Unconjugated Bile Salts Research Articles

Effect ofBacteroides melaninogenicus culture supernatant and deconjugated bile salt on lipid absorption

Lipid malabsorption is a common clinical manifestation of small bowel bacterial overgrowth. Its pathogenesis, however, remains controversial. Bacteroides melaninogenicus ssp. intermedius, an anaerobic bacterium, is commonly isolated from the upper bowel of patients with small intestinal bacterial overgrowth. The effects of a culture supernate of this organism and deoxycholate, an unconjugated bile salt, on intestinal oleic acid absorption were examined using a rat closed-loop model. The supernatant reduced the in vitro uptake of oleic acid by 19% (P< 0.001). Deoxycholate did not significantly reduce the lipid absorption. Combined supernate and deoxycholate did not have an additive effect on absorption of oleic acid. We conclude that anaerobic bacterial products may contribute to the malabsorption of lipid in the setting of bacterial overgrowth of the small bowel.

N-ethyl-tauroursodeoxycholic acid, a novel deconjugation-resistant bile salt analogue: Effects of acute feeding in the rat

The purpose of this study was to investigate the physicochemical/biological properties and the effects of acute administration of N-ethyl-tauroursodeoxycholic acid in bile-fistula rats. In vitro determination of high-performance liquid chromatography mobility, octanol/water partitioning, cholesterol solubilizing capacity, and sensitivity to enzyme deconjugation by bacteria and cholylglycine-hydroxylase were performed. In vivo determination of the following was also performed: (1) maximum secretory rate (SRmax) and choleretic/secretory properties during intravenous (IV) administration; (2) site/extent of absorption, effects on bile flow, lipid secretion, and biotransformations after intraduodenal infusion. N-ethyl-tauroursodeoxycholate has a lipophilicity slightly higher than tauroursodeoxycholate, close to taurocholate, and similar cholesterol solubilizing capacity. Deconjugation of N-ethyl-tauroursodeoxycholate was 3.4 ±2.1% after 72 hours, that of tauroursodeoxycholate was 100% after 24 hours. During IV infusion of 300 nmol/min/ 100 g, biliary secretion of N-ethyl-tauroursodeoxycholic and tauroursodeoxycholic acids averaged 185 ±76 (standard deviation) nmol/min/100 g and 221 ± 77 nmol/min/ 100 g (not significant). Increasing infusion rates caused progressive enhancement of bile flow and bile salt secretion until the SRmax was reached (1,305 ± 240 nmol/min/ 100 g for N-ethyl-tauroursodeoxycholic acid and 3,240 nmol/min/100 g for tauroursodeoxycholate). The two bile salts were similarly choleretic. IV feeding of N-ethyltauroursodeoxycholic promoted a greater lipid secretion than tauroursodeoxycholate. After intraduodenal feeding of 800 μmol, 38.8 ± 14.0% and 43.4 ± 12.4% of the two bile salts were recovered in bile. No unconjugated bile salts nor unusual metabolites were detected. Excluding the distal 30 cm of the ileum, only 2.3 ± 0.7% of N-ethyl-tauroursodeoxycholic and 4.0 ± 2.3% of tauroursodeoxycholic acid was absorbed. N-ethyl-tauroursodeoxycholic is a new, hydrophilic, deconjugation-resistant, synthetic bile salt, which, in acute studies in the rat, behaves as the natural analogue with respect to intestinal and hepatobiliary properties.

N -ethyl-tauroursodeoxycholic acid, a novel deconjugation-resistant bile salt analogue: Effects of acute feeding in the rat

