Bile salt micelle can sustain more cholesterol in the intermicellar aqueous phase than the maximal aqueous solubility

The intermicellar aqueous phase in equilibrium with micelle plays an important role in the uptake of sterol. To test the hypothesis whether cholesterol concentration in the intermicellar aqueous phase of a micellar solution is similar to its maximal aqueous solubility, cholesterol concentration in the intermicellar aqueous phase of a bile salt-cholesterol solution and maximal aqueous cholesterol solubility were quantitatively determined by capillary gas-liquid chromatography after filtration. Cholesterol concentration in the intermicellar aqueous phase increased linearly with cholesterol concentration in the micellar solution and reached 1.3 microM at its micellar solubility limit, while the maximal aqueous solubility of cholesterol was (1.2-1.4) x 10(-8) M. The intermicellar monomer concentration of taurocholate was 5.8 mM in which 26 x 10(-8) M cholesterol was solubilized. The results indicate the presence of a cholesterol concentration in the intermicellar aqueous phase that is significantly higher than its maximal aqueous solubility, which can be ascribed primarily to the presence of an intermicellar concentration of bile salt.     

Interaction of bile salt monomer and cholesterol in the aqueous phase

The purpose of the present study was to evaluate the possible interaction of bile salt monomer and cholesterol in the intermicellar aqueous phase. Cholesterol and taurocholate monomer concentrations in the intermicellar aqueous phase were determined using 0-20 mM taurocholate solutions saturated with cholesterol. Maximal solubilities of cholesterol in aqueous solutions having various concentrations of taurocholate, especially below its intermicellar monomer concentration (critical micellar concentration), were determined and compared with the intermicellar cholesterol concentration. The intermicellar monomer concentration of taurocholate was constant (6 mM) and independent of taurocholate concentrations. The cholesterol concentration in the intermicellar aqueous phase gradually increased, depending upon taurocholate concentrations, and became constant (1,3 microM) above 10 mM taurocholate. The solubility of cholesterol increased linearly with the taurocholate concentration even below the critical micellar concentration, and was 0.3 microM at 6 mM taurocholate, which was approx. 20-times higher than the aqueous solubility of cholesterol, but a fifth of the maximal intermicellar cholesterol concentration. The results indicate that the higher cholesterol concentration in the intermicellar aqueous phase compared to its aqueous solubility can be primarily ascribed to the interaction of cholesterol with bile salt monomers possibly forming bile salt-cholesterol dimers, and partly to the sustaining forces induced by numerous micelles.     

Studies on the mechanism of cholesterol uptake and on the effects of bile salts on this uptake by brush-border membranes isolated from rabbit small intestine

The effect of bile salts and other surfactants on the rate of incorporation of cholesterol into isolated brush-border membranes was tested. At constant cholesterol concentration, a stimulatory effect of taurocholate was noticed which increased as the bile salt concentration was raised to 20 mM. Taurodeoxycholate was as effective as taurocholate at concentrations of up to 5 mM and inhibited at higher concentrations. Glycocholate was only moderately stimulatory whereas cholate was nearly as effective as taurocholate at concentrations above 5 mM. Other surfactants such as sodium lauryl sulfate and Triton X-100 were very inhibitory at all concentrations tried whereas cetyltrimethyl ammonium chloride was stimulatory only at a very low range of concentrations. These micellizing agents all caused some disruption of the membranes and the greater effectiveness of taurocholate in stimulating sterol uptake was partly relatable to the weaker membrane solubilizing action of this bile salt. Preincubation of membranes with 20 mM taurocholate followed by washing and exposure to cholesterol-containing lipid suspensions lacking bile salt, did not enhance the incorporation of the sterol. In the absence of bile salt the incorporation of cholesterol was unaffected by stirring of the incubation mixtures. Increasing the cholesterol concentration in the mixed micelle while keeping the concentration of bile salt constant caused an increase in rate of sterol incorporation. This increased rate was seen whether the cholesterol suspension was turbid, i.e., contained non-micellized cholesterol, or whether it was optically-clear and contained only monomers and micelles. When the concentration of taurocholate and cholesterol were increased simultaneously such that the concentration ratio of these two components was kept constant, there resulted a corresponding increase in rate of cholesterol uptake. The initial rates of cholesterol incorporation from suspensions containing micellar and monomer forms of cholesterol were much larger than from solutions containing only monomers of the same concentration. The rates of incorporation of cholesterol and phosphatidylethanolamine from mixed micelles containing these lipids in equimolar concentrations were very different. The results as a whole suggest at least for those experimental conditions specified in this study, that uptake of cholesterol by isolated brush-border membranes involves both the monomer and micellar phases of the bulk lipid and that the interaction of the micelles with membrane does not likely involve a fusion process.     

