Vascularly Perfused Rat Small Intestine Research Articles

Pharmacokinetic Modelling of the Effect of Activated Charcoal on the Intestinal Secretion of Theophylline, using the Isolated Vascularly Perfused Rat Small Intestine

The effect of activated charcoal administration on the secretion of theophylline from the blood into the intestinal lumen has been examined by use of the rat isolated vascularly perfused small intestine. A closed two compartment model was used to analyse the vascular and luminal concentration-time curves obtained. An equation was derived to calculate the time-dependent intestinal clearance. From control experiments it was concluded that theophylline is secreted by a diffusional transport system through the intestinal wall. The intestinal clearance declined rapidly with time as a result of the concomitant increase in luminal theophylline concentration. After 120 min a steady state between the vascular and luminal perfusate was established. Administration of activated charcoal in the lumen had a profound effect on the kinetics of the drug. The vascular steady state concentration was depressed dramatically. The theophylline clearance remained nearly constant with time, because the blood to lumen concentration gradient was maximized. The maximal value for the intestinal theophylline clearance was estimated to be 0.88 mL min-1 and it equalled the value for the intestinal blood flow at the absorptive site. By use of the concept of absorptive site blood flow, the maximal effect of charcoal on systemic theophylline clearance could be adequately predicted for rats, dogs and man. Activated charcoal administration is only useful to enhance the systemic clearance of drugs or toxicants if that clearance is of the same order of magnitude as the absorptive site blood flow or lower.

Absorption of Hydroxyproline-Containing Peptides in Vascularly Perfused Rat Small Intestinein Situ

To assess the digestion and assimilation of gelatin and gelatin hydrolysates, the in situ absorption of typical hydroxyproline-containing dipeptides, Pro-Hyp, Hyp-Gly, Ser-Hyp Ala-Hyp, and pentadecapeptide, (Pro-Hyp-Gly)(5), was investigated in the rat small intestine. During vascular perfusion after the injection of Hyp-Gly, Pro-Hyp and (Pro-Hyp-Gly)(5) into the jejunum, peptide-form Hyp but not free-Hyp gradually increased in the perfusate. In contrast, in the case of Ser-Hyp and Ala-Hyp, both free- and peptide-form Hyp rapidly increased. The presence of these dipeptides and the pentadecapeptide in the perfusates was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using multiple reaction monitoring (MRM). Some digestive and absorbed forms from (Pro-Hyp-Gly)(5) were identified as Gly-(Pro-Hyp-Gly)(4), (Pro-Hyp-Gly)(4), Gly-(Pro-Hyp-Gly)(3), (Pro-Hyp-Gly)(3), Gly-(Pro-Hyp-Gly)(2), and (Pro-Hyp-Gly)(2) by MALDI-TOF/MS. The dipeptide hydrolase activity in intestinal mucosa toward Pro-Hyp and Hyp-Gly was extremely low, while Ser-Hyp and Ala-Hyp were substantially hydrolyzed in the cytosol. These results suggest that Hyp-peptides were resistant to intracellular hydrolysis and that a significant amount of these peptides was transported across the intestinal wall and may enter the portal circulation in an intact form.

P-Glycoprotein and an Unstirred Water Layer Barring Digoxin Absorption in the Vascularly Perfused Rat Small Intestine Preparation: Induction Studies with Pregnenolone-16α-carbonitrile

Digoxin, a substrate of P-glycoprotein (Pgp) and cytochrome P450 3a (Cyp3a), was used to illustrate the inductive effects of pregnenolone-16alpha-carbonitrile (PCN), a ligand of the pregnane X receptor, on the absorption and disposition of [3H]digoxin in the vascularly perfused rat small intestine preparation. Although increased Cyp3a protein was observed with Western blotting analysis after PCN treatment, metabolism of digoxin to the digoxigenin bis-digitoxoside metabolite in the rat small intestine remained insignificant (<4% dose). PCN pretreatment significantly decreased blood perfusate [3H]digoxin concentrations for both systemic and intraluminal administrations of [3H]digoxin due to increased Pgp levels. The apical secretion by Pgp increased at 90 min with PCN treatment, from 11.2 +/- 5.1% of dose to 20.1 +/- 8.6% of dose after systemic administration of [3H]digoxin; this increase was, however, statistically insignificant (P = 0.13) because of the high variability among preparations. When the composite data for the control and PCN-treated preparations were fit to published physiologically based pharmacokinetic models: the traditional model and the segregated flow model, suboptimal parameters were obtained. The data were further fit to expanded models with a bilayer membrane compartment housing the Pgp adjacent to the apical membrane, or an unstirred water layer (UWL) external to the apical membrane. The models with the UWL yielded improved fits and reasonable parameters associated with digoxin absorption, suggesting that the UWL posed as a barrier for digoxin absorption. Similar results were obtained with the segmental models (the segmental traditional model and the segmental segregated flow model) using the UWL, when heterogeneous distributions of Pgp in the duodenum, jejunum, and ileum were considered.

