Bile Salt Binding Research Articles

Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3.

Multidrug resistance protein 3 (MRP3) is an ATP-dependent transporter of 17beta-estradiol 17beta(d-glucuronide) (E(2)17betaG), leukotriene C(4) (LTC(4)), methotrexate, and the bile salts taurocholate and glycocholate. In the present study, the role of a highly conserved Trp residue at position 1242 on MRP3 transport function was examined by expressing wild-type MRP3 and Ala-, Cys-, Phe-, Tyr-, and Pro-substituted mutants in human embryonic kidney 293T cells. Four MRP3-Trp(1242) mutants showed significantly increased E(2)17betaG uptake, whereas transport by the Pro mutant was undetectable. Similarly, the Pro mutant did not transport LTC(4). By comparison, LTC(4) transport by the Ala, Cys, Phe, and Tyr mutants was reduced by approximately 35%. The Ala, Cys, Phe, and Tyr mutants all showed greatly reduced methotrexate and leucovorin transport, except the Tyr mutant, which transported leucovorin at levels comparable with wild-type MRP3. In contrast, the MRP3-Trp(1242) substitutions did not significantly affect taurocholate transport or taurocholate and glycocholate inhibition of E(2)17betaG uptake. Thus Trp(1242) substitutions markedly alter the substrate specificity of MRP3 but leave bile salt binding and transport intact.

The endothermic nature of the binding of anionic bile salts to a cationic adsorbent

The binding of a hydrophobic bile salt, sodium chenodeoxycholate, by a polyacrylamide with N,N,N-trimethylammonium dodecyl chloride (QPDA12) pendant groups was studied to evaluate the thermodynamic parameters associated with the binding. The Langmuir equation was used in interpreting the data obtained from HPLC measurements. An increase in binding affinity was observed with increasing temperature indicating that the driving force for binding is derived from an increase in entropy (ΔS° = 253 J mol–1K–1) despite the positive, or unfavourable, enthalpy change (ΔH° = 53 kJ mol–1). The positive thermodynamic parameters associated with the binding suggest that the disruption of hydrophobic and (or) ionic hydration associated with the bile salts and cationic polymer is the driving force for the binding.Key words: bile salts, endothermic binding, polymer sorbents, solvent randomization.

Amphiphilic block copolymers as bile acid sorbents: 2. Polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride): self-assembly and application to serum cholesterol reduction.

This paper presents morphological studies and preliminary bile salt binding properties of the new amphiphilic diblock copolymer polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride) (PS-b-PTMEACl)(1) (see Figure 1), a derivative of PS-b-poly(tert-butylacrylate) (PS-b-PtBuA). In an aqueous environment, PS-b-PTMEACl forms simple spheres (approximately 20 nm diameter), large compound micelles (>100 nm diameter), and larger, more complex architectures as presented and discussed below. The colloidal stability with respect to sodium chloride and as a function of particle concentration is also considered. Finally, PS-b-PTMEACl aggregates were prepared and tested as an alternative to the commercially available bile salt sequestrant resins that target coronary heart disease due to elevated cholesterol levels. Electron micrographs were employed to visualize the colloid-based polyelectrolyteminus signbiosurfactant interaction and chromatographic separation analytical methods were used to quantify the sequestration. The results indicate that although at this preliminary stage they require laborious preparation, self-assembled aggregates may present an interesting alternative to the clinically used bile salt sequestrants.

