Separation of bile acid methyl esters by high-performance liquid chromatography

Conjugated bile acids, namely glyco- and tauro-3α,6α-dihydroxy-5β-cholanoic acid (hyodeoxycholic acid), 3α,7α-dihydroxy-5β-cholanoic acid (chenodeoxycholic acid), 3α,6α,7α-trihydroxy-5β-cholanoic acid (hyocholic acid) and 3α-hydroxy-6-oxo-5β-cholanoic acid (6-keto-litocholic acid) were isolated from pig bile, and subsequently transformed into the corresponding methyl esters. Separation of the methyl esters of the isolated bile acids by high-performance liquid chromatography (HPLC) was accomplished on a ZORBAX-CN column (Dupont, Boston, MA) withn-hexane/2-propanol/methylene chloride (89∶6∶5, by vol) as the mobile phase containing traces (≈1%) of amyl alcohol and water as moderators. HPLC analysis of the methyl esters also showed the presence of methyl 3α-hydroxy-6-oxo-5α-cholanoate, which was probably produced in the course of alkaline hydrolysis of the conjugated bile acids.     

Composition of bile acids in ruminants

The bile acids found in sheep bile, beef bile, beef feces, sheep fetus bile, and beef fetus bile have been analyzed by using conventional techniques. Animals maintained on natural and purified diets were used. The bile acids are a complex mixture of isomeric hydroxy- and keto-5β-cholanoic acids which were substituted at one or several of the carbon atoms 3, 7, and 12. Cholic acid is the predominant bile acid found in these species. Deoxycholic acid was the major product formed from cholic acid when the animals were on a natural diet but the concentration of 3α, 12α-dihydroxy-7-keto-5β-cholanoic acid was elevated in the animals that were maintained on a high concentrated purified diet (without roughage). The fetus bile was found to contain nearly all of the bile acids found in the bile of the mature animal but in different concentrations.     

Bile acids in tissues: Binding of lithocholic acid to protein

Human liver contains two forms of lithocholic acid. One form is readily extractable by 95% ethanol/0.1% ammonia (soluble lithocholate, SL), while the other remains firmly bound to the residue (tissue-bound lithocholate, TBL). TBL could be hydrolytically released using clostridial cholanoylamino acid hydrolase, suggesting a peptide link between lithocholate and protein. With bovine serum albumin (BSA), lithocholic acid showed spontaneous amino group-modifying activity. When small molecular weight lysine (α-t-BOC-1-lysyl-β-naphthylamide) and arginine peptides (α-CBZ-di-arginyl-β-naphthylamide) were used in place of BSA, lithocholate bound specifically to the lysine peptide. The unusual affinity for lysine suggested that this amino acid might be involved as a residue in TBL. Synthesis of lithocholyl lysines and comparison with products of acid hydrolysis of TBL established ε-lithocholyl lysine as the predominant form in which lithocholic acid is found in tissue bound form. Supported in part by the Gomprecht Hepatitis Fund. The systematic nomenclature of bile acids referred to in this report by trivial names are as follows: Cholanic acid, 5β-cholan-24-oic acid; lithocholic acid, 3α-hydroxy-5β-cholan-24-oic acid; 3-epilithocholic acid, 3β-hydroxy-5β-cholan-24-oic acid; glycolithocholic acid, 3α-hydroxy-5β-cholan-24-oyl glycine; 3-ketocholanic acid, 3-keto-5β-cholan-24-oic acid; 12α-hydroxycholanic acid, 12α-hydroxy-5β-cholan-24-oic acid; chenodeoxycholic acid, 3α, 7α-dihydroxy-5β-cholan-24-oic acid; glycochenodeoxycholic acid, 3α, 7α-dihydroxy-5β-cholan-24-oyl glycine; deoxycholic acid, 3α, 12α-dihydroxy-5β-cholan-24-oic acid; glycodeoxycholic acid, 3α, 12α-dihydroxy-5β-cholan-24-oyl glycine; cholic acid, 3α, 7α, 12α-trihydroxy-5β-cholan-24-oic acid, glycocholic acid, 3α, 7α, 12α-trihydroxy-5β-cholan-24-oyl glycine; dehydrocholic acid, 3, 7, 12-triketo-5β-cholan-24-oic acid.     

