The assumed assimilation of cholesterol by Lactobacilli and Bifidobacterium bifidum is due to their bile salt-deconjugating activity.

To study the mechanism of the propsed assimilation of cholesterol, we cultured various strains of Lactobacillus acidophilus and a Bifidobacterium sp. in the presence of cholesterol and oxgall. During culturing, both cholesterol and bile salts were precipitated. Because of bacterial bile salt deconjugation, no conjugated bile salts were observed in either the culture fluids or the pellets. During incubation, the cell count and optical density decreased. The degree of precipitation of bile salts and of cholesterol was dependent on the culture conditions. If L. acidophilus RP32 was cultured under acidifying conditions, the degree of precipitation of deconjugated bile salts was higher than if the pH was maintained at 6.0. Under acidifying conditions, cholesterol was coprecipitated with the bile salts, whereas in pH-controlled cultures, no coprecipitation of cholesterol was observed. From control experiments with different mixtures of bile salts, it appeared that coprecipitation of cholesterol during culturing was a result of formation of deconjugated bile salts, which have a decreased solubility at pH values lower than 6.0. It is concluded that the removal of cholesterol from the culture medium by L. acidophilus RP32 and other species is not due to bacterial uptake of cholesterol, but results from bacterial bile salt-deconjugating activity.     

Involvement of Trihydroxyconjugated Bile Salts in Cholesterol Assimilation by Bifidobacteria

To determine the conditions of cholesterol assimilation, various strains of Bifidobacterium species were cultured in the presence of cholesterol and bile salts. During culturing, Bifidobacterium breve ATCC 15700 assimilates cholesterol in the presence of oxgall at pH values lower than 6. This strain was selected to study the influence of conjugated (taurocholic acid) and deconjugated (cholic acid) bile salts on cholesterol assimilation. B. breve ATCC 15700 assimilated cholesterol (up to 51%) when cultures were undertaken in the presence of taurocholic acid, whereas less than 13% of the initial amount of cholesterol was measured in the cells in the presence of cholic acid. Cultured in the presence of six individual di- or trihydroxyconjugated bile salts, bifidobacteria strains assimilated cholesterol. This assimilation appeared to be more important in the presence of trihydroxyconjugated bile salts (tauro- and glycocholic acids). It is concluded that trihydroxyconjugated bile salts are involved in the assimilation of cholesterol by bifidobacteria. Received: 20 June 1996 / Accepted: 19 July 1996     

Bile salt hydrolase and cholesterol removal effect by Bifidobacterium bifidum NRRL 1976

Summary Growing cells of Bifidobacterum bifidum NRRL 1976 exhibited an ability to remove cholesterol in the presence of bile salts. The cholesterol removal by Bifidobacterium bifidum was due to a co-precipitation together with unconjugated bile acids, which was linked to the bile salt hydrolase (BSH) activity of the cells at pH values lower than 5.0 and the cholesterol removed was partially recovered when the cells were washed with phosphate buffer at pH 7, while the remaining cholesterol was extracted from the cells. It is concluded that the removal of cholesterol from the growth medium by Bifidobacterium bifidum strain is due to both bacterial assimilation and precipitation of cholesterol.     

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes

The availability of different sources of cholesterol for bile acid synthesis by cultured chick embryo hepatocytes was studied. Mevalonolactone was taken up by the cells and converted to cholesterol, cholesterol ester and tauroconjugates of bile acids. The addition of mevalonolactone had little effect on the conversion of endogenous cholesterol to taurocholic acid; however, taurochenodeoxycholic acid synthesis was stimulated. 25-30% of the cholesterol synthesized from mevalonolactone was converted to taurochenodeoxycholic, taurocholic and two so-far unidentified bile acids. All bile acids were secreted into the incubation medium. When cholesterol was added as mixed liposomes with phosphatidylcholine, it was taken up by the cells and converted to bile acids. At low concentrations of liposomes, the greater part of the cholesterol which was taken up by the cells was converted to bile acids. At higher concentrations, considerable amounts of cholesterol and cholesterol ester accumulated inside the cells. When mevalonolactone and cholesterol liposomes was added together, both substrates were used simultaneously for bile acids synthesis. HDL cholesterol was the best substrate tested, yielding large amounts of two, so-far, unidentified bile acids (possibly allo-bile acids) and smaller amounts of taurocholic and taurochenodeoxycholic acid. Addition of HDL suppressed the conversion of endogenous cholesterol to taurocholic acid; taurochenodeoxycholic acid synthesis, however, was stimulated.     