Binding of bile salts to soluble and insoluble dietary fibers of seaweeds

Soluble and insoluble dietary fibers are recognized to have different physiological responses. Therefore, we worked out a new method for binding of bile salts to soluble and insoluble dietary fibers in seaweeds. Dried and pulverized Porphyra yezoensis, Undaria pinnatifida, Laminaria japonica, and Hizikia fusiformis were suspended in cholate, chenodeoxycholate, or deoxycholate solution, and incubated. After centrifugation and then precipitation with ethanol according to the analytical method of soluble and insoluble dietary fibers, the concentration of cholates not binding to both dietary fibers was measured by enzymatic colorimetry. The percentage of bile salts bound to insoluble dietary fiber was higher than that to soluble dietary fiber, because the amount of insoluble dietary fiber was higher in seaweeds. The binding ability of soluble dietary fiber seemed to be greater than that of insoluble dietary fiber. Adsorption of cholate to seaweeds was lower in comparison with chenodeoxycholate and deoxycholate. Thus, seaweed dietary fibers have a different capacity to adsorb cholates.

Bile Acid Sequestrants Based on Cationic Dextran Hydrogel Microspheres. 2. Influence of the Length of Alkyl Substituents at the Amino Groups of the Sorbents on the Sorption of Bile Salts

Cationic dextran hydrogel microspheres with pendant quaternary ammonium groups having alkyl substituents (C2–C12) at quaternary nitrogen were synthesized. The in vitro sorption of sodium salts of four bile acids (glycocholic, cholic, taurocholic, and deoxycholic acids) with these hydrogels was studied as a function of substituent alkyl chain length and bile acid hydrophobicity. Sorption experiments were performed in phosphate buffer solutions (pH 7.4) containing one bile salt (individual sorption) or mixtures of several bile salts (competitive sorption). Parameters for individual sorption were calculated taking into consideration the stoichiometric and cooperative binding of bile salts to oppositely charged polymer hydrogels. The results show that the increase in the length of the alkyl chain of the substituent leads to an increase in both ionization constant K0 and overall stability constant of binding K, but decreases the cooperativity parameter u. The competitive sorption studies indicate that the hydrogels display a good affinity for both dihydroxylic and trihydroxylic bile salts. The molar ratio of maximum amounts bound for the two types of bile acid is 2 to 1, which is much lower than those reported for other cationic polymers recommended as bile acid sequestrants. The binding constants for the sorption of bile salts by some dextran hydrogels are 20–30 times higher than those obtained for cholestyramine under similar sorption conditions. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:681–689, 2001

Bile Salt Anion Sorption by Polymeric Resins: Comparison of a Functionalized Polyacrylamide Resin with Cholestyramine

Cholestyramine and a cross-linked polyacrylamide resin with lateral alkyl quaternary ammonium groups (QPDA12) were used to study their ability to bind several bile salt anions (including the cholate, glycocholate, taurocholate, and chenodeoxycholate), individually and competitively, from phosphate buffer solutions at room temperature. The latter resin showed high affinities for all the bile salt anions examined, while cholestyramine exhibited a high affinity only for the more hydrophobic chenodeoxycholate. However, for the binding with cholestyramine, cooperative effects were more pronounced, leading to the enhancement of sorption at higher concentrations. The Langmuir equation and its modified versions were used in the interpretation of both individual and competitive binding of bile salts. The data from competitive binding studies indicated that the presence of the tightly bound chenodeoxycholate did not significantly diminish the ability of QPDA12 resin to bind cholate. However, for cholestyramine, the sorption of chenodeoxycholate increased the relative binding affinity for the more hydrophilic cholate, revealing a novel “cooperative” effect involving different bile salt anions. The latter results suggest that the observed higher affinity of QPDA12 is brought about predominantly through the hydrophobic interactions with the pendant alkyl groups rather than with the resin backbone.

