The roles of bile salts in the uptake of beta-carotene and retinol by rat everted gut sacs.

The effects of bile salts, Tween 20 and hexadecyltrimethylammonium-bromide on the uptake of beta-[3H]carotene and [3H]retinol by rat-everted gut sacs were studied in vitro under conditions simulating those present in the intestinal lumen during lipid absorption. 2. Micellar solutions significantly enhanced uptake over emulsions. Maximum uptake occurred at the critical micellar concentration of the bile salts mixture. At higher detergent concentrations beta-carotene uptake declined sharply; retinol absorption remained high. 3. In beta-carotene absorption bile salts functioned not only as micellar solubilizers but also may have been required for interaction with the cell membrane or as a transport carrier. In retinol uptake their primary function appeared only to be micellar solubilization. Both uptake and efflux of substrates were enhanced in bile salt micellar solutions compared to the other detergents. 4. Beta-carotene cleavage and conversion to retinyl esters occurred only in bile salts solutions. Retinol esterification was seen with all detergents. These effects increased as the tri/dihydroxy bile salts ratio was increased. 5. Beta-carotene uptake appeared to be reversible and passive at low concentrations. Retinol uptake was reversible, 7-30 times more rapid, and partially inhibited by 2,4-dinitrophenol at higher concentrations. An energy-requiring step may have been rate limiting.     

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 greater than 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 calculated glucose concentration profile in the intercellular spaces of everted jejunum of rat.

On the basis of experimental data from isolated and everted rat jejunum and on extension of Diamond and Bossert's mathematical model, the glucose concentration in the intercellular spaces has been calculated and has been found higher (less than 2 mM) than that present in the serosal space.     

Molecular and micellar associations in the pH-dependent stable and metastable dissolution of unconjugated bilirubin by bile salts

Unconjugated bilirubin (UCB) is almost insoluble in water at neutral pH, but appears in normal human gallbladder bile at concentrations up to 35 microM. We therefore determined whether conjugated bile salts could increase the dissolved concentration [( Bt]) of UCB over the pH range 3.0-11.0. Using crystalline UCB, [Bt] was higher with less ordered crystals, with increasing pH and bile salt concentration, and with taurocholate (TC) micelles compared to taurodehydrocholate (TDHC) dimers. Plots of [Bt] verus pH from pH 3.0-9.3 fit the equation, [Bt] = A(1 + K'1/[H]+ + K'1.K'2/[H+]2), where A = [Bt] at pH less than 4.0, and K'1 and K'2 are the two apparent ionization constants of UCB. Estimated pK'1 values in NaCl, TC, and TDHC were 6.8, 6.0, and 5.6, respectively; pK'2 was greater than or equal to 9.3 in each system. Acidification of disodium bilirubinate to pH less than 8.5 produced high, metastable [Bt] in 50 mM TC; this was absent in 0.15 M NaCl, and minor in 50 mM TDHC. In all solutions, maximum [Bt] of 60-65 mM was attained at pH greater than or equal to 10.5. This work helps explain the immense variation among reported [Bt] values, indicates that UCB monoanion predominates at the pH range of bile, and suggests that bile salt monomers, dimers, and micelles enhance the solubility of UCB in bile.     

Micelle formation of sodium chenodeoxycholate and solubilization into the micelles: comparison with other unconjugated bile salts

Micellization of sodium chenodeoxycholate (NaCDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle at 288.2, 298.2, 308.2, and 318.2 K. They were compared with those of three other unconjugated bile salts; sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium ursodeoxycholate (NaUDC). The I(1)/I(3) ratio of pyrene fluorescence and the solubility dependence of solution pH were employed to determine the CMC values. As the results, a certain concentration range for the CMC and a stepwise molecular aggregation for micellization were found reasonable. Using a stepwise association model of the bile salt anions, the mean aggregation number (n) of NaCDC micelles was found to increase with the total anion concentration, while the n values decreased with increasing temperature; 9.1, 8.1, 7.4, and 6.3 at 288.2, 298.2, 308.2, and 318.2 K, respectively, at 50 mmol dm(-3). The results from four unconjugated bile salts indicate that the number, location, and orientation of hydroxyl groups in the steroid nucleus are quite important for growth of the micelles. Activity of the counterion (Na(+)) was determined by a sodium ion selective electrode in order to confirm the low counterion binding to micelles. The solubilized amount of cholesterol into the aqueous bile salt solutions increased in the order of NaUDC相似文献   

Daily rhythmic change in the transport of histidine by everted sacs of rat small intestine

Fluctuations in the intestinal transport of L- and D-histidine were measured in rats on three feeding schedules under conventional lighting conditions, with a dark night. In rats fed ad libitum, the transport of L-histidine through the everted intestine showed a daily rhythmic change, being high at 4 p.m. and low in the early morning. In rats adapted to daytime feeding, the transport of L-histidine was highest at 6 a.m. and low at night. In starved rats, the rhythmicity was maintained for at least one day of fasting. Transport of D-histidine showed no daily fluctuation.     