The purpose of this study was to investigate the physicochemical/biological properties and the effects of acute administration of N-ethyl-tauroursodeoxycholic acid in bile-fistula rats. In vitro determination of high-performance liquid chromatography mobility, octanol/ water partitioning, cholesterol solubilizing capacity, and sensitivity to enzyme deconjugation by bacteria and cholylglycine-hydroxylase were performed. In vivo determination of the following was also performed: (1) maximum secretory rate (SRmax) and choleretic/secretory properties during intravenous (IV) administration; (2) site/ extent of absorption, effects on bile flow, lipid secretion, and biotransformations after intraduodenal infusion. N-ethyl-tauroursodeoxycholate has a lipophilicity slightly higher than tauroursodeoxycholate, close to taurocholate, and similar cholesterol solubilizing capacity. Deconjugation of N-ethyl-tauroursodeoxycholate was 3.4 +/- 2.1% after 72 hours, that of tauroursodeoxycholate was 100% after 24 hours. During IV infusion of 300 nmol/min/ 100g, biliary secretion of N-ethyl-tauroursodeoxycholic and tauroursodeoxycholic acids averaged 185 +/- 76 (standard deviation) nmol/min/100 g and 221 +/- 77 nmol/min/ 100 g (not significant). Increasing infusion rates caused progressive enhancement of bile flow and bile salt secretion until the SRmax was reached (1,305 +/- 240 nmol/min/ 100 g for N-ethyl-tauroursodeoxycholic acid and 3,240 nmol/min/100 g for tauroursodeoxycholate). The two bile salts were similarly choleretic. IV feeding of N-ethyl-tauroursodeoxycholic promoted a greater lipid secretion than tauroursodeoxycholate. After intraduodenal feeding of 800 mumol, 38.8 +/- 14.0% and 43.4 +/- 12.4% of the two bile salts were recovered in bile. No unconjugated bile salts nor unusual metabolites were detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of bile salt uptake into skate hepatocytes

The substrate specificity for the transporter that mediates the hepatic uptake of organic anions in freshly isolated hepatocytes of the elasmobranch little skate (Raja erinacea) was determined for bile salts and bile alcohols. The Na(+)-independent transport system exhibits a substrate specificity, which is different from the specificity of Na(+)-dependent bile salt transport in mammals. Unconjugated and conjugated di- and tri-hydroxylated bile salts inhibit uptake of cholyltaurine and cholate competitively. Inhibition is significantly greater with unconjugated as opposed to glycine- or taurine-conjugated bile salts. However, the number of hydroxyl groups in the steroid moiety of the bile salts has only minor influences on the inhibition by the unconjugated bile salts. Since the transport system seems to represent an archaic organic-anion transport system, other anions, such as dicarboxylates, amino acids and sulphate, were also tested, but had no inhibitory effect on bile salt uptake. To clarify whether bile alcohols, the physiological solutes in skate bile, share this transport system, cholyltaurine transport was studied after addition of 5 beta-cholestane-3 beta,5 alpha,6 beta-triol, 5 alpha-cholestan-3 beta-ol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. These bile alcohols inhibit cholyltaurine uptake non-competitively. In contrast, uptake of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, which is Na(+)-independent, is not inhibited by cholyltaurine. The findings further characterize a Na(+)-independent organic-anion transport system in skate liver cells, which is not shared by bile alcohols and has preference for unconjugated lipophilic bile salts.

Electrogenicity of Na-coupled bile salt transport in isolated rat hepatocytes.

The importance of membrane voltage in uptake of bile salts into hepatocytes is not known. Electrogenicity of the primary bile salt transport process, Na-bile salt cotransport, has been difficult to determine because the large K and Cl conductances of the sinusoidal membrane (GK and GCl, respectively) obscure any transport associated currents. In the present study hepatocytes were treated to reduce these membrane conductances and electrogenic entry of taurocholate and glycocholate was demonstrated. Intracellular voltage and resistance changes resulting from bile salt transport were measured in hepatocytes in which GK and GCl were blocked by impalement with Na acetate microelectrodes and external exposure to quinine (400 microM). This increased the cell input resistance from 153 +/- 17 to 230 +/- 17 M omega (n = 14, P < 0.001). Under these conditions, exposure to 100 microM of taurocholate or glycocholate produced Na-dependent depolarizations of 3.0 +/- 0.5 and 4.2 +/- 0.8 mV, respectively. These correspond to transport currents of 13.9 and 7.6 pA/cell, which are comparable to those predicted from known [3H]taurocholate uptake rates if one positive charge enters the cell with each bile salt molecule. Although uptake of these two bile salts was electrogenic, this was not the case for all bile salts. Na-dependent transport of taurodehydrocholate, which occurs at similar rates to that for taurocholate, produced no voltage change. The unconjugated bile salts cholate and ursodeoxycholate also produced no measurable voltage or resistance changes. In conclusion, Na-dependent uptake of taurocholate and glycocholate is electrogenic, whereas uptake of taurodehydrocholate, ursodeoxycholate, and cholate is predominantly electroneutral.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions.