Importance of Arginines 63 and 423 in Modulating the Bile Salt-dependent and Bile Salt-independent Hydrolytic Activities of Rat Carboxyl Ester Lipase

Previous studies using chemical modification approach have shown the importance of arginine residues in bile salt activation of carboxyl ester lipase (CEL) activity. However, the x-ray crystal structure of CEL failed to show the involvement of arginine residues in CEL-bile salt interaction. The current study used a site-specific mutagenesis approach to determine the role of arginine residues 63 and 423 in bile salt-dependent and bile salt-independent hydrolytic activities of rat CEL. Mutations of Arg(63) to Ala(63) (R63A) and Arg(423) to Gly(423) (R423G) resulted in enzymes with increased bile salt-independent hydrolytic activity against lysophosphatidylcholine, having 6.5- and 2-fold higher k(cat) values, respectively, in comparison to wild type CEL. In contrast, the R63A and R423A mutant enzymes displayed 5- and 11-fold decreases in k(cat), in comparison with wild type CEL, for bile salt-dependent cholesteryl ester hydrolysis. Although taurocholate induced similar changes in circular dichroism spectra for wild type, R63A, and R423G proteins, this bile salt was less efficient in protecting the mutant enzymes against thermal inactivation in comparison with control CEL. Lipid binding studies revealed less R63A and R423G mutant CEL were bound to 1,2-diolein monolayer at saturation compared with wild type CEL. These results, along with computer modeling of the CEL protein, indicated that Arg(63) and Arg(423) are not involved directly with monomeric bile salt binding. However, these residues participate in micellar bile salt modulation of CEL enzymatic activity through intramolecular hydrogen bonding with the C-terminal domain. These residues are also important, probably through similar intramolecular hydrogen bond formation, in stabilizing the enzyme in solution and at the lipid-water interface.

Protective effect of the bile salt hydrolase-active Lactobacillus reuteri against bile salt cytotoxicity.

Bacterial bile salt hydrolysis is considered a risk factor for the development of colon cancer because of the risk of forming harmful secondary bile salts after an initial deconjugation step. In this study, the influence of enhanced bacterial bile salt transformation by the bile salt hydrolase-active Lactobacillus reuteri was studied in batch culture using the microbial suspension of the Simulator of the Human Intestinal Microbial Ecosystem; (SHIME), which was supplemented with oxgall at 5 g/l or 30 g/l. Changes in the fermentative capacity of the microbial ecosystem and the (geno)toxic properties of the SHIME supernatants were investigated. Increasing concentrations of oxgall inhibited the fermentation. Transient cell toxicity was observed for samples supplemented with 5 g oxgall/l, while samples with 30 g oxgall/l exhibited toxicity. The results of the haemolysis test suggest that the detrimental effects were probably due to the membrane-damaging effects of bile salts. In all cases, the adverse effects could be counteracted by the addition of 7.5 +/- 0.5 log10 CFU L. reuteri/ml. Plausible mechanisms for the protective properties of L. reuteri could involve a precipitation of the deconjugated bile salts and a physical binding of bile salts by the bacterium, thereby making the harmful bile salts less bioavailable.

Cholestyramine Protection against Ochratoxin A Toxicity: Role of Ochratoxin A Sorption by the Resin and Bile Acid Enterohepatic Circulation

We have shown that the addition of cholestyramine (CHA, a resin known to bind bile salts in the gastrointestinal tract) to ochratoxin A (OTA)-contaminated rat diets reduced plasma levels of the toxin and prevented OTA-induced nephrotoxicity. To elucidate the mechanism of action of CHA, we carried out in vitro experiments to determine whether the resin may bind the toxin. For comparative purposes, binding of bile salts to the resin was also examined. Results showed that CHA binds both OTA and bile salts (taurodeoxycholate [TDC] and taurocholate [TCA]). Also, CHA showed greater affinity for OTA and TDC than for TCA. At 1mM concentration, 96% of OTA and 80% of TDC were bound to the resin, while for TCA binding was only 50%. However, saturation of the resin was reached at higher levels with bile acids compared to OTA (3.67 mmol/g resin for TCA and 3.71 mmol/g resin for TDC versus 2.85 mmol/g resin for OTA). To characterize the nature of the binding of the toxin to CHA, NaCl (0 to 200 mM) was added to a fixed amount of OTA or bile acids. As expected, TCA absorption was decreased by the addition of NaCl (<50 mM), indicating electrostatic binding. However, OTA and TDC sorption was decreased only at high concentrations of NaCl (>150 mM), suggesting a stronger binding to the resin than that shown with TCA. Sequential competitive studies demonstrated that CHA binds more OTA than TCA. The results of the in vivo study show the role of bile salts in OTA absorption. The toxin's plasma levels at 1 and 3 h after a single oral dose of OTA were significantly decreased in bile salt–depleted rats compared to the control. Thus, the alteration of the bile salt biliary pool and OTA enterohepatic circulation may be an additional mechanism of action of the resin against mycotoxin toxicity.