Rapid hydrolysis of bile acid conjugates using microwaves: Retention of absolute stereochemistry in the hydrolysis of (25R) 3α,7α,12α-trihydroxy-5β-cholestan-26-oyltaurine

In recent years, defects of bile acid synthesis caused by disorders of peroxisome biogenesis have led to increased interest in C₂₇ bile acids. In humans, while the majority of bile acids are C₂₄ carboxylic acids, the presence of increased concentrations of C₂₇ bile acids and their metabolites in hereditary diseases associated with peroxisomal dysfunction can serve as a useful marker for the intensity of the metabolic disorder. Our present studies describe an efficient method for the rapid hydrolysis of C₂₇ and C₂₄ bile acid conjugates using a commercial microwave oven. The advantages of this method include freedom from racemization, minimal activation, mild reaction conditions, and the highly stereocontrolled nature of the reaction, thus allowing for free bile acid recovery in high yield. For example, when (25R) 3α,7α,12α-trihydroxy-5β-cholestan-26-oyl taurine, a major compound present in the bile of Alligator mississippiensis, was deconjugated with 4% NaOH/diethylene glycol or 1 M LiOH/propylene glycol in the microwave oven for 4–6 min, 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid (THCA) was obtained in 81% yield with retention of configuration at C-25. It is suggested that present studies will be helpful in delineating the absolute stereochemistry of 3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA oxidase, the peroxisomal enzyme that catalyzes the first step in the oxidation of THCA.     

Biosynthesis of cholic acid in rat liver: Formation of cholic acid from 3α, 7α, 12α-trihydroxy- and 3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoic acids

Conversion of 3α,7α,12α-trihydroxy-5β-cholestanoic acid into 3α,7α,12α24-tetrahydroxy-5β-cholestanoic and cholic acids was catalyzed either by the mitochondrial fraction fortified with coenzyme A, ATP, MgCl₂ and NAD or by the combination of microsomal fraction and 100,000 x g supernatant fluid fortified with coenzyme A, ATP and NAD. 24-Hydroxylation and formation of cholic acid occurred at similar rates with the 25R- and the 25S-forms of 3α,7α,12α-trihydroxy-5β-cholestanoic acid. The 25R- and 25S-forms of 3α,7α,12α-trihydroxy-and 3α,7α,12α,24-tetrahydroxy-5β-cholestanoic acids were administered to bile fistula rats. Labeled cholic acid was isolated from the bile. The initial specific radioactivity of cholic acid was higher and the disappearance of radioactivity more rapid after administration of 3α,7α,12α-trihydroxy-5β-cholestanoic acid than of 3α,7α,12α,24-tetrahydroxy-5β-cholestanoic acid. The findings are discussed in relation to the assumed pathway for side chain cleavage in cholic acid biosynthesis.     

Effects of bile acid oxazolines on gallstone formation in prairie dogs

The effects of 2 bile acid analogs, chenodeoxy-oxazoline [2-(3α,7α-dihydroxy-24-nor-5β-cholanyl)-4,4-dimethyl-2-oxazoline] and ursodeoxy-oxazoline [2-(3α, 7β-dihydroxy-24-nor-5β-cholanyl)-4,4-dimethyl-2-oxazoline] were examined in the prairie dog model of cholesterol cholelithiasis. Gallstones and biliary cholesterol crystals were induced in 5 out of 6 male prairie dogs fed a semisynthetic diet containing 0.4% cholesterol for 8 weeks. Six animals maintained on a low cholesterol control diet (0.08% cholesterol) exhibited neither gallstones nor biliary cholesterol crystals. The addition of 0.06% chenodeoxy-oxazoline to the lithogenic diet did not prevent induced cholelithiasis or the appearance of cholesterol crystals in bile. In contrast, 0.06% dietary ursodeoxy-oxazoline prevented gallstones in 5 out of 6 prairie dogs (but cholesterol crystals were present in the bile of 4 of these animals). Histologically, most of the livers from the prairie dogs fed the cholesterol-supplemented semisynthetic diet showed bile duct proliferation, inflammatory infiltration and fibrosis along the portal tracts. These pathologic changes were generally not ameliorated by adding chenodeoxy-oxazoline or chenodeoxy-oxazoline plus chenodeoxycholic acid to the diet. Portal tract pathology was markedly reduced in most animals by adding ursodeoxy-oxazoline to the cholesterol-supplemented diet. The pathologic changes overall could best be correlated with the presence of gallstones, but not with the incidence of biliary cholesterol crystals.     