Effects of Lactobacillus acidophilus 43121 and a mixture of Lactobacillus casei and Bifidobacterium longum on the serum cholesterol level and fecal sterol excretion in hypercholesterolemia-induced pigs

The hypocholesterolemic effects of Lactobacillus acidophilus 43121 (43121) and a mixture of Lactobacillus casei and Bifidobacterium longum (MIX) were studied in hypercholesterolemia-induced pigs. Serum total cholesterol was decreased by supplementation of either 43121 or MIX, although, high-density lipoprotein cholesterol was not changed. The hypocholesterolemic effect of 43121 and MIX was mainly due to bile acid dehydroxylation, this effect being supplementation-time dependent.     

Diabetes and intestinal cholesterol uptake from bile salt solutions

A previously validated in vitro technique was used to determine the effect of diabetes mellitus on the intestinal uptake of cholesterol from various micellar bile salt solutions. The bile salts studied included cholic (C), taurocholic (TC), glycocolic (GC), chenodeoxycholic (CDC), taurochenodeoxycholic (TCDC), glycochenodeoxycholic (GCDC), deoxycholic (DC), taurodeoxycholic (TDC), and glycodeoxycholic (GDC). In control rats there was a reciprocal decline in cholesterol uptake with increasing concentrations of these nine bile acids, and cholesterol uptake was greater from the conjugated primary bile acids than from the unconjugated ones. With a 5 mM concentration of bile acids, the ratios of the uptake of 0.2 mM cholesterol in control rats were C = CDC = DC, TCDC greater than TC greater than TDC, and GC = GCDC greater than GDC; with 20 mM concentrations, the ratios of cholesterol uptake in control rats were C greater than CDC greater than DC, TC greater than TCDC greater than TDC, and GC = GCDC greater than GDC. In the diabetic animals cholesterol uptake was higher than in control rats when using 5 or 20 mM of each of the conjugated bile acids and with cholic acid. In contrast, cholesterol uptake was similar in diabetic and control animals when cholesterol was solubilized with 5 or 20 mM CDC or DC. These differences in cholesterol uptake using the various bile acids and the failure of CDC and DC to facilitate the enhanced uptake of cholesterol in diabetic animals remains unexplained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut

The objective of this study was to evaluate the effect of human gut-derived lactic acid bacteria and bifidobacteria on cholesterol levels in vitro. Continuous cultures inoculated with fecal material from healthy human volunteers with media supplemented with cholesterol and bile acids were used to enrich for potential cholesterol assimilators among the indigenous bacterial populations. Seven potential probiotics were found: Lactobacillus fermentum strains F53 and KC5b, Bifidobacterium infantis ATCC 15697, Streptococcus bovis ATCC 43143, Enterococcus durans DSM 20633, Enterococcus gallinarum, and Enterococcus faecalis. A comparative evaluation regarding the in vitro cholesterol reduction abilities of these strains along with commercial probiotics was undertaken. The degree of acid and bile tolerance of strains was also evaluated. The human isolate L. fermentum KC5b was able to maintain viability for 2 h at pH 2 and to grow in a medium with 4,000 mg of bile acids per liter. This strain was also able to remove a maximum of 14.8 mg of cholesterol per g (dry weight) of cells from the culture medium and therefore was regarded as a candidate probiotic.     

Identification of Gibberellin A3 and Abscisic acid from Artemisia capillaris Leaves

The hypocholesterolemic effects of Lactobacillus acidophilus 43121 (43121) and a mixture of Lactobacillus casei and Bifidobacterium longum (MIX) were studied in hypercholesterolemia-induced pigs. Serum total cholesterol was decreased by supplementation of either 43121 or MIX, although, high-density lipoprotein cholesterol was not changed. The hypocholesterolemic effect of 43121 and MIX was mainly due to bile acid dehydroxylation, this effect being supplementation-time dependent.     

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.     