Cholesterol Enhances Membrane-Damaging Properties of Model Bile by Increasing the Intervesicular-Intermixed Micellar Concentration of Hydrophobic Bile Salts

Bile salts are potent detergents that, at concentrations attained in bile and intestine, can disrupt cell membranes. Hepatic secretion of vesicles containing lecithin and cholesterol appears to be critical in preventing bile salt damage to hepatobiliary epithelia. We hypothesize that the protective effect of biliary lipids results from lowering of the bile salt intervesicular intermixed micellar bile salt concentration (IMMC) to which epithelial membranes are exposed. We further hypothesize that increases in biliary cholesterol, by reducing association of bile salts with vesicles and mixed micelles, may increase bile toxicity by raising the bile salt IMMC.Method.Large unilamellar lecithin vesicles (100 nm) with varying cholesterol:lecithin molar ratios (C:L) of 0, 0.5, and 1 were added to taurochenodeoxycholate (TCDCA), taurocholate (TCA), or taurodeoxycholate (TDCA) in Tris-buffered saline, pH 7.4. Human erythrocyte ghosts (model target membrane), prepared by osmotic hemolysis and resealed with [14C]inulin trapped inside, were added and incubated at 37°C for 30 min and 4 h. Plasma membrane disruption was quantified by [14C]inulin release and bile salt IMMC was determined by ultrafiltration.Results.Membrane disruption started at a concentration of 0.5 mM for TDCA, 1 mM for TCDCA, and 2 mM for TCA and was complete within 4 h at concentrations of 1, 2, and 4 mM, respectively. Addition of 2 mM lecithin to 2 mM TDCA, 4 mM TCDCA, or 5 mM TCA reduced or eliminated membrane leakage and lowered the IMMC. For TDCA and TCDCA, the protective effect of vesicles was entirely attributable to reduction in IMMC; in contrast for TCA, the protective effect exceeded that which would have been expected based solely on reduction of the IMMC. Inclusion of cholesterol attenuated the binding of bile salts to vesicles and raised the IMMC, thereby reducing the protective effect of lecithin over the time course of these studies. Although there was loss of phospholipid and cholesterol from the erythrocyte membranes on addition of bile acids even in the presence of vesicles, the ratio of cholesterol to phospholipid in the erythrocyte membrane did not change.Conclusion.Lecithin protects against membrane disruption by hydrophobic bile salts by lowering the IMMC. Cholesterol added to lecithin raises the bile salt IMMC and reduces or eliminates this protective effect. This mechanism of potentiation of bile salt toxicity by cholesterol may be an important contributor to the pathogenesis of gallbladder disease in cholesterol cholelithiasis.

Molecular cloning of the bile salt-dependent lipase of ferret lactating mammary gland: an overview of functional residues

Ferret lactating mammary gland bile salt-dependent lipase (BSDL, EC 3.1.1.-) has been cloned by RT-PCR. The open reading frame consists of 1869 nucleotides which encode 623 amino acids of the functional enzyme. When compared to other species, the greatest homology is observed between residues 1 and 484, with little or no homology at the C-terminal end where seven repeated segments of similar sequence are located. Ferret mammary gland BSDL retains residues involved in the active site and the tentative heparin binding site at similar positions in comparison to other milk or pancreatic BSDL. Other important items, such as binding peptide to chaperone molecular, phosphorylation site(s) or bile salt binding sites, were also tentatively located in well conserved regions of seven available BSDL sequences.