Effect of 7-methylated bile acids and bile alcohols on cholesterol metabolism in hamsters

The effect of 7-methyl substituted bile acid and bile alcohol analogues on cholesterol metabolism was studied in the hamster. Animals were fed chow plus 0.1% cholesterol supplemented with 0.1% of one of the following steroids: chenodeoxycholic acid, 7-methyl-chenodeoxy-cholic acid, 7β-methyl-24-nor-5β-cholestane-3α,7α,25-triol, cholic acid, 7-methyl-cholic acid, or 7β-methyl-24-nor-5β-cholestane-3α,7α,12α,25-tetrol. Cholesterol absorption was determined from fecal analysis after feeding of radiolabeled cholesterol and β-sitosterol. Of the six compounds studied, chenodeoxycholic acid and 7-methyl-chenodeoxycholic acid decreased intestinal cholesterol absorption (17% and 31% decrease, respectively). Only 7-methyl-chenodeoxycholic acid decreased serum cholesterol concentration (29% decrease), but there were no analogous changes of liver and biliary cholesterol concentration and cholesterol saturation of bile. Total fecal neutral sterol excretion was increased in the groups fed chenodeoxycholic acid and 7-methyl-chenodeoxycholic acid. In addition, the production of coprostanol was increased in both groups. These data suggest that 7-methyl-chenodeoxycholic acid resembles chenodeoxycholic acid in its effect on cholesterol metabolism and may be a potential candidate for further studies of its gallstone-dissolving properties.     

Intracerebrally injected monohydroxy and other C24 steroid acids as demyelinating agents in the guinea pig

Sodium salts of lithocholic acid (3α-hydroxy-5β-cholanoic acid), 5β-cholanoic acid, Δ⁵-cholenoic acid and 3-keto-5β-cholanoic acid injected intracerebrally into guinea pigs in doses of 1 mg or higher produced periventricular demyelination. 24-¹⁴C-sodium lithocholate was rapidly released from the brain (only traces remained 2 hr after injection) if injected in quantities ranging from 2 μg to 5 mg. This rapid elimination is believed to account for the relatively high dose of lithocholate required for producing demyelination, and may also account for the limited demyelinating capacity of the other acids injected intracerebrally.     

Differential effects of ursodeoxycholic acid and ursocholic acid on the formation of biliary cholesterol crystals in mice

The preventive effect of 3α,7β,12α-trihydroxy-5β-cholanoic acid (ursocholic acid) and ursodeoxycholic acid on the formation of biliary cholesterol crystals was studied in mice. Cholesterol crystals developed with 80% incidence after feeding for five weeks a lithogenic diet containing 0.5% cholesterol and 0.25% sodium cholate. When 0.25% ursocholic acid or ursodeoxycholic acid was added to the lithogenic diet, the incidence as well as the grade (severity) of the gallstones were reduced. Plasma and liver cholesterol levels were decreased by ursodeoxycholic acid but not by ursocholic acid. Gallbladder cholesterol and phospholipid levels were decreased by both bile acids. The biliary bile acid level was decreased by ursocholic acid but not by ursodeoxycholic acid. After feeding ursocholic acid, its level in the bile was about 25% and the levels of cholic acid and β-muricholic acid decreased. Fecal sterol excretion was not changed by ursocholic acid, but was increased by ursodeoxycholic acid. After feeding ursocholic acid, fecal excretion of deoxycholic acid, cholic acid, and ursocholic acid increased. No differences were found between mice, with or without gallstones, in plasma and liver cholesterol levels, biliary phospholipid and bile acid levels, fecal sterol and bile acid levels, and biliary and fecal bile acid composition. The results suggest that the lower incidence of crystal formation after treatment with ursocholic acid is probably by a different mechanism than with ursodeoxycholic acid. In the mouse model, ursodeoxycholic acid exerts its effect at least partially, by decreasing cholesterol absorption. Ursocholic acid is well absorbed and excreted into bile and transformed into deoxycholic acid by the intestinal microflora in mice.     