The effect of tyrosine conjugation on the critical micellar concentration of free and glycine-conjugated bile salts

We investigated the effect of conjugation with the aromatic amino acid tyrosine on the critical micellar concentration (CMC) of bile salts. The CMC values were determined by surface tension and by dye solubilization. The surface tension measurement employed the Du Nouy ring detachment method and the dye solubilization measurement utilized a water-insoluble dye, 1-O-tolylazo-2-naphthol. We compared the CMC values of the sodium salts of cholyltyrosine (cholylTyr), deoxycholyltyrosine (deoxycholylTyr), deoxycholylglycyltyrosine (deoxycholylGlyTyr) chenodeoxycholyltyrosine (chenodeoxycholylTyr), chenodeoxycholylglycyltyrosine (chenodeoxycholylGlyTyr), cholyldiglycyltyrosine (cholylGlyGlyTyr) and cholylglycyltyrosine (cholylGlyTyr) with their respective glycine conjugated bile salts. Both techniques of CMC determination indicated that tyrosine conjugation to free and glycine-conjugated bile salts reduced their CMC significantly.     

Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats

Human milk contains prebiotic oligosaccharides, which stimulate the growth of intestinal bifidobacteria and lactobacilli. It is unclear whether the prebiotic capacity of human milk contributes to the larger bile salt pool size and the more efficient fat absorption in infants fed human milk compared with formula. We determined the effect of prebiotic oligosaccharides on bile salt metabolism in rats. Rats were fed a control diet or an isocaloric diet containing a mixture of galactooligosaccharides (GOS), long-chain fructooligosaccharides (lcFOS), and acidified oligosaccharides (AOS) for 3 wk. We determined synthesis rate, pool size, and fractional turnover rate (FTR) of the primary bile salt cholate by using stable isotope dilution methodology. We quantified bile flow and biliary bile salt secretion rates through bile cannulation. Prebiotic intervention resulted in significant changes in fecal and colonic flora: the proportion of lactobacilli increased 344% (P < 0.01) in colon content and 139% (P < 0.01) in feces compared with the control group. The number of bifidobacteria also increased 366% (P < 0.01) in colon content and 282% in feces after the prebiotic treatment. Furthermore, pH in both colon and feces decreased significantly with 1.0 and 0.5 pH point, respectively. However, despite this alteration of intestinal bacterial flora, no significant effect on relevant parameters of bile salt metabolism and cholate kinetics was found. The present data in rats do not support the hypothesis that prebiotics naturally present in human milk contribute to a larger bile salt pool size or altered bile salt pool kinetics.     

The influx of uric acid and other purines into everted jejunal sacs of the rat and hamster.

The in vitro transport of [2-14-C]uric acid, [8-14-C]hypoxanthine, and [8-14-C]xanthine, each dissolved in Krebs--Ringer bicarbonate buffer, was studied with everted jejunal sacs from rat and hamster. No evidence could be obtained for the development of a concentration gradient between the intracellular fluid and the incubation medium or between the sac contents and the incubation medium, for any of the three oxypurines. Inhibitiors of active transport, such as anaerobiosis for dinitrophenol, had no significant effect on the rate of transport. A large percentage of hypoxanthine and xanthine was oxidized to urine acid in the sac-wall homogenate, sac contents, and incubation medium during the course of the incubation. This oxidation could be prevented by addition of allopurinol (3 mM) to the incubation medium, but concentration gradients were still not obtained. No active transport mechanism could be demonstrated for uric acid, hypoxanthine, or xanthine in rat or hamster jejunum.     