In vitro binding of bile salt to rhubarb stalk powder

A new fiber source was developed by blanching, drying and grinding the fresh stalks of rhubarb, an underutilized and universal crop. The product was found to contain 74% total dietary fiber (66% insoluble and 8% soluble dietary fiber), on dry weight basis. Preliminary studies involving experimental animals and hypercholesterolemic subjects revealed that this fiber source is potentially hypolipidemic, though the underlying mechanism still remains unknown. To investigate its mechanism of action, this study was undertaken to determine its potential to complex with bile salts in vitro. Comparison of various fiber sources showed that the rhubarb fiber had the maximum ability to bind taurocholate, and bound 11 and 2.5 fold more bile salt than cellulose and wheat bran respectively. The binding increased linearly with increasing rhubarb fiber concentration. Increasing the bile salt concentration also increased the binding in a dose dependent manner and showed a saturation at a concentration greater than 10mM. The rhubarb fiber had a binding capacity of 40 μmol of taurocholate per gram. Cholate competed taurocholate for binding indicating that conjugation of bile salt was not a mandatory requirement for the binding Based on the chemical composition of rhubarb stalk powder, the bile salt binding property appears to be due to a combination of factors present in this product such as its high fiber content (insoluble with pectin), hydrophilic nature and high ash content. The ability to bind bile salts might be responsible for its hypocholesterolemic action observed in experimental animals and humans.

Transbilayer movement of fully ionized taurine-conjugated bile salts depends upon bile salt concentration, hydrophobicity, and membrane cholesterol content.

Taurine-conjugated bile salts mediate rapid transmembrane flux of divalent cations, irrespective of whether bile salts and divalent cations are initially on the same or opposite side of the membrane. We therefore hypothesized that ionized bile salts can equilibrate between membrane hemileaflets. We quantitated bile salt binding to large unilamellar egg yolk phosphatidylcholine (EYPC) +/- cholesterol (Ch) vesicles under conditions in which one or both hemileaflets were initially exposed to bile salts. At unbound taurodeoxycholate (TDC) concentrations >0.2 mM, the dependence of binding on TDC concentration after 30 min was indistinguishable for vesicles prepared by either method and did not change from 30 minutes to 24 h. At unbound TDC concentrations <0.1 mM, the ratio of bound/free TDC to EYPC vesicles doubled over a single exponential time course. Equilibration times were greater for the more hydrophilic bile salts taurocholate and tauroursodeoxycholate, for EYPC/Ch vesicles, and at lower temperatures. For glycine-conjugated bile salts, time-dependent changes in binding did not occur, consistent with more rapid equilibration of the small fraction of the protonated form. We conclude that fully ionized conjugated bile salts translocate between lipid bilayer hemileaflets, in contrast to previous observations that equilibration of fully ionized unconjugated bile salts occurs at a negligible rate in small unilamellar vesicles. The rate of "flip-flop" increases with increases in intramembrane bile salt concentration and hydrophobicity but decreases with cholesterol content and lower temperature. We speculate that physiologically, even in the absence of a specific membrane transporter, bile salts can gain access to intracellular compartments and mediate increases in divalent cation flux that may underlie cytotoxicity.

The crystal structure of bovine bile salt activated lipase: insights into the bile salt activation mechanism.