Effect of chitosan feeding on intestinal bile acid metabolism in rats

The effect of chitosan feeding (for 21 days) on intestinal bile acids was studied in male rats. Serum cholesterol levels in rats fed a commercial diet low in cholesterol were decreased by chitosan supplementation. Chitosan inhibited the transformation of cholesterol to coprostanol without causing a qualitative change in fecal excretion of these neutral sterols. Increased fiber consumption did not increase fecal excretion of bile acids, but caused a marked change in fecal bile acid composition. Litcholic acid increased sigificantly, deoxycholic acid increased to a leasser extent, whereas hyodeoxycholic acid and the 6β-isomer and 5-epimeric 3α-hydroxy-6-keto-cholanoic acid(s) decreased. The pH in the cecum and colon became elevated by chitosan feeding which affected the conversion of primary bile acids to secondary bile acids in the large intestine. In the cecum, chitosan feeding increased the concentration of α-,β-, and ω-muricholic acids, and lithocholic acid. However, the levels of hyodeoxycholic acid and its 6β-isomer, of monohydroxy-monoketo-cholanoic acids, and of 3α, 6ξ, 7ξ-trihydroxy-cholanoic acid decreased. The data suggest that chitosan feeding affects the metabolism of intestinal bile acids in rats.     

Metabolism and cholestatic effect of 3α-hydroxy-7ζ-methyl-5β-cholanoic acid

3α-Hydroxy-7ζ-methyl-5β-cholanoic acid (7ζ-methyl-LA) was infused intravenously into bile fistula hamsters to investigate its metabolism and effect on the bile flow as compared with lithocholic acid. Following infusion of the labeled bile acids, bile was collected quantitatively to allow measurement of bile flow and bile acid composition. More than 80% of radioactivity was recovered in bile within 4 hr. 7ζ-Methyl-LA and lithocholic acid in bile were present as the taurine and glycine conjugates; no free bile acids were detected. 7ζ-Methyl-LA was neither hydroxylated nor metabolized to any measurable extent, though lithocholic acid was 7α-hydroxylated to chenodeoxycholic acid (30–45%). At the infusion rate at which lithocholic acid induced a severe cholestasis (264 nmol/min), 7ζ-methyl-LA did not decrease the bile flow. In fact, the infusion of 7ζ-methyl-LA produced a mild choleresis under conditions where endogenous bile acid excretion was not changed appreciably compared to control infusions with albumin. It is concluded that 7ζ-methyl-LA is not metabolized in the hamster but is conjugated with taurine and glycine, and that the introduction of a methyl group at the 7-position of lithocholic acid appears to alleviate the cholestatic effect of lithocholic acid in the hamster.     

Studies on the 12α and 26-hydroxylation of bile alcohols by rabbit liver microsomes

12α-Hydroxylation of two C₂₇-steroids by rabbit liver microsomes was studied. Optimal assay conditions were determined with 7α-hydroxy-4-cholesten-3-one and 5β-cholestane-3α,7α-diol as substrates. The rate of 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was found to be greater than that of 5β-cholestane-3α,7α-diol by ca. 60%. Microsomal 26-hydroxylation of 5β-cholestane-3α7α-diol was also measured, and the ratio of 26-hydroxylation to 12α-hydroxylation of 5β-cholestane-3α,7α-diol was found to be ca. 0.4. Rabbit liver 12-αhydroxylase was more active than that of three other species (man, rat, monkey), explaining in part the predominance of cholic acid in rabbit bile.     