Route-dependent metabolism of morphine in the vascularly perfused rat small intestine preparation.

1. To compare the disposition of tracer morphine ([3H]M) following systemic and intraduodenal administration in the recirculating, rat small intestine preparation in absence or presence of verapamil (V), an inhibitor of P-glycoprotein. 2. To develop a physiological model to explain the observations. A bolus dose of [3H]M was added to the reservoir or injected into the duodenum of the rat small intestine preparation. V (200 microM in reservoir) was either absent (control studies) or present. Intestinal microsomal, incubation studies were performed to evaluate the effect of V on morphine glucuronidation. After systemic administration, [3H]M was not metabolized but was exsorbed into lumen. By contrast, both [3H]M and the 3beta-glucuronide metabolite, [3H]M3G, appeared in reservoir and lumen after intraduodenal administration. A physiologically-based model that encompassed absorption, metabolism and secretion was able to describe the route-dependent glucuronidation of M. The presence of V resulted in diminished levels of M3G in perfusate and lumen and mirrored the observation of decreased glucuronidation in microsomal incubations. Verapamil appeared to be an inhibitor of glucuronidation and not secretion of M. M was secreted and absorbed by the rat small intestine. Route-dependent glucuronidation of M was explained by physiological modeling when M was poorly partitioned in intestinal tissue, with a low influx clearance from blood and a even poorer efflux clearance from tissue. The poor efflux rendered a much greater metabolism of M that was initially absorbed from the lumen. V increased the extent of M absorption through inhibition of M glucuronidation.

First-Pass Effect: Significance of the Intestine for Absorption and Metabolism

ABSTRACTThe occurrence of low systemic availability due to significant metabolism or poor absorption of orally administered drugs has been well recognized. Three rate controlling factors affecting the oral absorption: unstirred water layer, membrane limitation, or flow limitation, have been identified. These are much affected by the physicochemical properties of the drug: pKA, water/lipid solubility, structural mimicry to endogenous substrates for transport proteins, and the physiology of the GI tract. Drug metabolizing enzymes are found to be present in the intestine, albeit the content is lower than that found in liver. The presence of pre-absorptive versus post-absorptive intestinal metabolism is presently discussed in experimental sets of data with luminal and systemic administration of the drugs in the vascularly perfused rat small intestine preparation. The effect of the anterior anatomical placement of the intestine and its contribution to metabolism, in relation to that for the liver, has been examined in our laboratory by the perfused intestine-liver preparation. The effect of concentration and flow have been studied and general principles governing drug absorption and metabolism in the intestine and the subsequent effects on the liver have been discussed.

The absorption and first-pass metabolism of [14C]-1,3-dinitrobenzene in the isolated vascularly perfused rat small intestine.

We tested the hypothesis that the small intestine is capable of the first-pass, reductive metabolism of xenobiotics. A simplified version of the isolated vascularly perfused rat small intestine was developed to test this hypothesis with 1,3-dinitrobenzene (1,3-DNB) as a model xenobiotic. Both 3-nitroaniline (3-NA) and 3-nitroacetanilide (3-NAA) were formed and absorbed following intralumenal doses of 1,3-DNB (1.8 or 4.2 mumol) to isolated vascularly perfused rat small intestine. Dose, fasting, or antibiotic pretreatment had no effect on the absorption and metabolism of 1,3-DNB in this model system. The failure of antibiotic pretreatment to alter the metabolism of 1,3-DNA indicated that 1,3-DNB metabolism was mammalian rather than microfloral in origin. All data from experiments initiated with lumenal 1,3-DNB were fit to a pharmacokinetic model (model A). ANOVA analysis revealed that dose, fasting, or antibiotic pretreatment had no statistically significant effect on the model-dependent parameters. 3-NA (1.5 mumol) was administered to the lumen of isolated vascularly perfused rat small intestine to evaluate model A predictions for the absorption and metabolism of this metabolite. All data from experiments initiated with 3-NA were fit to a pharmacokinetic model (model B). Comparison of corresponding model-dependent pharmacokinetic parameters (i.e. those parameters which describe the same processes in models A and B) revealed quantitative differences. Evidence for significant quantitative differences in the pharmacokinetics or metabolism of formed versus preformed 3-NA in rat small intestine may require better definition of the rate constants used to describe tissue and lumenal processes or identification and incorporation of the remaining unidentified metabolites into the models.

