Absorption and presystemic glucuronidation of 1-naphthol in the vascularly fluorocarbon emulsion perfused rat small intestine

Summary Using the isolated vascularly fluorocarbon emulsion perfused rat small intestine some factors which determine the extent of the intestinal glucuronidation of 1-naphthol to 1-naphthol--d-glucuronide were studied. Increasing the luminal 1-naphthol concentration resulted in a concomitant increase in the 1-naphthol appearance in the vascular perfusate. In contrast, the total appearance of 1-naphthol--d-glucuronide increased less than proportional to the increase in the luminal 1-naphthol concentration. About 88% of the total amount of 1-naphthol--d-glucuronide excreted was released into the vascular perfusate. The capacity-limited intestinal glucuronide efflux is most likely due to saturation of the excretory mechanism for 1-naphthol--d-glucuronide. Decreasing the vascular flow rate influenced both the appearance of 1-naphthol and 1-naphtol--d-glucuronide in the vascular perfusate, whereas the appearance of 1-naphthol--d-glucuronide in the luminal perfusate was essentially flow-independent. A noradrenaline-induced change in the haemodynamic state of the vascular bed (with the total flow kept constant) resulted in a marked decrease in the 1-naphthol vascular concentration. The vascular 1-naphthol--d-glucuronide concentration was only slightly affected. These results indicate that changes in blood flow and blood flow distribution within the intestinal wall can affect the extent of presystemic intestinal metabolism by interfering with the absorption of the parent compound and the efflux of formed conjugates. These parameters can be of paramount importance for causing variable intestinal first-pass effects of drugs in vivo. Send offprint requests to M. H. de Vries at the above address     

Integration of hepatic drug transporters and phase II metabolizing enzymes: Mechanisms of hepatic excretion of sulfate, glucuronide, and glutathione metabolites

The liver is the primary site of drug metabolism in the body. Typically, metabolic conversion of a drug results in inactivation, detoxification, and enhanced likelihood for excretion in urine or feces. Sulfation, glucuronidation, and glutathione conjugation represent the three most prevalent classes of phase II metabolism, which may occur directly on the parent compounds that contain appropriate structural motifs, or, as is usually the case, on functional groups added or exposed by phase I oxidation. These three conjugation reactions increase the molecular weight and water solubility of the compound, in addition to adding a negative charge to the molecule. As a result of these changes in the physicochemical properties, phase II conjugates tend to have very poor membrane permeability, and necessitate carrier-mediated transport for biliary or hepatic basolateral excretion into sinusoidal blood for eventual excretion into urine. This review summarizes sulfation, glucuronidation, and glutathione conjugation reactions, as well as recent progress in elucidating the hepatic transport mechanisms responsible for the excretion of these conjugates from the liver. The discussion focuses on alterations of metabolism and transport by chemical modulators, and disease states, as well as pharmacodynamic and toxicological implications of hepatic metabolism and/or transport modulation for certain active phase II conjugates. A brief discussion of issues that must be considered in the design and interpretation of phase II metabolite transport studies follows.     

Route of metabolization and detoxication of paralytic shellfish toxins in humans

Paralytic shellfish toxins (PST) are a collection of over 26 structurally related imidazoline guanidinium derivatives produced by marine dinoflagellates and freshwater cyanobacteria.Glucuronidation of drugs by UDP-glucuronosyltransferase (UGT) is the major phase II conjugation reaction in mammalian liver.In this study, using human liver microsomes, the in vitro paralytic shellfish toxins oxidation and sequential glucuronidation are achieved. Neosaxitoxin (neoSTX), Gonyautoxin 3/2 epimers (GTX3/GTX2) and Saxitoxin (STX) are used as starting enzymatic substrates. The enzymatic reaction final product metabolites are identified by using HPLC-FLD and HPLC/ESI-IT/MS. Four metabolites from GTX3/GTX2 epimers precursors, three of neoSTX and two of STX are clearly identified after incubating with UDPGA/NADPH and fresh liver microsomes. The glucuronic-Paralytic Shellfish Toxins were completely hydrolysed by treatment with β-glucuronidase. All toxin analogs were identified comparing their HPLC retention time with those of analytical standard reference samples and further confirmed by HPLC/ESI-IT/MS. Paralytic Shellfish Toxins (PST) were widely metabolized by human microsomes and less than 15% of the original PST, incubated as substrate, stayed behind at the end of the incubation.The apparent V_max and Km formation values for the respective glucuronides of neoSTX, GTX3/GTX2 epimers and STX were determined. The V_max formation values for Glucuronic-GTX3 and Glucuronic-GTX2 were lower than Glucuronic-neoSTX and Glucuronic-STX (6.8 ± 1.9 × 10⁻³; 8.3 ± 2.8 × 10⁻³ and 9.7 ± 2.8 × 10⁻³ pmol/min/mg protein respectively). Km of the glucuronidation reaction for GTX3/GTX2 epimers was less than that of glucuronidation of neoSTX and STX (20.2 ± 0.12; 27.06 ± 0.23 and 32.02 ± 0.64 μM respectively).In conclusion, these data show for the first time, direct evidence for the sequential oxidation and glucuronidation of PST in vitro, both being the initial detoxication reactions for the excretion of these toxins in humans. The PST oxidation and glucuronidation pathway showed here, is the hepatic conversion of its properly glucuronic-PST synthesized, and the sequential route of PST detoxication in human.     