Anomalous enantioselectivity in the sharpless asymmetric dihydroxylation reaction of 24-nor-5β-cholest-23-ene-3α,7α,12α-triol: Synthesis of substrates for studies of cholesterol side-chain oxidation

Recently we described a block in bile acid synthesis in cerebrotendinous xanthomatosis (CTX), a lipid storage disease related to an inborn error of bile acid metabolism. In this disease a defect in hepatic microsomal (24S) hydroxylation blocks the transformation of 5β-cholestane-3α,7α,12α,25-tetrol into (24S) 5β-cholestane-3α,7α,12α,24,25-pentol and cholic acid. Mitochondrial cholesterol 27-hydroxylation has also been reported to be abnormal in CTX subjects, but the relative importance of the enzymatic defect in this alternative microsomal pathway (namely, the 24S hydroxylation of 5β-cholestane-3α,7α,12α,25-tetrol relative to the abnormality in mitochondrial 27-hydroxylase) has not been established in CTX. To delineate the sequence of side-chain hydroxylations and the enzymatic block in bile acid synthesis, we synthesized the (23 R and 23 S) 24-nor-5β-cholestane-3α,7α,12α,23,25-pentols utilizing a modified Sharpless asymmetric dihydroxylation reaction on 24-nor-5β-cholest-23-ene-3α,7α,12α-triol, a C₂₆ analog of the naturally occurring C₂₇ bile alcohol, 5β-cholest-24-ene-3α,7α,12α-triol. Stereospecific conversion of the unsaturated 24-nor triol to the corresponding chiral compounds (23R and 23S), 24-nor-5β-cholestane-3α,7α,12α,23,25-pentols, was quantitative. However, conversion of the unsaturated 24-nor triol to the chiral nor-pentols had absolute stereochemistry opposite to the products predicted by the Sharpless steric model. The absolute configurations and enantiomeric excess of the C₂₆ nor-pentols and the C₂₇ pentols (synthesized from 5β-cholest-24-ene-3α,7α,12α-triol for comparison) were confirmed by nuclear magnetic resonance and lanthanide-induced circular dichroism Cotton effect measurements. These results may contribute to a better understanding of the role of the 24S-hydroxylation vs. 27-hydroxylation step in cholic acid biosynthesis. Presented in part at the 216th American Chemical Society National Meeting (Medicinal Chemistry Division, Abs. 368), Boston, MA, August 21–26, 1998.     

Bile acids LVII. Analysis of bile acids by high pressure liquid chromatography and mass spectrometry

Several high pressure liquid chromatographic methods for the separation of conjugated and free bile acids are presented. A mixture of synthetic conjugated bile acids has been separated by reverse-phase systems consisting of either a Waters Associates’ “fatty-acid analysis” or a μBondapak/C₁₈ column eluted with a mixture of 2-propanol/potassium phosphate buffer (pH 2.5 or 7.0). The major conjugated bile acids present in the gallbladder bile of obese subjects have been analyzed each in less than 30 min and quantitated with a U.V. detector set at 193 nm. Some of the 5α- and 5β-isomers of conjugated bile salts could be resolved in straight-phase systems on Corasil II or μPorasil columns. Mass spectra of the conjugated bile acids obtained by electron impact were characteristic of the type of amino acid attached to the side chain, and the number of hydroxyl substituents on the nucleus. Most of the isomers could readily be differentiated by the relative intensities of the fragment ions.     

Rapid analysis of oxidized cholesterol derivatives by high-performance liquid chromatography combined with diode-array ultraviolet and evaporative laser light-scattering detection