Taurocholate- and taurochenodeoxycholate-lecithin micelles: The equilibrium of bile salt between aqueous phase and micelle

The equilibrium of bile salt between aqueous phase and mixed micelle was studied in solutions of pure bile salt and lecithin comparing taurocholate and taurochenodeoxycholate. The relationship between bile salt concentration in the aqueous phase and the ratio of bile salt/lecithin in the mixed micelle was determined by equilibrium dialysis on serial dilutions of these solutions. Extrapolation of this relationship to zero mixed-micellar bile salt permitted calculation of the critical micelle concentration (CMC) of the mixed micelle. For taurocholate, taurochenodeoxycholate, and an equimolar mix of these two bile salts, the mixed micelle CMC's were 3.1 mM, 0.47 mM, and 0.89 mM respectively. In the most concentrated solutions, aqueous phase bile salt concentration surpassed the CMC of the simple bile salt micelle by more than four-fold indicating the presence of simple micelles as well as mixed micelles. At all dilutions taurochenodeoxycholate had a much greater affinity for the mixed micelle than did taurocholate. This last finding may be the reason for the superior cholesterol solubilizing capacity of taurochenodeoxycholate-lecithin solutions compared to taurocholate-lecithin solutions.     

The intermicellar bile salt concentration in equilibrium with the mixed-micelles of human bile.

The intermicellar bile salt concentration in equilibrium with the bile salt-lecithin-cholesterol mixed-micelle has been studied in human bile. Equilibrium-dialysis, used to measure the biliary intermicellar bile salt concentration, has been validated as an applicable method by studying the cholate-lecithin mixed-micelle, for which intermicellar bile salt concentration values have previously been reported. The intermicellar bile salt concentration of bile was essentially independent of ionic strength in the range 0.05-0.15 M chloride. Simple dilution of bile lowered the intermicellar bile salt concentration (about 2/3 reduction for each two-fold dilution). This reduction occurred because of a simultaneous decrease in the molar ration of bile salt/phospholipid in the micelle. Dilution of micelles with micellar bile salt/phospholipid held constant did not affect the intermicellar bile salt concentration. The relationship between intermicellar bile salt concentration and micellar bile salt/phospholipid, defined in the dilution studies, was linear in the range of study. For a composite of five biles, this relationship was described by the equation: intermicellar bile salt concentration = 1.27 (bile salt/phsopholipid) + 0.538. Data obtained on an artificial bile agreed closely with the results obtained on bile suggesting that the other constituents of bile did not affect this analysis. These findings may be helpful in understanding the process of micellar cholesterol solubilization in bile.     

Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.     

Distinct effects of three bile salts on cholesterol solubilization by oleate-monoolein-bile salt micelles.

Micellar cholesterol solubilities in bile salt-monoolein-oleic acid systems have been determined. Whatever the bile salt/oleyl compounds ratio, taurochenodeoxycholate solubilizes more cholesterol than taurocholate and much more than tauroursodeoxycholate. At pH 6.7, the cholesterol solubility limit is about the same with either oleate or monoolein. Cholesterol solubility falls in oleate-bile acid mixtures as the pH is raised. The capacity for supersaturation with cholesterol is greater for bile salt-monoolein than for bile salt-oleate micelles. For the latter it decreases as pH increases.     