Bifidobacteria Strain Behavior Toward Cholesterol: Coprecipitation with Bile Salts and Assimilation

Resting cells and growing cells of bifidobacteria strains exhibited an ability to remove cholesterol in the presence of bile salts. In resting cell assays, the removed cholesterol was precipitated in the presence of cholic acid at pH values lower than 5.4. However, this precipitated cholesterol was redissolved when the pellets were washed with phosphate buffer, pH 7, and no cholesterol was found in the cells. It appears that this precipitation is a transient phenomenon. In the case of growing cells, the removed cholesterol was partially recovered when cells were washed with phosphate buffer, pH 7, while the remaining cholesterol was extracted from the cells. Cultured in the presence of radiolabeled free or esterified cholesterol, bifidobacteria strains were able to assimilate esterified cholesterol. It is concluded that the removal of cholesterol from the growth medium by bifidobacteria strains is due to both bacterial assimilation and precipitation of cholesterol. Received: 8 February 1996/Accepted: 11 March 1996     

Active taurocholic acid flux through hepatoma cells increases the cellular pool of unesterified cholesterol derived from lipoproteins

The effect of bile acid flux on the fate of lipoprotein-derived cholesterol was studied in bile acid-transporting McNtcp.18 hepatoma cells. The intracellular unesterified cholesterol (UC) concentration rose when McNtcp.18 cells grown in the presence of either high density lipoproteins (HDL) or low density lipoproteins (LDL) were incubated with taurocholic acid (TCA). This effect was more pronounced when the exogenous source of cholesterol was HDL. The presence of TCA in the culture medium of McNtcp.18 cells had no discernible effect on the uptake of cholesteryl esters (CE) from either lipoprotein. TCA treatment of cells preincubated with either lipoprotein did not affect cholesterol synthesis but antagonized the stimulation of cholesterol esterification in cells that were incubated with LDL. The CE concentration in cells treated with TCA was decreased, relative to cells not incubated with TCA, suggesting that cellular CE stores were also hydrolyzed. The TCA treatment reduced the amount of total cholesterol released into the medium by the lipoprotein-treated cells, which was coincident with the reduction in the amount of apolipoprotein B in the culture medium. However, the proportion of UC released into the medium by the lipoprotein-treated cells was increased in cells capable of active bile acid transport. The results indicate that active bile acid flux through hepatoma cells increases the cellular pool of UC derived from lipoproteins. The UC released by the cells into the culture medium under this condition may represent cholesterol destined for direct biliary secretion.     

Effects of internal biliary bypass on the regulation of hepatic cholesterol 7alpha-hydroxylase activity in rats.

In order to re-evaluate the importance of the enterohepatic circulation of bile acid in the regulation of the activities of hepatic cholesterol 7alpha-hydroxylase, bile acid metabolism was examined in internal biliary bypass models of rats. A polyethylene tube was inserted into the common bile duct and another side of the tube was placed in the duodenum (DD), lower jejunum (JD), cecum (CeD), or transverse colon (CoD) as internal biliary bypass models and in the urinary bladder as an external biliary drainage (ED). After bile diversion for 7 days in each group, hepatic cholesterol 7alpha-hydroxylase activities, bile acid concentrations in bile, serum, and portal vein, biliary bile acid compositions, and intestinal absorption rates of infused labeled taurocholic acid were analyzed. Hepatic cholesterol 7alpha-hydroxylase activity was similar in the JD group compared with the DD group, however, it was significantly up-regulated in the CeD (227% of the DD group), CoD (312%), and ED groups (316%). Biliary, serum, and portal bile acid concentrations were not significantly changed in the DD, JD, and CeD groups but those were significantly lower in the CoD and ED groups compared with the DD group. The proportion of the secondary bile acids was significantly increased in the CeD group and was decreased in the CoD and ED groups. The absorption rate of taurocholic acid was almost 100%, 56%, and 23% in the JD, CeD, and the CoD group, respectively. As the cholesterol 7alpha-hydroxylase activity was not significantly changed in the JD group and the predominance of secondary bile acids did not suppress the enzyme activity in the CeD group, the luminal factor, which is absorbed in the presence of bile acids, and the bile acid metabolites are not likely the regulatory factor. The cholesterol 7alpha-hydroxylase activity seems to be primarily regulated by the intestinal absorption of bile acids and partly by the intestinal mucosal factor which is linked to the intestinal bile acid absorption.     

