic sinusoidal circulation in a receptor-mediated paracrine fashion. (+info)
Effects of continuous and intermittent feeding on biliary lipid outputs in man: application for measurements of intestinal absorption of cholesterol and bile acids.
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Hepatic outputs of biliary lipids can be measured by intestinal perfusion techniques, either during constant infusion of liquid formula into the duodenum or through-out a 24-hour period during which time three meals are given along with an overnight fast. The purpose of this study was to compare these two methods for estimating secretion of biliary lipids. In 21 subjects, mean hourly outputs measured during continuous feeding were highly comparable to those during intermittent feeding, showing that the constant infusion technique gave valid estimations of overall daily secretion rates of biliary lipids. On the other hand, the intermittent mode of feeding showed phasic changes of outputs and composition of biliary lipids in response to feeding and fasting apart from total outputs over 24 hours. However, it takes longer to complete and requires the use of a meal marker. By combining the intestinal perfusion technique with measurements of fecal excretion of neutral steroids and bile acids, this method may be used to estimate absorption of cholesterol and bile acids from the intestine. Thus, these measurements allow quantification of a number of parameters of the enterohepatic circulation. (+info)
Charcoal treatment and risk of escape ovulation in oral contraceptive users.
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Ovulatory potential was studied during the use of two oral contraceptive pill preparations, after repeated mid-cycle administration of activated charcoal. Eleven women used monophasic pills containing gestodene, 75 microgram, plus ethinyl oestradiol, 30 microgram, or norethisterone acetate, 1 mg, plus ethinyl oestradiol, 30 microgram, for 4 months each, in randomized order. During both pill treatments the third cycle was a control cycle, and during the fourth cycle of both pill types, 5 g of activated charcoal was ingested four times a day, starting 3 h after pill intake, on cycle days 12, 13 and 14. Ovarian activity was monitored by intravaginal ultrasonography of follicles and by measurements of serum concentrations of LH, FSH, oestradiol and progesterone throughout the control and charcoal-treatment cycles of both pill treatments. None of the women ovulated. Follicular activity seen in two women did not correlate with charcoal administration. It is concluded that the possible enterohepatic recirculation of gestodene and norethisterone is not of clinical importance. Repeated charcoal treatment, when administered 3 h after but at least 12 h before pill intake, can be used to treat diarrhoea in women taking oral contraceptives. (+info)