Calcium binding by monosulfate esters of taurochenodeoxycholate

The effect of sulfate esterification of the 3 alpha- or 7 alpha-hydroxyl groups of taurochenodeoxycholate on calcium binding was studied at 0.154 M NaCl in the presence and absence of phosphatidylcholine using a calcium electrode. For comparison, similar studies were made with taurochenodeoxycholate, taurodeoxycholate, and taurocholate. No high affinity calcium binding was demonstrable for any of these bile salts in pre-micellar solutions. Taurine-conjugated bile salts have greater affinity for calcium when in a micellar form. At elevated bile salt concentrations, the calcium binding of unsulfated dihydroxy taurine conjugates was similar to that of the monosulfate esters of taurochenodeoxycholate. The presence of phosphatidylcholine decreased calcium binding of the unsulfated dihydroxy bile salts and slightly increased calcium binding by taurocholate. However, the addition of phosphatidylcholine to monosulfate esters of taurochenodeoxycholate results in large increments in calcium binding. The results indicate that increased calcium binding due to the presence of phosphatidylcholine in bile salt solutions depends, in part, on the hydrophilicity of the bile salt and that the interaction of monosulfate esters of taurochenodeoxycholate with phosphatidylcholine leads to the formation of a high affinity calcium binding site.     

Absence of negative feedback control of bile acid biosynthesis in cultured rat hepatocytes

The effect of individual bile acids on bile acid synthesis was studied in primary hepatocyte cultures. Relative rates of bile acid synthesis were measured as the conversion of lipoprotein [4-14C]cholesterol into 4-14C-labeled bile acids. Additions to the culture media of cholate, taurocholate, glycocholate, chenodeoxycholate, taurochenodeoxycholate, glycochenodeoxycholate, deoxycholate, and taurodeoxycholate (10-200 microM) did not inhibit bile acid synthesis. The addition of cholate (100 microM) to the medium raised the intracellular level of cholate 10-fold, documenting effective uptake of added bile acid by cultured hepatocytes. The addition of 200 microM taurocholate to cultured hepatocytes prelabeled with [4-14C]cholesterol did not result in inhibition of bile acid synthesis. Taurocholate (10-200 microM) also failed to inhibit bile acid synthesis in suspensions of freshly isolated hepatocytes after 2, 4, and 6 h of incubation. Surprisingly, the addition of taurocholate and taurochenodeoxycholate (10-200 microM) stimulated taurocholate synthesis from [2-14C]mevalonate-labeled cholesterol (p less than 0.05). Neither taurocholate nor taurochenodeoxycholate directly inhibited cholesterol 7 alpha-hydroxylase activity in the microsomes prepared from cholestyramine-fed rats. By contrast, 7-ketocholesterol and 20 alpha-hydroxycholesterol strongly inhibited cholesterol 7 alpha-hydroxylase activity at low concentrations (10 microM). In conclusion, these data strongly suggest that bile acids, at the level of the hepatocyte, do not directly inhibit bile acid synthesis from exogenous or endogenous cholesterol even at concentrations 3-6-fold higher than those found in rat portal blood.     

Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase in the rat

Specific activities of the hepatic microsomal enzymes 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase and cholesterol 7alpha-hydroxylase were studied in rats fed sterols and bile acids. The administration of bile acids (taurocholate, taurodeoxycholate, taurochenodeoxycholate) at a level of 1% of the diet for 1 wk reduced the activity of HMG CoA reductase. Taurocholate and taurodeoxycholate, but not taurochenodeoxycholate, inhibited cholesterol 7alpha-hydroxylase. Dietary sitosterol produced increases in the specific activity of HMG CoA reductase (3.6-fold) and cholesterol 7alpha-hydroxylase (1.4-fold), and biliary cholesterol concentrations in this group more than doubled. Compared with controls fed the stock diet, the simultaneous administration of sitosterol and taurochenodeoxycholate resulted in a 60% decrease of HMG CoA reductase activity and no change in cholesterol 7alpha-hydroxylase activity or biliary cholesterol concentration. Rats fed sitosterol plus taurocholate had nearly normal HMG CoA reductase activity, but cholesterol 7alpha-hydroxylase was inhibited and biliary cholesterol remained high. Bile acid secretion rates and biliary bile acid composition were similar in controls and sterol-fed animals. In all groups receiving bile acids, biliary secretion of bile acids was nearly doubled and bile acid composition was shifted in the direction of the administered bile acid. It is concluded that the composition of the bile acid pool influences the hepatic concentrations of the rate-controlling enzymes of bile acid synthesis.     

Effects of monosulfate esters of taurochenodeoxycholate on bile flow and biliary lipids in hamsters

The effect of the 3 alpha- and 7 alpha-monosulfate esters of taurochenodeoxycholate on bile flow and biliary lipids was compared to the effect of unsulfated taurochenodeoxycholate. Test bile salts were infused directly into the portal circulation through a catheter introduced into the splenic pulp. Recovery of unsulfated and sulfated bile salts was complete; no biotransformation of any of the administered compounds was noted. Equivalent choleresis was noted in response to administration of each of the test bile salts. Of particular interest, the biliary cholesterol and phospholipid content was tightly linked to biliary bile salt monosulfates; the slope of the line describing the relationship between bile salts and lipids was similar to that for the unsulfated bile salt. The critical micellar concentration of the 3 alpha- and 7 alpha-monosulfate esters was 19 mM and 18 mM, respectively. Sulfation of taurochenodeoxycholate, therefore, does not impair its bile secretory function. Despite a higher critical micellar concentration, biliary lipid excretion with monosulfate esters is equivalent to that seen with unsulfated bile salt. The role of hydrophobic/hydrophilic balance in the promotion of biliary lipid excretion may need to be redefined.     

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.     

Some aspects of mechanism of inhibition of cholesterol absorption by beta-sitosterol

Mixed bile salt micelle solubilized either cholesterol or beta-sitosterol to a comparable extent. When added simultaneously, beta-sitosterol restricted the micellar solubility of cholesterol. beta-Sitosterol also reduced the cholesterol content in the aqueous (micellar) phase of the intestinal contents of rats, the extent of reduction being comparable with that observed in vitro. The intestinal uptake of cholesterol in vivo was equivalent to the micellar incorporation of cholesterol both in vitro and in vivo. beta-Sitosterol had no inhibitory effect on cholesterol absorption from the micellar solution in jejunal loops in situ, whereas the rate of beta-sitosterol uptake was only about one-fifth that of cholesterol. The intestinal uptake of beta-sitosterol intubated into the stomach of rats was about one-fifth that of cholesterol. The intestinal brush-border membrane discriminated these sterols. These results suggest that the restriction of the micellar solubility of cholesterol, rather than the inhibition of uptake from brush-border membrane, is the major determinant for the interference of beta-sitosterol with cholesterol absorption.     

Some aspects of mechanism of inhibition of cholesterol absorption by β-sitosterol

(1) Mixed bile salt micelle solubilized either cholesterol or β-sitosterol to a comparable extent. When added simultaneously, β-sitosterol restricted the micellar solubility of cholesterol. (2) β-Sitosterol also reduced the cholesterol content in the aqueous (micellar) phase of the intestinal contents of rats, the extent of reduction being comparable with that observed in vitro. The intestinal uptake of cholesterol in vivo was equivalent to the micellar incorporation of cholesterol both in vitro and in vivo. (3) β-Sitosterol had no inhibitory effect on cholesterol absorption from the micellar solution in jejunal loops in situ, whereas the rate of β-sitosterol uptake was only about one-fifth that of cholesterol. (4) The intestinal uptake of β-sitosterol intubated into the stomach of rats was about one-fifth that of cholesterol. The intestinal brush-border membrane discriminated these sterols. These results suggest that the restriction of the micellar solubility of cholesterol, rather than the inhibition of uptake from brush-border membrane, is the major determinant for the interference of β-sitosterol with cholesterol absorption.     