The intestinally located pancreatic enzyme, bile salt activated lipase (BAL), possesses unique activities for digesting different kinds of lipids. It also differs from other lipases in a requirement of bile salts for activity. A structure-based explanation for these unique properties has not been reached so far due to the absence of a three-dimensional structure. The crystal structures of bovine BAL and its complex with taurocholate have been determined at 2.8 A resolution. The overall structure of BAL belongs to the alpha/beta hydrolase fold family. Two bile salt binding sites were found in each BAL molecule within the BAL-taurocholate complex structure. One of these sites is located close to a hairpin loop near the active site. Upon the binding of taurocholate, this loop becomes less mobile and assumes a different conformation. The other bile salt binding site is located remote from the active site. In both structures, BAL forms similar dimers with the active sites facing each other. Bile salts activate BAL by binding to a relatively short ten-residue loop near the active site, and stabilize the loop in an open conformation. Presumably, this conformational change leads to the formation of the substrate-binding site, as suggested from kinetic data. The BAL dimer observed in the crystal structure may also play a functional role under physiological conditions.

Molecular Assembly of Cholesterol-Bearing Poly(allylamine) for Binding Bile Salts in Water

Molecular Assembly of Cholesterol-Bearing Poly(allylamine) for Binding Bile Salts in Water

Adaptive regulation of hepatic bile salt transport: role of bile salt hydrophobicity and microtubule-dependent vesicular pathway

Background/Aims: The hepatic transport of bile salts can be regulated by changes in bile salt pool size and/or in the flux of bile salts through the liver. Prolonged bile salt pool depletion is associated with down-regulation of maximum taurocholate transport and decreased canalicular membrane specific bile salt binding sites. This study was undertaken to investigate; a) whether adaptive down-regulation of maximum hepatic bile salt transport occurs to the same extent for bile acids of different hydrophobicity; and b) the role of microtubule-dependent vesicular pathway in the adaptive changes of bile salt transport capacity. Methods: Male rats were subjected to 24-h or 48-h external biliary diversion to induce bile salt pool depletion. Basal bile flow, bile salt secretion and lipid secretion, maximum secretory rate of three bile salts of different hydrophobicity (tauroursodeoxycholate, taurocholate and taurochenodeoxycholate) and changes in the biliary excretion of two markers of the microtubule-dependent vesicular pathway (horseradish peroxidase and polyethyleneglycol molecular weight-900) were measured in control and bile salt-depleted rats. Taurocholate-stimulated horseradish peroxidase biliary excretion was also assessed in order to define whether the restoration of bile salt flux across the hepatocytes increased the excretion of this marker in bile salt-depleted rats. Results: The reduction in the maximum secretory rate of the three bile salts under study observed after prolonged biliary diversion was clearly related to their hydrophobicity, with greater reduction for taurochenodeoxycholate and smaller reduction for tauroursodeoxycholate, compared with taurocholate. The biliary excretion of vesicular transport markers was significantly reduced in bile salt-depleted rats. However, when stimulated by taurocholate, biliary excretion of horseradish peroxidase was similar to controls. Conclusions: The magnitude of the decrease of the hepatic bile salt maximum transport capacity seen after bile salt pool depletion is directly related to the hydrophobicity of the bile salt infused. A functionally depressed vesicular transport pathway appears to be also a contributing factor to this phenomenon.

The binding of cholesterol and bile salts to recombinant rat liver fatty acid-binding protein.

The physiological role of liver fatty acid-binding protein (L-FABP) has yet to be clarified. An important feature of this member of the family of intracellular lipid-binding proteins is the wide range of compounds that have been identified as potential physiological ligands. By using recombinant L-FABP, the binding of cholesterol, bile salts and their derivatives has been investigated under conditions that allow a direct comparison of the binding affinities of these ligands for fatty acids. The results demonstrate an inability of L-FABP to bind cholesterol, although the anionic derivative, cholesteryl sulphate, will bind under similar assay conditions. Of the bile salts examined, lithocholate and taurolithocholate sulphate showed the greatest binding to L-FABP. It is proposed that an important function of L-FABP is to bind certain physiological amphipathic anions, thus preventing the "free' concentrations of these compounds from exceeding their critical micelle concentration, which could result in cell damage.

Influence of phospholipid on bile salt binding to calcium hydroxyapatite and on the poisoning of nascent hydroxyapatite crystals.