The principles governing the in vitro solubility of the common natural conjugated and unconjugated bile acids and salts in relation to pH, micelle formation, and Ca2+ concentration are considered from a theoretical standpoint and then correlated first with experimental observations on model systems and second with the formation of precipitates containing bile acids in health and disease. In vitro, taurine-conjugated bile acids are soluble at strongly acidic pH; glycine-conjugated bile acids are poorly soluble at moderately acidic pH; and many of the common, natural unconjugated bile acids are insoluble at neutral pH. For both glycine-conjugated and unconjugated bile acids, solubility rises exponentially, with increasing pH, until the concentration of the anion reaches the critical micellization concentration (CMC) when micelle formation occurs and solubility becomes practically unlimited. In vivo, in health, conjugated bile acids are present in micellar form in the biliary and intestinal tract. Unconjugated bile acids formed in the large intestine remain at low monomeric concentrations because of the acidic pH of the proximal colon, binding to bacteria, and absorption across the intestinal mucosa. In diseases in which proximal small intestinal content is abnormally acidic, precipitation of glycine-conjugated bile acids (in protonated form) occurs. Increased bacterial formation of unconjugated bile acids occurs with stasis in the biliary tract and small intestine; in the intestine, unconjugated bile acids precipitate in the protonated form. If the precipitates aggregate, an enterolith may be formed. In vitro, the calcium salts of taurine conjugates are highly water soluble, whereas the calcium salts of glycine conjugates and unconjugated bile acids possess limited aqueous solubility that is strongly influenced by bile acid structure. Precipitation occurs extremely slowly from supersaturated solutions of glycine-conjugated bile acids because of metastability, whereas super-saturated solutions of unconjugated bile acids rapidly form precipitates of the calcium salt. In systems containing Ca2+ ions and unconjugated bile acids, pH is important, since it is the key determinant of the anion concentration. For bile acids with relatively soluble calcium salts (or with a low CMC), the concentration of the anion will reach the CMC and micelles will form, thus precluding formation of the insoluble calcium salt. For bile acids, with relatively insoluble calcium salts (or with a high CMC), the effect of increasing pH is to cause the anion to reach the solubility product of the calcium salt before reaching the CMC so that precipitation of the calcium salt occurs instead of micelle formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Intestinal taurine and the enterohepatic circulation of taurocholic acid in the cat.

Taurine’s most well defined role is the conjugation of bile salts in the liver. After synthesis from cholesterol, bile salts are conjugated with taurine and/or glycine before secretion into the bile canaliculi1. When taurine is limiting, most mammals have the ability to conjugate bile salts with glycine, exceptions being the dog2 and the cat3,4, which conjugate bile salts almost exclusively with taurine. The percentage of total bile salts conjugated with taurine is determined by both the hepatic taurine concentration and the affinity of the bile salt conjugase for glycine and taurine2,5-7. Taurine is the preferred substrate in most species with 90 percent taurine conjugation occurring in the rat when hepatic taurine and glycine concentrations are equal7. Hepatic taurine depletion in rats, caused by the infusion of cholic acid7 or by feeding guanidinoethanesulfonic acid5, results in a substantial increase in the amount of bile salts conjugated with glycine. In species that cannot conjugate bile salts with glycine (the dog and cat), hepatic taurine depletion leads instead to an increase in the proportion of unconjugated bile salts, the majority being free cholic acid2,3.

Conjugates of ursodeoxycholate protect against cytotoxicity of more hydrophobic bile salts: In vitro studies in rat hepatocytes and human erythrocytes