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.     

Physicochemical and physiological properties of 5alpha-cyprinol sulfate,the toxic bile salt of cyprinid fish

5alpha-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5alpha-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was approximately 4 mM. At concentrations >1 mM, 5alpha-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5alpha-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax congruent with 0.5 micromol/min/kg) as compared with taurocholate. 5alpha-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5alpha-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5alpha-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5alpha-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit. These studies indicate that 5alpha-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish.     

Structural alterations in lecithin-cholesterol vesicles following interactions with monomeric and micellar bile salts: physical-chemical basis for subselection of biliary lecithin species and aggregative states of biliary lipids during bile formation

Using complementary physical-chemical methods including turbidimetry, quasielastic light scattering, gel filtration, and phase analysis, we examined the interactions between dilute concentrations of the common bile salt, taurochenodeoxycholate (TCDC), and uni- and multilamellar vesicles (MLVs) composed of defined molecular species of lecithin (L) and varying contents of cholesterol (Ch). Dissolution rates of MLVs with micellar TCDC, as assessed by turbidimetry, were more rapid with vesicles composed of sn-1 palmitoyl species, typical of biliary L, compared with those composed of the more hydrophobic sn-1 stearoyl species. Incorporation of Ch retarded MLV dissolution rates in proportion to the Ch content, and only at high Ch contents were dissolution rates appreciably influenced by the sn-2 fatty acid composition of L. When MLVs contained Ch in amounts characteristic of intracellular membranes (Ch/L approximately 0.1), the dissolution rates of the individual L species by TCDC accurately predicted the steady state L composition of human bile. TCDC interacted with small unilamellar L/Ch vesicles (SUVs) at concentrations well below, as well as appreciably above, its critical micellar concentration. In accordance with the TCDC-egg yolk L-H2O phase diagram, perimicellar concentrations of TCDC interacted with SUVs to form aggregates that were approximately twice the size of the SUVs. These were consistent with the formation of a dispersed hexagonal (rod-like) phase, which co-existed with aqueous bile salt (BS) monomers and either micellar or unilamellar SUV phases. Micellar TCDC completely solubilized SUVs as mixed micelles, putatively via this transient hexagonal phase. With modest Ch-supersaturation, dissolution was followed by the reemergence of a new vesicle population that coexisted metastably with mixed micelles. With high Ch supersaturation, TCDC extracted L and Ch molecules from SUVs in different proportions to form Ch-supersaturated mixed micelles and Ch-enriched SUVs, in accordance with the metastable phase diagram. These experiments are consistent with the hypothesis that sn-1 palmitoyl L species are subselected for bile, in part, by physical-chemical interactions of intracellular BS concentrations with Ch-poor membranes and that the subsequent evolution of Ch-rich vesicles and Ch-saturated mixed micelles occurs via a transitional hexagonal (rod) phase. These liquid-crystalline states are likely to be transient in Ch-unsaturated biles, but may persist in Ch-supersaturated human biles because of their high Ch contents which retard or inhibit these phase transitions.     

