Characterization of lipophilic drug binding to rat intestinal fatty acid binding protein

Intestinal fatty acid binding protein (I-FABP) is present at high levels in the absorptive cells of the intestine (enterocytes) where it plays a role in the intracellular solubilization of fatty acids (FA). However, I-FABP has also been shown to bind to a range of non-FA ligands, including some lipophilic drug molecules, albeit with generally lower affinity than FA. The significance of these lower affinity interactions with exogenous compounds is not known. In this manuscript, we describe further characterization of drug-rat I-FABP binding interactions using a thermal-shift assay. A structural explanation of the observed affinity of rat I-FABP for different drugs based on spectroscopic data and modeling experiments is presented. In addition, immunocytochemistry has been used to probe the expression of I-FABP in a cell culture model reflective of the absorptive cells of the small intestine. Taken together, these data suggest a possible role for I-FABP in the disposition of some lipophilic drugs within the enterocyte.

Examination of the role of intestinal Fatty acid-binding protein in drug absorption using a parallel artificial membrane permeability assay

Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally diverse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein—intestinal fatty acid-binding protein (I-FABP). Binding to I-FABP significantly enhanced the transport of lipophilic drug molecules across a model membrane, and the degree of transport enhancement was related to both drug lipophilicity and I-FABP binding affinity. These data suggest that intracellular lipid-binding proteins such as I-FABP may enhance the membrane transport of lipophilic xenobiotics and facilitate drug access to the enterocyte cytoplasm and cytoplasmic organelles.

The interaction of lipophilic drugs with intestinal fatty acid-binding protein

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen {approx} bezafibrate > S-ibuprofen >> nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.

The effect of vitamin : a deficiency on glucose uptake in the rat

This thesis describes an investigation of the effects of vitamin A deficiency on gut function, The central hypothesis to be tested was that acute vitamin A deficiency affects glucose uptake from the small intestine- The hypothesis was tested using a system involving perfusion of isolated segments of the small intestine in the anaesthetized rat. The system was used to study effects on glucose uptake under steady-state conditions. In the initial part of the study, experiments were diverted towards setting up the system for measuring steady-state uptake, and determining the relative contributions of active uptake and diffusion. Phenol red was found to be a reliable non-absorbable marker for determining net water movement. Phlorizin, generally at 1 mmol/L, was used as a competitive (reversible) inhibitor of active uptake. It is difficult however to confirm complete inhibition of active uptake by phlorizin because of the limited solubility of the inhibitor. The kinetics of glucose uptake f ram intra-luminal maltose were found to be, in general, not significantly different from those applying to the uptake of glucose from an equivalent glucose solution. Maltase activity in the perfused gut segment was found to be sufficient to hydrolyse most of the maltose (80 per cent or more) in the solution being perfused, a much greater proportion than was absorbed. Glucose absorptive capacity, measured on an intestinal dry weight basis, was greatest in the duodenum and progressively less in the jejunum and ileum. The rate of water uptake f ran the gut was increased by the presence of glucose in the lumen, and was linked to glucose uptake as shown by the inhibition of water uptake by phlorizin. Uptake of glucose by solvent drag was demonstrated by showing an increased rate of glucose uptake when the rate of water uptake was increased by perfusing a solution of reduced osmotic pressure. In the experiment a low intra-luminal glucose concentration was used to preclude net uptake by diffusion and active uptake was blocked with phlorizin. This process was further investigated using streptozotocin-diabetic rats in which the diabetes establishes a hyperosomotic blood with hyperglycaemia. Uptake by solvent drag was more obvious in diabetic animals. A back-diffusion (exsorption) of glucose from the tissues to the lumen was also shown; the rate being proportional to plasma glucose concentration. Vitamin A deficiency was established in weanling rats after 6-7 weeks feeding on a diet based on wheat starch, coconut oil, and casein washed with hot ethanol, together with vitamins and minerals. The vitamin A deficiency led to classic eye signs and was reversed by the addition to the diet of retinoic acid (5 g/g diet). Vitamin A deficiency decreased intestinal mucus production (dry weight) but had no detectable effect on the histology of the villous epithelium as shown under the light microscope. Using perfusion experiments it was shown that vitamin A deficiency had no significant effect on the rate of active uptake of glucose, but that deficiency increased the rate of passive uptake.