Intraduodenal infusion of hydrophobic bile salts to bile-fistula rats leads within hours to severe hepatocellular necrosis and cholestasis; simultaneous administration of conjugates of ursodeoxycholate, either intraduodenally or intravenously, reduces or prevents liver injury. To evaluate the short-term protective effects of ursodeoxycholate at the cellular level, we incubated primary monolayer cultures of adult rat hepatocytes or freshly isolated washed human erythrocytes for 1 to 240 min with varying defined concentrations of different bile salts in the presence or absence of ursodeoxycholate. Cytolysis was quantified by measuring the release into the medium of cytosolic lactate dehydrogenase (hepatocytes) or hemoglobin (erythrocytes). In both systems, cytolysis increased sigmoidally with increasing bile salt concentration, and the relative toxicity of different bile salts proceeded in the following order: tauroursodeoxycholate was less toxic than taurocholate, which was less toxic than taurodeoxycholate. Taurochenodeoxycholate was more toxic to erythrocytes than taurodeoxycholate; the two were equally toxic to rat hepatocytes. Unconjugated bile salts were more toxic than their conjugates. The addition of tauroursodeoxycholate to taurochenodeoxycholate or taurodeoxycholate led to time-dependent and concentration-dependent reduction or elimination of the toxicity of the more hydrophobic component. Protection was evident within minutes. With respect to hemolysis, at pH 8.5 glyco was less protective than tauroursodeoxycholate, and free ursodeoxycholate was only minimally protective. We conclude that the hepatocytotoxicity of hydrophobic bile salts at millimolar concentrations is markedly reduced in the presence of tauroursodeoxycholate. Conjugates of ursodeoxycholate also prevented disruption of erythrocytes by bile salts, suggesting that protection does not depend on liver-specific pathways of bile salt uptake, compartmentation, transport or metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescent derivatives of bile salts. II. Suitability of NBD-amino derivatives of bile salts for the study of biological transport.

Interaction of unconjugated and taurine-conjugated NBD-amino-dihydroxy-5 beta-cholan-24-oic acids bearing the fluorophor in the 3 alpha, 3 beta, 7 alpha, 7 beta, 12 alpha, or 12 beta position with albumin results in a small hypsochromic shift of the emission maximum and an increase in quantum yield, suggesting binding by hydrophobic interactions. The different unconjugated fluorescent bile salt derivatives are metabolized by intact rat liver in different ways. The unconjugated 3 beta-NBD-amino derivative is completely transformed to its taurine conjugate and secreted as such, whereas the 3 alpha-NBD-amino derivative is completely transformed to a polar fluorescent compound not identical with its taurine conjugate. The unconjugated 7 alpha- and 7 beta-NBD-amino derivatives are only partially conjugated with taurine and mainly secreted in unmetabolized form. The unconjugated 12 alpha- and 12 beta-NBD-amino derivatives are not at all transformed to their taurine conjugates, but are partially metabolized to unidentified compounds. They are predominantly secreted as the unmetabolized compounds. In contrast to the unconjugated derivatives, all taurine-conjugated fluorescent bile salt derivatives are secreted into bile unmetabolized. With the exception of the 3 alpha-compound, all synthesized taurine-conjugated fluorescent derivatives interfere with the secretion of cholyltaurine. Differential photoaffinity labeling studies using (7,7-azo-3 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2'-[2'-3H(N)]aminoethanesulfonate as a photolabile derivative revealed that in liver cells all fluorescent bile salt derivatives interact with the same polypeptides as the physiological bile salts. The hepatobiliary transport of taurine-conjugated NBD-amino bile salt derivatives is, due to hydrophobic interactions, accompanied by an increase in fluorescence intensity which is favorable for the study of biological bile salt transport by fluorescence microscopy.

Sensitivity ofHelicobacter pylori to different bile salts

The sensitivity of Helicobacter pylori to different unconjugated or conjugated bile acids both on BHI blood agar and in BHI broth supplemented with starch, horse serum or egg yolk was studied. Bile salts were more toxic in the medium containing starch than in the media containing horse serum or egg yolk, and unconjugated bile salts were more toxic than the conjugated salts. Deoxycholic acid was the most toxic of the bile acids studied. Ox bile was bacteriostatic at the 2% level. Bile Salts 3 mixture was bactericidal at a concentration of 0.25 mmol.