Pancreatic cholesterol esterases. 3. Kinetic characterization of cholesterol ester resynthesis by the pancreatic cholesterol esterases

The ability of cholesterol esterase to catalyze the synthesis of cholesterol esters has been considered to be of limited physiological significance because of its bile salt requirements for activity, though detailed kinetic studies have not been reported. This study was performed to determine the taurocholate, pH, and substrate requirements for optimal cholesterol ester synthesis catalyzed by various pancreatic lipolytic enzymes, including the bovine 67- and 72-kDa cholesterol esterases, human 100-kDa cholesterol esterase, and human 52-kDa triglyceride lipase. In contrast to current beliefs, cholesterol esterase exhibits a bile salt independent as well as a bile salt dependent synthetic pathway. For the bovine pancreatic 67- and 72-kDa cholesterol esterases, the bile salt independent pathway is optimal at pH 6.0-6.5 and is stimulated by micromolar concentrations of taurocholate. For the bile salt dependent synthetic reaction for the 67-kDa enzyme, increasing the taurocholate concentration from 0 to 1.0 mM results in a progressive shift in the pH optimum from pH 6.0-6.5 to pH 4.5 or lower. In contrast, cholesterol ester hydrolysis by the 67-, 72-, and 100-kDa enzymes was characterized by pH optima from 5.5 to 6.5 at all taurocholate concentrations. Optimum hydrolytic activity for these three enzyme forms occurred with 10 mM taurocholate. Since hydrolysis is minimal at low taurocholate concentrations, the rate of synthesis actually exceeds hydrolysis when the taurocholate concentration is less than 1.0 mM. The 52-kDa enzyme exhibits very low cholesterol ester synthetic and hydrolytic activities, and for this enzyme both activities are bile salt independent. Thus, our data show that cholesterol esterase has both bile salt independent and bile salt dependent cholesterol ester synthetic activities and that it may catalyze the net synthesis of cholesterol esters under physiological conditions.     

Taurocholate uptake by adult rat hepatocytes in primary culture

Adult rat hepatocytes were cultured on Petri dishes for 25--30 h prior to measuring their ability to transport taurocholate. A rapid uptake of the bile acid (25 muM) was observed: about 20% was accumulated in the cells within 15 min. The taurocholate transport was saturable with an apparent Km of 28 +/- 10 muM and a maximal velocity V of 0.07 +/- 0.02 nmol/(micrograms DNA x min). Uptake was shown to be energy dependent as it was inhibited about 65% by antimycin A (20 micrograms/ml). The monohydroxylated bile acid taurolithocholate and the dihydroxylated taurochenodeoxycholate inhibited taurocholate transport to about 30 and 40% resp. of the control. The transport process was strongly dependent on sodium ions. It is concluded that the characteristics of taurocholate uptake into adult rat hepatocytes are very similar either in freshly prepared cells or in hepatocytes which are cultured on Petri dishes for 25--30 h.     

Spectrophotometric determination of the critical micellar concentration of bile salts using bilirubin monoglucuronide as a micellar probe. Utility of derivative spectroscopy.

We have developed a simple biologically non-invasive method for determining the critical micellar concentration (CMC) of bile salts using pure naturally occurring bilirubin IX alpha monoglucuronide (BMG), an important bile pigment present in virtually all mammalian biles. This methodology employs visible absorbance spectroscopy of BMG in bile salts over a range of bile salt concentrations that include the reported CMC. Using 100 microM-BMG in 0.4 M-imidazole buffer at pH 7.8, we calculated that the CMC for sodium taurochenodeoxycholate is between 2.5 and 3.0 mM based on: (1) an abrupt change in lambda max. in this concentration range, (2) a precipitous decrease in the amplitude of the absorbance shoulder at 450 nm, (3) a sudden decrease in the second derivative absorbance of BMG at 400 nm and an increase in absorbance at 470 nm, (4) a sharp change in the 4th derivative absorbance at 375 and 395 nm. In contrast, sodium taurocholate, a bile salt that reportedly does not have a CMC but continuously self-associates over a wide concentration range, exhibited none of these changes. The use of derivative spectroscopy enhances the ability to detect the CMC changes and also indicates the number of BMG species in solution and their relative energy states.