Glucuronidation of bilirubin and the occurrence of pigment gallstones in patients with chronic haemolytic diseases

A group of thirty-seven patients with increased haem catabolism has been studied to gain insight in their bilirubin conjugating capacity. Bilirubin UDP-glucuronyl transferase activity (GlcATa) in the liver and bilirubin monoconjugates in bile were measured and the hepatic bilirubin clearance was calculated from the radio-chromium-survival data. In the present group, 41% of the patients clearly had a deficiency in bilirubin conjugation similar to what is classically found in Gilbert's syndrome. The association may facilitate detection of these patients as serum bilirubin levels were higher (65.8 microM +/- 19) (m +/- 1 SD) in the fifteen patients with associated Gilbert's syndrome versus thirteen having only haemolysis (43.6 microM +/- 15). A fair correlation was found between the percentage of monoconjugates in bile and the GlcATa levels in the liver as well as with the calculated hepatic bilirubin clearance, although some discrepancies exist. Using these determinations, a clearcut separation from normal values was not obtained, suggesting at least in the present group of patients that Gilbert' syndrome represents only one end of a continuum of bilirubin conjugation rates and not a separate entity. Pigment stones in the gall-bladder were documented in 51% of the patients and usually at an early age. There was no relationship towards sex, serum bilirubin, GlcATa in liver, total bilirubin or monoconjugates in bile. Age played some role as well as the type of haemolysis as all patients with congenital dyserythropoiesis (n = 4) or acquired haemolysis (n = 3) had lithiasis. Moderate chronic cholecystitis was present, whereas an accumulation of iron and bile pigment was evident in the liver.     

Plasma concentrations of lamotrigine and its 2-N-glucuronide metabolite during pregnancy in women with epilepsy

Objective: To further characterize pregnancy-induced alterations in the pharmacokinetics of lamotrigine (LTG).
Methods: Fifteen women treated with LTG were studied during 17 pregnancies. Complete trough blood samples from all trimesters and baseline > 1 month after delivery were available for 12 pregnancies (Group A), whereas, five contributed with samples only from the third trimester and baseline (Group B). High-performance liquid chromatography (HPLC) was used to determine LTG plasma concentrations, and liquid chromatography-mass spectrometry to assay the main metabolite 2-N-lamotrigine glucuronide (2-N-GLUC) in plasma.
Results: In group A, the mean dose/plasma concentration ratio (D/C) of LTG at baseline after pregnancy was 66.5 ± 17.9 (± SD) L/day and approximately 250% higher in late pregnancy. The mean lamotrigine-2-N-glucuronide/lamotrigine plasma concentration ratio (2-N-GLUC/LTG) was 0.349 ± 0.141 (± SD) at baseline and 147% higher in late pregnancy. Taking group A and B together, the 2-N-GLUC/LTG ratio was 175% higher in the third trimester compared to baseline.
Conclusion: Our study confirms a significant decline in LTG plasma levels during pregnancy in women on monotherapy with LTG. An increased 2-N-GLUC/LTG ratio suggests that this decline may be related to an increased metabolism of LTG by glucuronidation.     

Role of gut in xenobiotic metabolism

The gastrointestinal tract forms the first line of defense in the body against the main load of xenobiotics. The gastrointestinal mucosa has several mechanisms through which the xenobiotics are modified. The monooxygenase activities in most species are relatively low in the mucosa as compared to the liver, but conjugation, for example, via glucuronide formation proceeds efficiently. UDP-glucuronosyltransferase activities can exceed those in the liver. Glutathione S-transferase activity is also high. The biotransformation activities are readily inducible in the mucosa and this is, at least partly, responsible for the oral-aboral gradient seen in enzyme activities. In rainbow trout glutathione S-transferase is, however, significantly higher at the aboral third than in two oral segments, although in rats the intestinal glutathione S-transferase shows a clear oral-aboral gradient. The gradient is independent of the presence of microflora at least in the case of carboxylesterase and glutathione S-transferase. A similar gradient can also be found from the gut lumen, in both germ-free and specific pathogen-free rats. The cells in the middle of the villi appear to be most responsive under the influence of inducers. The readily occurring induction in the mucosa provides a suitable model for studies on biological effects to defined compounds and mixtures.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of this 65th birthday     