THE ABSORPTION AND FIRST‐PASS METABOLISM OF [14C]-1, 3-DINITROBENZENE IN THE ISOLATED VASCULARLY PERFUSED RAT SMALL INTESTINE

We tested the hypothesis that the small intestine is capable of the first-pass, reductive metabolism of xenobiotics. A simplified version of the isolated vascularly perfused rat small intestine was developed to test this hypothesis with 1, 3-dinitrobenzene (1, 3-DNB) as a model xenobiotic. Both 3-nitroaniline (3-NA) and 3-nitroacetanilide (3-NAA) were formed and absorbed following intralumenal doses of 1, 3-DNB (1·8 or 4·2 μmol) to isolated vascularly perfused rat small intestine. Dose, fasting, or antibiotic pretreatment had no effect on the absorption and metabolism of 1, 3-DNB in this model system. The failure of antibiotic pretreatment to alter the metabolism of 1, 3-DNB indicated that 1, 3-DNB metabolism was mammalian rather than microfloral in origin. All data from experiments initiated with lumenal 1, 3-DNB were fit to a pharmacokinetic model (model A). ANOVA analysis revealed that dose, fasting, or antibiotic pretreatment had no statistically significant effect on the model-dependent parameters. 3-NA (1·5 μmol) was administered to the lumen of isolated vascularly perfused rat small intestine to evaluate model A predictions for the absorption and metabolism of this metabolite. All data from experiments initiated with 3-NA were fit to a pharmacokinetic model (model B). Comparison of corresponding model-dependent pharmacokinetic parameters (i.e. those parameters which describe the same processes in models A and B) revealed quantitative differences. Evidence for significant quantitative differences in the pharmacokinetics or metabolism of formed versus preformed 3-NA in rat small intestine may require better definition of the rate constants used to describe tissue and lumenal processes or identification and incorporation of the remaining unidentified metabolites into the models.

Effects of dexamethasone on glutamine metabolism in the isolated vascularly perfused rat small intestine

Post-stress metabolism is associated with a large glutamine (Gln) efflux from muscle and an increased Gln utilization by the small intestine. Both appear to be modulated by corticosteroids. The present investigation was performed to better characterize the mechanism of corticoid action on Gln metabolism in an isolated preparation of vascularly perfused rat small intestine. In all perfusions, a synthetic perfusate free from blood components was used with only 0.6 mM Gln and 10 mM glucose as substrates. Irrespective of dexamethasone concentrations in the vascular perfusate (none, 0.25 mg l-1, or 2.5 mg l-1, isolated intestines from normal rats exhibited unchanged extraction rates of Gln (-85 +/- 8, -89 +/- 10, and -87 +/- 16 nmol min-1 g-1) and unchanged production rates of alanine (43 +/- 9, 40 +/- 7, and 51 +/- 5 nmol min-1 g-1) and ammonia (49 +/- 15, 45 +/- 13, and 54 +/- 13 nmol min-1 g-1). Similarly, when intestines were vascularly perfused 2 or 9 days after dexamethasone injection (0.45 mg kg-1 BW), net Gln uptake also remained unchanged (-88 +/- 16 and -84 +/- 11 nmol min-1 g-1). There was, however, a shift in nitrogenous products of Gln metabolism from ammonia (-31% and -38%) to alanine (+16% and +64%). Thus, the failure of dexamethasone to increase Gln uptake in the isolated rat intestine may indicate that rather than acting directly on the mucosa, dexamethasone could regulate intestinal Gln consumption in vivo by indirect mechanisms possibly involving extramucosal tissues. Dexamethasone pretreatment may modulate the pattern of nitrogenous products in portal venous blood presented to the liver and thus support enhanced nitrogen loss through ureagenesis by metabolic cooperation between gut and liver.

Absorption of Inorganic, Trivalent Chromium from the Vascularly Perfused Rat Small Intestine

The absorption of inorganic, trivalent chromium (Cr) by the rat small intestine was investigated by quantifying three components of the absorptive process: 1) Cr uptake from a test meal, 2) Cr transport into the circulation and 3) Cr retention by the intestine. An in vitro, in situ double-perfusion technique was used in which the intestinal vasculature, from the superior mesenteric artery to the portal vein, and the intestinal lumen, from the duodenum to the ileum, were perfused simultaneously. The vasculature was perfused with a synthetic "plasma" (vascular perfusate) while the lumen was perfused with a nutrient-rich solution (intestinal perfusate) at concentrations of trivalent Cr of 0.2-20 mumol/l (10-1000 ppb). Dose-response curves, in which Cr transport, retention and uptake were plotted against the luminal Cr concentration, revealed that Cr absorption is a nonsaturable process. Regardless of the Cr concentration of the intestinal perfusate, 5.90 +/- 0.33% (mean +/- SEM) of the test dose was taken up from the meal, 5.52 +/- 0.33% was transported into the vascular perfusate and 0.38 +/- 0.03% was retained by the small intestine. Based on the criterion of saturability, it was concluded that inorganic, trivalent Cr is absorbed by the nonmediated process of passive diffusion in the small intestine of rats fed a Cr-adequate diet (1.44 micrograms Cr/g diet).