Bile salts stimulate glycoprotein release by guinea pig gallbladderin vitro

Alterations in the composition of bile during cholesterol gallstone formation appear to be responsible for increased release of gallbladder mucin, a potent cholesterol nucleating agent. We investigated the effects of bile salts on release of radiolabeled glycoproteins by explants of guinea pig gallbladder in organ culture. Bile salts, in concentrations of 1 to 5 mmol/L, caused a dose-dependent release of [3H]-glycoproteins with a range of potencies in this order: chenodeoxycholate greater than deoxycholate much greater than cholate greater than ursodeoxycholate = control. Chenodeoxycholate and deoxycholate were significantly more potent than cholate (p less than 0.0001). Unconjugated and taurine-conjugated bile salts were of similar potency. Bile salts also caused increased release of glycoproteins from explants of guinea pig gastric antrum and colon. The bile salts released after bile salt exposure included mucin and lower molecular weight glycoproteins from the gallbladder. Release of glycoproteins in response to bile salts was not inhibited by indomethacin, atropine or propranolol, nor was it dependent on extracellular calcium or microtubules. Glycoprotein release in response to bile salts was associated with membrane damage as indicated by a dose-dependent leakage of the cytoplasmic enzyme lactate dehydrogenase, although light microscopy did not reveal structural damage to epithelial cells. We conclude that hydrophobic bile salts stimulate gallbladder glycoprotein release in vitro by a detergent effect on the plasma membrane rather than by a receptor-mediated secretory pathway.

Quantitative estimation of the hydrophilic-hydrophobic balance of mixed bile salt solutions.

This paper describes the derivation of a bile salt monomeric hydrophobicity index that quantitatively defines the composite hydrophilic-hydrophobic balance of a mixture of bile salts. The index is based on the logarithms of bile salt capacity factors determined using reversed phase high performance liquid chromatography (HPLC) (stationary phase octadecyl silane; mobile phase methanol-water 70:30 w/w, ionic strength 0.15). It has been standardized arbitrarily to set indices of taurocholate and taurolithocholate to 0 and 1, respectively. Indices of tauroursodeoxycholate, taurohyodeoxycholate, taurochenodeoxycholate, and taurodeoxycholate were found to be -0.47, -0.35, +0.46, and +0.59, respectively. Whereas capacity factors and hydrophobicity indices of taurine-conjugated bile salts were constant for pH 2.8-9.0, the hydrophilic-hydrophobic balance of glycine-conjugated and unconjugated bile salts was strongly influenced by pH. At alkaline pH (greater than 8.5), hydrophobicity indices of fully ionized unconjugated (n = 4) and glycine-conjugated (n = 6) bile salts differed by only 0.14 +/- 0.02 and 0.05 +/- 0.01, respectively, from those of the corresponding taurine conjugates. At acid pH (less than 3.5) the hydrophobicity indices of four unconjugated bile acids (protonated form) exceeded those of the corresponding salts (ionized form) by 0.76 +/- 0.04; indices of six glycine-conjugated bile acids exceeded those of the corresponding salts by only 0.26 +/- 0.03. Capacity factors of the salt forms of cholate and its conjugates increased dramatically with increasing ionic strength of the mobile phase; retention of the protonated forms (cholic and glycocholic acids) was only minimally influenced by ionic strength. Thus the difference in hydrophilic-hydrophobic balance between a bile acid and its corresponding salt decreases with increasing ionic strength. Examples are given of calculation of hydrophobicity indices for biliary bile salts (fully ionized) from four species under conditions of intact enterohepatic circulation. Mean values, from least to most hydrophobic, were: rat (-0.31) less than dog (0.11) less than hamster (0.22) less than human (0.32). This study provides a rational basis for calculating the hydrophilic-hydrophobic balance of mixed bile salt solutions over a broad range of pH.

Reversal of pigment gallstone disease in a canine model.

Unlike dietary-induced cholesterol gallstones, which may disappear spontaneously when the lithogenic diet is withdrawn, little is known about the natural history of pigment gallstones. We examined whether pigment gallstone disease, which can be uniformly induced in the dog by six weeks of a methionine-deficient diet, can be reversed by return to normal diet. As previously reported, all dogs develop pigment gallstones as well as significant increases in biliary total calcium, free ionized calcium, and cholesterol concentrations after six weeks of a lithogenic diet. These changes are accompanied by a significant increase in the concentration of unconjugated bile salts in bile. In addition, histologic changes in the gallbladder wall occur that are consistent with a moderate degree of chronic cholecystitis. This study clearly demonstrates that return to a normal diet for six weeks allows bile composition to normalize, gallstones to disappear in 50% of dogs, and gallbladder histologic changes to return toward normal. Thus, it would appear that pigment gallstone disease in this model may be reversible, at least early during its course. Although the relevance of these findings to pigment gallstones in humans must be established, the potential for nonoperative treatment of pigment gallstones should not be discounted.