Cholesterol Assimilation by Lactic Acid Bacteria and Bifidobacteria Isolated from the Human Gut

The objective of this study was to evaluate the effect of human gut-derived lactic acid bacteria and bifidobacteria on cholesterol levels in vitro. Continuous cultures inoculated with fecal material from healthy human volunteers with media supplemented with cholesterol and bile acids were used to enrich for potential cholesterol assimilators among the indigenous bacterial populations. Seven potential probiotics were found: Lactobacillus fermentum strains F53 and KC5b, Bifidobacterium infantis ATCC 15697, Streptococcus bovis ATCC 43143, Enterococcus durans DSM 20633, Enterococcus gallinarum, and Enterococcus faecalis. A comparative evaluation regarding the in vitro cholesterol reduction abilities of these strains along with commercial probiotics was undertaken. The degree of acid and bile tolerance of strains was also evaluated. The human isolate L. fermentum KC5b was able to maintain viability for 2 h at pH 2 and to grow in a medium with 4,000 mg of bile acids per liter. This strain was also able to remove a maximum of 14.8 mg of cholesterol per g (dry weight) of cells from the culture medium and therefore was regarded as a candidate probiotic.     

Synthesis and properties of bile acid phosphoramidites 5'-tethered to antisense oligodeoxynucleotides against HCV.

Recently, we synthesized antisense oligonucleotides (AS-ODNs) directed against the non-coding-region (NCR) and the adjacent core region of the hepatitis C virus (HCV) RNA. Backbone modifications like phosphorothioates, methyl- and benzylphosphonates were introduced three at each end of the sequence. For improvement of liver specific drug targeting and/or hepatocellular uptake efficient AS-ODNs were covalently conjugated to biomolecules such as cholesterol or bile acids. The use of base-labile alkylphosphonates afforded mild conditions for deprotection of bile acid conjugated AS-ODNs. Here, we describe a convenient synthesis of new cholic acid and taurocholic acid phosphoramidites. Derivatization to taurocholic acid was effected directly before phosphitylation reaction, which is the last step of the phosphoramidite synthesis. These building blocks were coupled to the 5'-position of AS-ODNs in the last step of solid-phase synthesis. After mild deprotection, purification and characterization the properties of these modified AS-ODNs like their lipophilicity or their ability to form stable duplices to DNA and RNA were investigated. Enhanced lipophilicity and formation of stable duplices and heteroduplices makes bile acid conjugated AS-ODNs interesting as antiviral antisense therapeutics against HCV.     

Polysorbate 80 improves the adhesion and survival of yogurt starters with cholesterol uptake abilities

The goal of this study is to improve the adhesion and survival of yogurt bacteria with probiotic traits by using polysorbate 80, a food additive emulsifier commonly found in milk derivative products. Polysorbate 80 was used at 1% (w/v), and its effects on yogurt bacteria's survival under simulated digestive conditions, cholesterol uptake activities, bile salt hydrolase (BSH) activity, and adhesion to HT-29 culture were studied. In the presence of 1% polysorbate 80, both starters demonstrated better cholesterol uptake and BSH activities, as well as higher bacterial survival at pH 2.5, particularly in associated cultures. In the presence of 0.3 % bile or cholic acid, polysorbate 80 reduced the drop in L. bulgaricus's survival load. However, the carbon source had a greater impact on S. thermophilus bile tolerance than the food additive emulsifier. Oleic acid was incorporated into both bacterial membranes when grown in the presence of bile and polysorbate 80, resulting in a higher unsaturated/saturated fatty acid ratio. In the presence of polysorbate 80, S. thermophilus adhered to HT-29 cells 2.3-fold better, while L. bulgaricus's adhesion remained unchanged. We suggest that polysorbate 80 may have a protective effect on cell survival under simulated digestive stress as well as a role in yogurt bacteria adhesion to the intestines, giving these bacteria more opportunities to exert their purported cholesterol-removal activities.     

Factors affecting motility and morphology of Cryptosporidium sporozoites in vitro

In vitro motility and morphology of Cryptosporidium sporozoites were examined in the presence of various solutions. Crude preparations of the bile salt, taurocholic acid, maintained both motility and morphology in a dose-dependent manner. These effects appeared to be due to the taurocholic acid itself, and not simply due to pH variations, osmotic factors, or contaminants. Lysis of sporozoites was also observed and was found to be dependent on pH, with acidic conditions (pH less than 6.2) triggering the lysis.     