Extensive evidence of the deleterious biological effects of oxidized 5-cholesten-3β-ol (cholesterol) derivatives has led to great interest in their detection. We observed that known oxidized cholesterol derivatives can be rapidly quantitated by combining reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorption and evaporative laser light-scattering (ELSD) detection. Using a 20 × 0.46 cm C18 HPLC column and methanol/acetonitrile (60:40, vol/vol) as the mobile phase at 1.0 mL/min, 10 species of oxidized cholesterol derivative were measured by UV (205, 234, and 280 nm) while 5-cholestan-5α,6α-epoxy-3β-ol (5α-epoxycholesterol), 5-cholestan-5β,6β-epoxy-3β-ol (5β-epoxycholesterol), and 5-cholestan-3β,5α,6β-triol (cholestanetriol) were detected by only ELSD. The minimal limits of detection ranged from 100 to 500 ng depending on sterol and detector. The response was linear in the range 0–1000 or 0–2000 ng depending on detector. These oxidized cholesterol derivatives were also identified by HPLC/mass spectrometry analysis combined with UV detector. Heated tallow contained cholestanetriol, 5-cholesten-3β,7α-diol (7α-hydroxycholesterol), 5-cholesten-3β,7β-diol (7β-hydroxycholesterol), 5-cholesten-3β-ol-7-one (7-ketocholesterol), 5α- and 5β-epoxycholesterols under the developed analysis condition. Photooxidized cholesterol had cholestanetriol, 7α- and 7β-hydroxycholesterols and 3,5-cholestadien-7-one. On the other hand, 7α- and 7β-hydroxycholesterols, 7-ketocholesterol, 5α- and 5β-epoxycholesterols and 3,5-cholestadien-7-one were observed in copper-oxidized low-density lipoprotein. Thus, this developed HPLC analysis method could be applied to identification of oxidized cholesterol derivatives in food and biological specimen.     

Synthesis and metabolism of sodium 3α,7α-dihydroxy-25,26-bishomo-5β-cholane-26-sulfonate in the hamster

This paper reports the chemical synthesis of a new bile acid analogue, namely sodium 3α,7α-dihydroxy-25,26-bishomo-5β-cholane-26-sulfonate (bishomoCDC-sul) from chenodeoxycholic acid and describes its metabolism in the hamster. The structure of the new compound was confirmed by proton and carbon-13 nuclear magnetic resonance spectroscopy. After intravenous infusion of [³H]-labeled sulfonate into bile fistula hamsters, it was extracted by the liver and secreted into the bile; more than 65% of the radioactivity was recovered in the bile within 1 h. Following intraduodenal administration of the [³H]sulfonate and [¹⁴C]chenodeoxycholyltaurine, both compounds were excreted into the bile more slowly; only 41 and 43% of the radioactivity, respectively, were recovered in the bile during the four-hour experimental period. In contrast, when the labeled compounds were injected into the terminal ileum, both the sulfonate and chenodeoxycholyltaurine were repidly absorbed and secreted into the bile; 84 and 97%, respectively, of the radioactivity were recovered during a four-hour period. Chromatographic analysis demonstrated that in these short-term experiments most (>95%) of the sulfonate was secreted into the bile without biotrasformation regardless of the route of administration. When infused intravenously at increasing doses, bishomoCDC-sul induced cholestasis at an infusion rate of 1 μmol/min/kg. These results suggest that sodium 3α,7α-dihydroxy-25,26-bishomo-5β-cholane-26-sulfonate was absorbed from the terminal ileum by active transport, extracted by the liver, and secreted into the bile in a manner similar to that of the natural bile acids.     

Functionalization of unactivated carbons in 3α,6- and 3α,24-dihydroxy-5β-cholane derivatives by dimethyldioxirane

Direct remote functionalization of unactivated carbons by dimethyldioxirane (DMDO) was examined for 3α,6- and 3α,24-dihydroxy-5β-cholane derivatives. DMDO oxidation of stereoisomeric methyl 3α,6-diacetoxy-5β-cholanoates caused the direct, unexpected 14α- and 17α-hydroxylations, in analogy with that of the 5α-H analogs, regardless of the differences in stereochemical configuration of the A/B-ring junction and of the acetoxyl groups at C-3 and C-6. On the other hand, the ester derivatives of 3α,24-dihydroxy-5β-cholane with DMDO were transformed into the corresponding 5β-, 14α-, and 17α-hydroxy compounds, whereas the ether derivatives yielded the 5β-hydroxy, 3-oxo, and C-24 oxidized products, accompanied by their dehydrated ones.     