Effect of infusion of bile salts into the mesenteric artery in situ on jejunal mucosal transport function in dogs.

We studied the effects of increased circulating levels of bile salts on jejunal mucosal function in dogs. In situ luminal perfusion of a 30-cm proximal jejunal segment was performed while deoxycholate, cholate, taurodeoxycholate, or taurocholate solutions were directly added to the mesenteric arterial supply, reaching the intestinal wall at successive concentrations of 5, 8, 12, and 22 mumol/L. The transport rates of water, sodium, glucose, fructose, glycine, and lysine were measured. The mucosa of the experimental loop was assayed for ATPase activity, and examined by light and electron microscopy. Deoxycholate at 8 microM in the blood supply of the perfused jejunal segment was associated with a significant (p less than 0.02) reduction in the absorption rates of water, sodium, glucose, and glycine, and inhibition of mucosal Na+, K+-ATPase. The absorption of fructose and lysine, and brush border enzyme activities, were not affected. Cholate had a similar effect at 12 microM. There were no obvious histological alterations, but electron microscopy showed swelling of mitochondria in the enterocytes. The reduction in mucosal transport, the inhibition of mucosal Na+, K+-ATPase, and the mitochondrial swelling were reversed after discontinuation of the bile salt infusion. The taurine conjugates at 22 microM depressed transport of water and sodium only, and did not inhibit Na+, K+-ATPase. Our study indicates that increased circulating concentrations of unconjugated bile salts, particularly deoxycholate, may impair Na+, K+-ATPase-related jejunal mucosal function.

Effect of Infusion of Bile Salts into the Mesenteric Artery in Situ on Jejunal Mucosal Transport Function in Dogs

Summary:We studied the effects of increased circulating levels of bile salts on jejunal mucosal function in dogs. In situ luminal perfusion of a 30‐cm proximal jejunal segment was performed while deoxycholate, cholate, tauro‐deoxycholate, or taurocholate solutions were directly added to the mesenteric arterial supply, reaching the intestinal wall at successive concentrations of 5, 8, 12, and 22 μmol/L. The transport rates of water, sodium, glucose, fructose, glycine, and lysine were measured. The mucosa of the experimental loop was assayed for ATPase activity, and examined by light and electron microscopy. Deoxycholate at 8 μM in the blood supply of the perfused jejunal segment was associated with a significant (p &lt; 0.02) reduction in the absorption rates of water, sodium, glucose, and glycine, and inhibition of mucosal Na+,K+ ‐ATPase. The absorption of fructose and lysine, and brush border enzyme activities, were not affected. Cholate had a similar effect at 12 μM. There were no obvious histological alterations, but electron microscopy showed swelling of mitochondria in the enterocytes. The reduction in mucosal transport, the inhibition of mucosal Na+,K + ‐ATPase, and the mitochondrial swelling were reversed after discontinuation of the bile salt infusion. The taurine conjugates at 22 μM depressed transport of water and sodium only, and did not inhibit Na, K + ‐ATPase. Our study indicates that increased circulating concentrations of unconjugated bile salts, particularly deoxycholate, may impair Na+, K + ‐ATPase‐related jejunal mucosal function.

Study of biologically relevant physical-chemical properties of bile salts by reverse-phase liquid chromatography

The study of chromatographic behaviour of bile salts with reverse-phase HPLC columns (C18 and C8) and buffered water — methanol eluent systems indicated that the octanol-water partition coefficient and the micellar cholesterol-solubilizing capacity are linearly correlated with the chromatographic mobilities if eluents with pH>7 and 0.154 M saline concentration are used (correlation coefficients 0.98 and 0.86 respectively). The critical micellar concentration was poorly correlated with the chromatographic mobility under the various conditions tested. The pKa values of bile salts were determined by the changes of mobility with the pH of eluent. Values in the range 4.92–5.06 and 4.27–4.47 were found respectively for unconjugated and glycineconjugated bile salt, in agreement with previous estimates. Values in the range 0.74–1.02 represent the first direct evaluation for taurine-conjugates.