Glycine-conjugated, dihydroxy bile salts inhibit calcium hydroxyapatite (HAP) formation by binding to and poisoning nascent crystal embryos. Their taurine-conjugated counterparts bind less well to hydroxyapatite and do not inhibit its formation; but more hydrophobic, synthetic analogs of the taurine conjugated bile salts are inhibitors of hydroxyapatite formation. Because hydrophobicity is an important determinant of the ability of bile salts to inhibit hydroxyapatite crystal growth, experiments were performed to study the effect of the physiologically important mixed micelles of bile salt and phospholipid. Taurodeoxycholate/phosphatidylcholine (10:1) mixed micelles bound to HAP at lower total lipid concentrations than did pure taurodeoxycholate. At low total lipid concentrations, phosphatidylcholine (PC) binding appeared to predominate, suggesting that PC had a higher affinity than did taurodeoxycholate (TDC) for the HAP surface. Although glycodeoxycholate (3 mM) significantly (> 95%) inhibited hydroxyapatite precipitation, higher concentrations of taurodeoxycholate, either alone or mixed with phosphatidylcholine, did not affect hydroxyapatite formation. These results suggest that biliary phospholipids do not modulate the ability of bile salts to inhibit hydroxyapatite crystal growth.

Bile acid measurements using a cholestyramine-coated TSM acoustic wave sensor.

A novel chemical sensor for bile acids is described. A 10-MHz piezoelectric crystal operating in the thickness-shear mode (TSM) is coated on one side with cholestyramine resin and mounted in a batch-mode sensor block which exposes the coating to solution. After sample injection, the binding process is observed in real time as a drop in frequency as the bile salt binds to the coating, reaching > 90% completion within 10 min with most of the binding occurring within the first minute. Linear calibration curves are generated with sensitivity increasing in the order cholate approximately glycocholate < taurocholate < < taurodeoxycholate. Detection limits in water are in the range 0.2-9 nmol and are better than those observed in phosphate buffer. A multistep regeneration protocol allows the coating to be reused more than 400 times over a period of several months. Precision for replicate injections is approximately 10% RSD and depends on the reproducibility of the regeneration and injection steps. In terms of the binding process, hydrophobic interactions are observed to be of importance in the ability of bile salts to displace other counterions. However, anions with greater charge density also appear to compete effectively for binding sites on the resin. In particular, at equimolar concentrations of citrate and bile salt, the trivalent citrate anion reduces the amount of bile salt binding by approximately 40%. This suggests that the efficiency of cholestyramine-based bile salt sequestering drugs used in the reduction of hypercholesterolemia may be improved by eliminating citric acid as an excipient and avoiding the use of fruit juices during ingestion.

Mathematical Model and Dimensional Analysis of Glycocholate Binding to Cholestyramine Resin: Implications for in Vivo Resin Performance

In large doses, cholestyramine resin lowers blood serum cholesterol by binding bile salts in the intestinal lumen and thus increases the fecal excretion of bile salts. In order to gain a better understanding of the low in vivo potency of cholestyramine, mathematical models estimating the amount of glycocholate bound per gram of cholestyramine and the free glycocholate concentration were derived and employ the capacity-corrected molar selectivity coefficient. Predictions of the quantity of glycocholate bound per gram of cholestyramine and of the free glycocholate concentration matched observed values (r2 = 0.993 and r2 = 0.998, respectively) over a wide range of conditions. Simulated binding studies indicated the relative importance of several biopharma-ceutical parameters for improved resin in vivo performance. Increasing resin selectivity of glycocholate over chloride has greatest therapeutic impact if bile salt sequestering is most important in the upper portion of the intestines. Furthermore, ion exchange phenomena was subjected to dimensional analysis and revealed the controlling factors as components of two dimensionless numbers, GC* and CI*. Placing physiologic limits on values of GC* and CI* suggests requisite selectivity properties of more potent bile acid sequestrants and dosing strategies to optimize current resin therapy.