Effects of feeding chenodeoxycholic acid on metabolism of cholesterol and bile acids in germ-free rats

The aim of this investigation was to study the influence of chenodeoxycholic acid administration on cholesterol and bile acid synthesis in germ-free rats. Seven rats were fed a basal diet and 2 groups of 4 rats received the same diet supplemented with 0.4 and 1% chenodeoxycholic acid, respectively. After 6 weeks, feces were collected in one 3- and one 4-day pool for analysis of cholesterol and bile acids. When the sampling period was finished, the rats were killed and the liver microsomal fractions isolated. The activities of HMG CoA reductase and cholesterol 7α-hydroxylase were determined, the 7α-hydroxylase by a mass fragmentographic method. The 2 dominating bile acids in the untreated rats were cholic acid and β-muricholic acid. During treatment with chenodeoxycholic acid, 60–70% of this bile acid was converted into α- and β-muricholic acid, indicating a high activity of the 6β-hydroxylase. The excretion of cholic acid was almost completely inhibited and the 7α-hydroxylase activity was decreased ca 75% in the rats fed 1% chenodeoxycholic acid. The activity of the hepatic HMG CoA reductase was unchanged. The fecal excretion of cholesterol increased 2–3 times. An accumulation of cholesterol was seen in the rats treated with 1% chenodeoxycholic acid, which was probably a result of the decreased catabolism of cholesterol to bile acids.     

New Nortriterpenoid and Ceramides From Stems and Leaves of Cultivated <Emphasis Type="Italic">Triumfetta cordifolia</Emphasis> A Rich (Tiliaceae)

To highlight the role of plants in traditional healing, the leaves and the stems of cultivated Triumfetta cordifolia were phytochemically studied yielding a new nor-ursane type (1), a new ceramide (2) and a new piperidinic ceramide derivative (3) named, respectively, 2α,19α-dihydroxy-3-oxo-23-nor-urs-12-en-28-oic acid, (2R)-2-hydroxy-N-[(2S,3S,4R,26E)-1,3,4-trihydroxy-26-triaconten-2-yl] tetradecanamide and (2R,8Z)-2-hydroxy-{(2S,3R,5R,6S)-3,5-dihydroxy-6-[(1E,5Z)-hexadeca-1,5-dienyl]-2-(β-d-glucopyranosyloxy)methyl piperidine-1-yl} tetracos-8-enamide (3). These were obtained together with lupeol (4), stigmasterol (5), 3-O-β-d-glucopyranoside of β-sitosterol (6), tormentic acid (7) from stems and heptadecanoic acid (8), β-carotene (9), oleanolic acid (10), and 24-hydroxytormentic acid (11) from leaves. The structures were determined on the basis of NMR data (¹H-, ¹³C-, 2D-NMR analyses), mass spectrometry and confirmed by chemical transformations as well as comparison of spectral data with those reported in the literature. The FRAP method was used to evaluate the antioxidant activity of fractions collected from flash chromatography and isolated compounds. Among the fractions, four reduced Fe^III-TPTZ to Fe^II-TPTZ while isolated pure compounds showed no activity.     

Structure-retention correlation of isomeric bile acids in inclusion high-performance liquid chromatography with methyl β-cyclodextrin

The structure-retention correlation of various C₂₄ bile acid isomers was studied by the addition of methyl β-cyclodextrin (Me-β-CD) to mobile phases in reversed-phase high-performance liquid chromatography (HPLC). The compounds examined include a series of monosubstituted bile acids related to cholanoic acids differing from one another in the position and configuration of an oxygen-containing function (hydroxyl or oxo group) at the position C-3, C-6, C-7, or C-12 and the stereochemistry of the A/B-ring fusion (trans 5α-H and cis 5β-H) in the steroid nucleus. The inclusion HPLC with Me-β-CD was also applied to biologically important 4β- and 6-hydroxylated bile acids substituted by three to four hydroxyl groups in the 5β-steroid nucleus. These bile acid samples were converted into their fluorescence prelabeled 24-pyrenacyl ester derivatives and chromatographed on a Capcell Pak C₁₈ column eluted with methanol-water mixtures in the presence or absence of 5 mM Me-β-CD. The effects of Me-β-CD on the retentions of each compound were correlated quantitatively to the decreasing rate of capacity factors and the relative strength of host-guest inter-actions. On the basis of the retention data, specific and nonspecific hydrogen-bonding interactions between the bile acids and the Me-β-CD were discussed.