Bile-mediated aminoglycoside sensitivity in Lactobacillus species likely results from increased membrane permeability attributable to cholic acid

Few studies have been conducted on antimicrobial resistance in lactobacilli, presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 type strains and isolates representing 14 species by using agar disk diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i) they were more susceptible (up to 256-fold in some cases) to the deconjugated bile acid cholic acid than to the conjugate taurocholic or taurodeoxycholic acid, and (ii) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain, Lactobacillus plantarum WCFS1, at increasing concentrations of ox gall (0 to 30.3 mg/ml) displayed corresponding decreases in resistance to all of the aminoglycosides tested and ethidium bromide. This effect was clinically relevant, with the gentamicin MIC decreasing from >1,000 to 4 mug/ml in just 3.8 mg of ox gall per ml. In uptake studies at pH 6.5, [G-3H]gentamicin accumulation increased over control levels when cells of this strain were exposed to bile acids or reserpine but not when they were exposed to carbonyl cyanide m-chlorophenylhydrazone. The effect was dramatic, particularly with cholic acid, increasing up to 18-fold, whereas only modest increases, 3- and 5-fold, could be achieved with taurocholic acid and ox gall, respectively. Since L. plantarum, particularly strain WCFS1, is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that mainly cholic acid, but not taurocholic acid, effectively permeabilizes the membrane to aminoglycosides. However, at pHs approaching neutral conditions in the intestinal lumen, aminoglycoside resistance due to membrane impermeability may be complemented by a potential efflux mechanism.     

Factors Affecting Motility and Morphology of Cryptosporidium Sporozoites In Vitro1

In vitro motility and morphology of Cryptosporidium sporozoites were examined in the presence of various solutions. Crude preparations of the bile salt, taurocholic acid, maintained both motility and morphology in a dose-dependent manner. These effects appeared to be due to the taurocholic acid itself, and not simply due to pH variations, osmotic factors, or contaminants. Lysis of sporozoites was also observed and was found to be dependent on pH, with acidic conditions (pH < 6.2) triggering the lysis.     

Immunochemical studies on the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum

Antisera to lipoteichoic acid of Bifidobacterium bifidum subsp. pennsylvanicum were obtained by injecting lipoteichoic acid/methylated BSA complexes into rabbits. Precipitin tests showed that the glycerol phosphate backbone is primarily responsible for serological specificity while the polysaccharide part of the molecule plays a minor role. Whole cells of B. bifidum subsp. pennsylvanicum were capable of absorbing antibodies, indicating the presence of lipoteichoic acid (14% of the total content) at or near the bacterial surface. Cross-reactivity with strains of the genera Bifidobacterium and Lactobacillus was tested using absorption of antiserum by whole bacteria and reactivity of phenol extracts. The results indicated that lipoteichoic acid is a common antigen within the genus Bifidobacterium. The cross-reactivity with the lactobacilli tested was very low.     

Biodiversity of human faecal bacteria isolated from phytic acid enriched chemostat fermenters

BACKGROUND: Myo-inositol hexaphosphate (IP6) or phytic acid is found mostly in cereals and legumes and is thought to possess anti-carcinogenic properties. AIM: To isolate and identify faecal bacteria capable of phytic acid metabolism and to assess the effectiveness of prebiotics (dietary oligosaccharides, metabolised by selective colonic bacteria) in preserving the integrity of phytic acid. METHODS: Faecal samples from three volunteers were used in continuous culture experiments under varying conditions of pH, substrate concentration and dilution rates, seventy three different isolates cultured at steady state were then screened for phytic acid metabolism and identified through partial sequencing of their 16S rRNA genes (16S ribosomal ribonucleic acid). Utilisation of phytic acid was also assessed in a continuous culture system enriched with prebiotic fructooligosaccharides (FOS). RESULTS: Bacteroides spp., Clostridium spp. and facultatively anaerobic bacteria generally appeared to maintain viable counts in the presence of phytic acid. Bifidobacterium spp. and Lactobacillus spp. appeared less able to maintain viable counts in the presence of phytic acid. These results were confirmed by an increase in viable counts of Bacteroides spp., Clostridium spp. and a decrease in viable counts of Bifidobacterium spp. and Lactobacillus spp. once phytic acid was introduced to a FOS enriched continuous culture. CONCLUSIONS: The phytate metabolising biodiversity from the human large intestine does not appear to encompass major bacterial genera associated with beneficial or benign health effects (e.g. Lactobacillus spp. and Bifidobacterium spp).