Effect of albumin on the solubility of cholesterol in bile

Despite the fact that a considerable amount of albumin is present in bile, little is known about the effect of albumin on micellar solubility of cholesterol. The effect of albumin on solubility of cholesterol in various micellar bile salt solutions was studied using Millipore filtration after equilibration. In addition, partitioning of cholesterol from micellar solution was studied using a polyethylene disc method. Decrease of the solubility of cholesterol by the presence of albumin was observed only in unconjugated bile salt solution. The lowering effect of albumin on the cholesterol solubility was found to be proportional to the hydrophobicity of bile salt. In contrast, albumin had almost no effect on cholesterol solubility, either in conjugated bile salt solution or in micellar bile salt solution containing phosphatidylcholine. Addition of albumin enhanced the partitioning of cholesterol out of the micelles in sodium chenodeoxycholate solution as a result of decreased micellar solubility and increased the aqueous solubility of cholesterol in the presence of albumin. Therefore, conjugated bile salt and phosphatidylcholine exert a buffering action on the albumin-induced adverse effect on cholesterol solubility, thus stabilising bile against inadvertent precipitation of cholesterol.

Giardia lamblia: The role of conjugated and unconjugated bile salts in killing by human milk

Killing of Giardia lamblia trophozoites by nonimmune human milk in vitro is dependent upon the presence of cholate which activates the milk bile salt-stimulated lipase to cleave fatty acids from milk triglycerides. In the present studies, conjugated bile salts, which predominate in vivo, displayed striking differences from unconjugated bile salts in ability to support killing by milk. Human milk killed >99% of the parasites in the presence of cholate, but not glycocholate or taurocholate. In contrast, after brief sonication which disrupts milk fat globules, milk killed G. lamblia after addition of either conjugated or unconjugated bile salts. Whereas cholate stimulated milk lipase to cleave triglycerides of either unsonicated or sonicated human milk, glycocholate or taurocholate stimulated lipolysis only in sonicated milk. Since the concentration of bile salts in the small intestine fluctuates, the effect of this variable on killing was examined. Each bile salt at and above its critical micellar concentration increased Giardia survival of human milk probably because it sequestered released fatty acids in micelles. This partial protection could be overcome by increasing the milk concentration. Human hepatic and gall bladder bile and artificial bile also activated human milk to kill at low concentrations but partly protected the parasite at higher concentrations. These studies show that conjugated bile salts can activate the bile salt-stimulated lipase of sonicated human milk to release fatty acids; and kill G. lamblia. Conversely, bile salts in concentrations above their critical micellar concentration sequester fatty acids and interfere with killing. Thus, nonimmune host secretions such as milk and bile may affect the course of infection by G. lamblia.

The enterohepatic circulation of methotrexate in vivo: inhibition by bile salt.

We examined the enterohepatic circulation of methotrexate (MTX) in the rat in vivo and determined the effect of the unconjugated bile salt, cholate, on the process. MTX (70 mg/kg body weight) was administered i. v. and the bile salt (1 mM) was delivered through intestinal perfusion. In the control group 38.43% +/- 4% of the administered dose of MTX appeared in bile 2 h after administration of the drug. In the bile salt-treated group 21.4% +/- 3.7% of the administered doses of MTX appeared in bile, which was significantly lower (P less than 0.01) than the proportion in the control group. The liver content of MTX was depressed by 23% in the bile salt-treated group compared with the control group. This study demonstrates, in vivo, the important role that the enterohepatic circulation plays in exposing the small intestine to toxic levels of MTX and shows that the unconjugated bile salt, cholate, inhibits the process.

Binding of bile salts to fibre-enriched wheat fibre.

A commercial product of fibre-enriched wheat fibre (Fiberform R) was tested for its binding of bile salts in vitro. The wheat fibre preparation was standardized and through enzymatic digestion of protein and starch contained 78 per cent fibre (w/w). Fibre-enriched wheat fibre bound with high capacity both conjugated and unconjugated bile salts. Binding was saturable, reversible and showed no specificity towards tauro- or glycine-conjugated bile salts. Binding was rapid, dependent on pH, was enhanced by the presence of high salt concentrations and partially inhibited by 6 M urea. This indicated that binding was a combination of hydrophobic and hydrophilic interactions.