Relationship between alcohol abuse upper gastro-intestinal haemorrhage and nutritional status during acute hospital admission

This thesis is concerned with the effect of alcohol consumption on the pathogenesis of bleeding from the upper gastrointestinal tract via nutritional pathways. Altered nutritional status is a frequently recognised clinical accompaniement of heavy alcohol consumption in hospitalized patients. Similarly, upper gastrointestinal bleeding is frequently accompanied by the presence of heavy alcohol consumption. Nevertheless, the clinical quantification of alcohol intake is often descriptive, so that a link between alcohol use and upper gastrointestinal haemorrhage via nutritional mechanisms has been only generally defined. In the literature review, the methods of defining alcohol use and abuse, using interview, biochemical and haematological techniques are noted. The relationship between alcohol abuse and nutrient imbalances is reviewed, especially in relation to possible effects on the gastrointestinal tract, appetite and eating habits. A further section reviews the relationship between alcohol use and anatomical lesions of the upper gastrointestinal tract likely to lead to bleeding. Following the chapter in which the methods used in this thesis are described. Chapter 4 seeks to describe the study population and its subgroups in this thesis in relation to interview, biochemical and haematological methods. Alcohol use is defined in relation to (1) a clinical classification of heavy or light drinking, based on a questionnaire administered in Casualty, (2) a quantified method of determining alcohol consumption during a subsequent ward dietetic assessment, (3) in relation to a biochemical definition (recent drinking and non-drinking), and a classification of (1) and (2) called, for the purposes of this thesis, 'alcohol abusers' and 'nonabusers'. Heavy, regular and recent drinkers and alcohol abusers tend to be male and younger than light, infrequent and nonrecent drinkers and nonabusers. Chapter 5 relates the nutritional status of those patients admitted acutely to hospital in relation to the groups defined in Chapter 4, Nutritional status is defined in terms of food intake, anthropometry, biochemical and haematological parameters. Different methods of defining alcohol use give rise to different patterns of nutritional impairment. Chapter 6 relates the nutritional status of those patients admitted acutely to hospital in relation to the presence or absence of an endoscopically defined site of upper gastrointestinal bleeding. A difference is seen between those bleeding from a Mailory-weiss tear and other sites of bleeding, similarly, biochemical differences in nutritional status emerge between those patients who presented in shock, and those who did not. Chapter 7 explores the relationships between biochemical markers of nutritional status and haemostatic variables in the groups of abusers/non-abusers, the various sites of primary bleeding/controls, and shock/non-shock. Serum copper appears to be related to altered haemostasis in a manner not apparently described elsewhere.

The influence of specific luminal factors on the colonic epithelium : high-dose butyrate and physical changes suppress early carcinogenic events in rats

INTRODUCTION Although luminal delivery of butyrate is one putative mechanism by which biology of the colonic epithelium might be influenced by changes in luminal contents, there is a paucity of supportive cause–effect evidence. This study aimed to directly establish whether distal colonic butyrate delivery is able to alter the response of the distal colonic epithelium to a carcinogen.
METHODS Groups of male Sprague-Dawley rats with chronically intubated colons received infusions of 80 mM butyrate or 0.9 percent saline into distal colon two or five times daily. Three weeks after exposure to azoxymethane (15 mg/kg subcutaneously), the density of aberrant crypts was quantified in distal colon.
RESULTS Infusions of 0.5 ml twice daily, whether containing saline or butyrate, decreased the number of aberrant crypt foci by 45 percent compared with rats receiving no infusions (P = 0.004, analysis of variance). Similar results were obtained when infusions were restricted to the postinitiation phase. When infusions were increased to 1 ml five times daily, saline infusions similarly suppressed aberrant crypt formation (38 percent), but butyrate infusions suppressed it to a greater degree (by 64 percent; P = 0.02 compared with saline infusion, t-test).
CONCLUSIONS High levels of butyrate delivery to the distal colonic lumen alter the epithelial response to a carcinogen in otherwise healthy rats. This finding directly supports the notion that the effects of butyrate on cells in vitro do occur in vivo provided a sufficient dose is delivered. The effect of infusion of liquid per se on the epithelial response highlights the potential impact physical changes alone can have on the colonic epithelium.

Intestinal microbiota associated with differential feed conversion efficiency in chickens

Analysis of model systems, for example in mice, has shown that the microbiota in the gastrointestinal tract can play an important role in the efficiency of energy extraction from diets. The study reported here aimed to determine whether there are correlations between gastrointestinal tract microbiota population structure and energy use in chickens. Efficiency in converting food into muscle mass has a significant impact on the intensive animal production industries, where feed represents the major portion of production costs. Despite extensive breeding and selection efforts, there are still large differences in the growth performance of animals fed identical diets and reared under the same conditions. Variability in growth performance presents management difficulties and causes economic loss. An understanding of possible microbiota drivers of these differences has potentially important benefits for industry. In this study, differences in cecal and jejunal microbiota between broiler chickens with extreme feed conversion capabilities were analysed in order to identify candidate bacteria that may influence growth performance. The jejunal microbiota was largely dominated by lactobacilli (over 99% of jejunal sequences) and showed no difference between the birds with high and low feed conversion ratios. The cecal microbial community displayed higher diversity, and 24 unclassified bacterial species were found to be significantly (<0.05) differentially abundant between high and low performing birds. Such differentially abundant bacteria represent target populations that could potentially be modified with prebiotics and probiotics in order to improve animal growth performance.