A ready-to-use activated charcoal mixture. Adsorption studies in vitro and in dogs: its influence on the intestinal secretion of theophylline in a rat model.

A practical, ready-to-use preparation of activated charcoal (AZU mixture) for application in toxicology has been formulated. To establish its efficacy, the formulation was tested in vitro and in dogs. The in vitro adsorption capacity was compared to that of freshly prepared charcoal suspension in water (CW) and to Carbomix. Langmuir adsorption coefficients demonstrated small but clinically insignificant differences in adsorption capacity between the preparations. The laxative sodium sulfate did not reduce the adsorption capacity of charcoal in vitro. Dogs were given 60 mg of paracetamol per kg as an oral solution followed by 5 g of activated charcoal preparation. The area under the plasma concentration versus time curve (control 2955 +/- 353 mg.min-1.l-1) was significantly reduced following CW (921 +/- 453) and AZU (786 +/- 270). The premixed AZU charcoal formulation is efficacious, inexpensive and overcomes the problems of bed-side preparation. An isolated vascularly perfused rat small intestine can be used to describe the effect of activated charcoal on the intestinal secretion of theophylline.

Utilization of vascular leucyl-leucine (LEULEU), alanyl-glutamine (ALAGLN), and glycyl-glutamine (GLYGLN) by the isolated vascularly perfused rat small intestine : [formula omitted], I. Müller, A. Raible, D. Vieillard-Baron, D. Bauder, P. Fürst, F. Hartmann. Medizinische Universitätsklinik I, D-7400 Tübingen und Institute of Biol. Chem. and Nutrition, Universität Hohenheim, D-7000 Stuttgart

Utilization of vascular leucyl-leucine (LEULEU), alanyl-glutamine (ALAGLN), and glycyl-glutamine (GLYGLN) by the isolated vascularly perfused rat small intestine : [formula omitted], I. Müller, A. Raible, D. Vieillard-Baron, D. Bauder, P. Fürst, F. Hartmann. Medizinische Universitätsklinik I, D-7400 Tübingen und Institute of Biol. Chem. and Nutrition, Universität Hohenheim, D-7000 Stuttgart

Absorption and Metabolism of Adenine, Adenosine-5'-Monophosphate, Adenosine and Hypoxanthine by the Isolated Vascularly Perfused Rat Small Intestine

Intestinal vascular perfusion and in vivo live animal experiments were conducted in order to evaluate the nature and extent of the intestinal metabolism of adenine, adenosine, adenosine-5′-monophosphate (AMP) and hypoxanthine in the rat. Radiolabeled purine substrates were administered intralumenally. Intestinal contents, tissue and/or portal flow were collected and evaluated for resultant purine metabolites by liquid and paper chromatography and paper electrophoresis. Adenosine, AMP and hypoxanthine were quantitatively metabolized to end products (primarily uric acid) within 15 minutes after administration. In contrast, the metabolism of adenine to uric acid was considerably slower. Up to 20% of the administered adenine was recovered unmetabolized in the portal vasculature. Nonetheless uric acid was the primary metabolite recovered from the portal circulation in the isolated intestine regardless of the purine substrate or concentration administered. Since lumenal inosine concentrations rose sharply with increasing doses of AMP, either transport or metabolism of inosine is a rate-limiting step in the intestinal metabolism of purines to uric acid in the rat. Finally, the large percentage of the radiolabel in uric acid recovered in the lumen is consistent with the hypothesis that the intestine is an extrarenal site for purine excretion.

Pasteur effect in the in vitro vascularly perfused rat small intestine

Isolated small intestine perfused in vitro with media with low oxygen concentration was found to contain low levels of ATP when compared with rat small intestine in vivo. The addition of fluorocarbon FC 75 to an erythrocyte-free perfusion medium was found to result in a high phosphate potential and a low rate of lactate production from glucose in isolated perfused small intestine, resembling the in vivo condition. This allowed the demonstration of a Pasteur effect in that replacement of oxygen by nitrogen (or the addition of 2,4-dinitrophenol) led to a rapid increase of the rate of glycolysis, and a decrease of the ATP concentration in the tissue