Oxalate uptake by everted sacs of rat colon. Regional differences and the effects of pH and ricinoleic acid

Hyperoxaluria is a complication of disorders associated with steatorrhea. The colon is the presumed site of enhanced oxalate absorption in patients with steatorrhea. We performed studies of colonic mucosal oxalate uptake in everted sacs of rat colon to determine the kinetics of colonic oxalate transport and to evaluate the effect of both pH and ricinoleic acid, a hydroxy fatty acid, on colonic oxalate uptake. Our study demonstrated that oxalate is transported throughout the colon by passive diffusion. Tissue uptake increased linearly with increasing oxalate concentrations and was unaffected by metabolic inhibitors, oxygen deprivation, or temperature changes. There were pH-dependent regional differences of oxalate uptake both in the presence and absence of ricinoleic acid. In the absence of ricinoleic acid, the highest oxalate uptake occurred at the lower pH values (5.4 and 6.4). In the presence of ricinoleic acid oxalate uptake was enhanced at the higher pH values (7.4 and 8.4); a finding most likely related to decreased solubility of ricinoleic acid at pH 5.4 and 6.4. Intraluminal pH is an important determinant of colonic oxalate uptake in the presence and absence of ricinoleic acid.     

Synthesis and biliary excretion of tyrosine-conjugated bile salts in Wistar rats

Tyrosine-labelled free and glycine-conjugated bile acids were synthesized and radiolabelled with 125I to high purity. The synthetic method utilized excess tyrosine methyl ester hydrochloride (1.4 equiv.) and bile acid (one equiv.) via dicyclohexylcarbodiimide (1.4 equiv.) with yields of 90-93% for tyrosine bile acid conjugates and glycyltyrosine conjugates and 56-60% yields for the glycylglycyltyrosine conjugates. All of the eight iodinated tyrosine bile acids tested were rapidly excreted into bile following intravenous injection. In bile duct-cannulated rats with ligated renal pedicles under pentobarbital anaesthesia the percentages of injected dose recovered from bile within 20 min were as follows: cholylglycine ([14C]cholylGly), 81.2 +/- 1.3%; taurocholate ([14C]taurocholate), 94.3 +/- 1.0%; cholyltyrosine (125I-cholylTyr), 85.5 +/- 3.3%; deoxycholyltyrosine (125I-deoxycholylTyr), 87.9 +/- 6.3%; chenodeoxycholyltyrosine (125I-chenodeoxycholylTyr), 93.4 +/- 2.9; cholylglycyltyrosine (125I-cholylGlyTyr), 95.7 +/- 6.7%; deoxycholylglycyltyrosine (125I-deoxylcholylGlyTyr), 92.5 +/- 3.2%; chenodeoxycholylglycyltyrosine (125I-chenodeoxycholylGlyTyr), 94.1 +/- 3.1%; cholyldiglycyltyrosine (125I-cholylGlyGlyTyr), 85.2 +/- 3.6%, and deoxycholyldiglycyltyrosine (125I-deoxycholylGlyGlyTyr), 85.5 +/- 2.7%. Values are means +/- SD. Thus the biliary excretion of 125I-chenodeoxycholylGlyTyr, 125I-chenodeoxycholylTyr, 125I-deoxycholylGlyTyr and 125I-cholylGlyTyr was similar to that of [14C]taurocholate, the major naturally occurring bile acid in the rat, and the biliary excretion of all the tyrosine conjugates was similar to or exceeded that of [14C]cholylglycine. Conjugation with tyrosine enhanced the efficiency of plasma-to-bile transport of most naturally occurring bile acids. Comparison of glycyltyrosine conjugates with glycylglycyltyrosine conjugates suggests that any additional benefit derived by elongation of the side-chain is probably negated by obscuring the 12 alpha-hydroxyl function on the steroid nucleus in the bile acid glycylglycyltyrosine conjugates.     

The absorption of antibody by the duodenum and jejunum in young rats

Experiments were performed to test the capacity of parts of the small intestine of rats aged27–29 days to absorb homologous antibody and transmit it to the circulation. No antibody absorption occurs after the oral administration of immune serum to rats of this age, for the postnatal transfer of immunity normally terminates at about20–21 days.
Antibody is readily absorbed and transmitted to the circulation from homologous immune serum introduced into the duodenum of27–28 day animals, after removal of the duodenal contents by flushing out with warm saline. Absorption and transmission occurs in some animals even if the duodenal contents are not previously removed. The animals used in these experiments were left with their mothers until shortly before the operation.
No transmission of antibody occurred when comparable experiments were performed on young rats aged27–28 days that had been weaned at the usual age of20–21 days.
Similar experiments were performed on the first segment of the jejunum. The results obtained are discussed in relation to the pinocytic activity of the epithelial components of the duodenum-jejunum.     

Vectorial transport of unconjugated and conjugated bile salts by monolayers of LLC-PK1 cells doubly transfected with human NTCP and BSEP or with rat Ntcp and Bsep

Na(+)-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a K(m) value of 20 microM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.