Toxicokinetics of p-tert-octylphenol in male Wistar rats

Only weak oestrogenic activity has been reported for p-alkylphenols compared with the physiological hormone 17β-estradiol. Despite the low potency, there is concern that due to bioaccumulation oestrogenically efficient blood levels could be reached in humans exposed to trace levels of p-alkylphenols. To address these concerns, toxicokinetic studies with p-tert-octylphenol [OP; p-(1,1,3,3-tetramethylbutyl)-phenol] as a model compound have been conducted in male Wistar rats. OP blood concentrations were determined by GC-MS in rats receiving either single oral (gavage) applications of 50 or 200mg OP/kg body wt or a single intravenous injection of 5mg/kg body wt. The OP blood concentration was ∼1970ng/ml immediately after a single intravenous application, decreased rapidly within 30 min, and was no longer detectable 6–8h after application. The curve of blood concentration vs time was used to calculate an elimination half-life of 310min. OP was detected in blood as early as 10min after gavage administration, indicating rapid initial uptake from the gastrointestinal tract; maximal blood levels reached 40 and 130ng/ml after applications of 50 and 200mg/kg, respectively. Using the area under the curve (AUC) of blood concentration vs time, low oral bioavailabilities of 2 and 10% were calculated for the 50 and 200mg/kg groups, respectively. OP toxicokinetics after repeated administration was investigated in male Wistar rats receiving daily gavage administrations of 50 or 200mg OP/kg body wt for 14 consecutive days. Profiles of OP blood concentration vs time determined on day 1 and day 14 were similar, indicating that repeated oral gavage administration did not lead to increased blood concentrations. Another group of rats received OP via drinking water saturated with OP (∼8mg/l, corresponding to a mean daily dose of ∼800μg/kg) over a period of up to 28 days. OP was not detected in any blood sample from animals treated via drinking water (detection limit was 1–5ng/ml blood). OP concentrations were also analysed in tissues obtained from the repeated gavage (14 days) and drinking water groups (14 and 28 days). In the 50mg/kg group, low OP concentrations were detected in fat and liver from some animals at average concentrations of 10 and 7ng/g tissue, respectively. OP was not detected in the other tissues analysed from this group. In the 200mg/kg group, OP was found in all tissues analysed except testes (fat, liver, kidney, muscle, brain and lung had average concentrations of 1285, 87, 71, 43, 9 and 7ng/g tissue, respectively). OP was not detected in tissues of animals receiving OP via drinking water for 14 or 28 days, except in muscle and kidney tissue of one single animal receiving OP for 14 days. Using rat liver fractions it was demonstrated that OP was conjugated via glucuronidation and sulphation in vitro. A V _max of 11.24 nmol/(min * mg microsomal protein) and a K _m of 8.77μmol/l were calculated for enzyme-catalysed OP glucuronidation. For enzyme-catalysed sulphation, a V _max of 2.85nmol/(minT15*mg protein) and a K _m of 11.35μmol/l were calculated. The results indicate that OP does not bioaccumulate in rats receiving low oral doses, in agreement with the hypothesis of a rapid first-pass elimination of OP by the liver after oral ingestion, via glucuronidation and sulphation. Only if these detoxification pathways are saturated may excessive doses lead to bioaccumulation. Received: 22 March 1996/Accepted: 12 June 1996     

Activity of rat UGT1A1 towards benzo[a]pyrene phenols and dihydrodiols

Four UDP-glucuronosyltransferases from the rat UGT1A family were tested for activity towards benzo[a]pyrene phenols and dihydrodiols. UGT1A1 and UGT1A7 were found to be broadly active towards BaP metabolites. Antisera recognizing rat UGT1A1 and UGT1A7 were used to assess UGT levels in relation to UGT activity towards benzo[a]pyrene-7,8-dihydrodiol (BPD). The rank BPD UGT activities were liver = intestine  kidney, whereas UGT1A1 was highest in liver and UGT1A7 was highest in intestine. Phenobarbital, an inducer of hepatic UGT1A1, only slightly increased BPD UGT activity, whereas UGT1A7 inducers more potently increased the activity. Inhibition studies using the differential UGT1A1 inhibitor, bilirubin, suggest that UGT1A1 is not a major contributor to the constitutive BPD glucuronidating activity of control rat liver microsomes. These data suggest that multiple UGT1A enzymes contribute to glucuronidation of BPD and other BaP metabolites, and that their relative contributions depend on tissue- and environmental-specific factors.