Comparative studies of bile salts. 5α-Chimaerol, a new bile alcohol from the white sucker Catostomus commersoni Lacépède

1. G.l.c. examination of bile alcohols prepared from the sucker Catostomus commersoni Lacépède (family Catostomidae) showed that although 5α-cyprinol (5α-cholestane-3α,7α,12α,26,27-pentol) was a minor constituent, the principal bile alcohol was an undescribed substance, probably present in the bile as the C-26 sulphate ester, whose i.r., n.m.r. and mass spectra agreed with the structure 5α-cholestane-3α,7α,12α,24,26-pentol. 2. M_D studies suggest that this 5α-chimaerol is the 24(+), 25S enantiomer and that 5β-chimaerol (chimaerol) from Chimaera monstrosa bile also has the 24(+), 25S configuration. These findings imply that bile alcohol biosynthesis in suckers and chimaeras includes stereospecific oxidation of cholesterol at C-26. 3. C. commersoni bile acids (present in minor amounts) probably consist largely of 3α,7α,12α-trihydroxy-5α-cholan-24-oic acid (allocholic acid). 4. 5α-Chimaerol sulphate and 5α-cyprinol sulphate are probably biochemically equivalent as bile salts, and can be considered as arising by parallel evolution.     

Bile salts of germ-free domestic fowl and pigs

1. The bile of germ-free domestic fowl contains taurine conjugates of 3α,7α-dihydroxy-5β-cholan-24-oic acid (chenodeoxycholic acid), 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid (cholic acid) and its 5α-epimer (allocholic acid): that of germ-free pigs contains glycine and taurine conjugates of chenodeoxycholic acid, 3α,6α-dihydroxy-5β-cholan-24-oic acid (hyodeoxycholic acid), 3α,6α,7α-trihydroxy-5β-cholan-24-oic acid (hyocholic acid) and (probably) cholic acid. Keto acids were not found. 2. Allocholic acid and hyodeoxycholic acid are thus proved to be primary bile acids in intact animals. 3. The evolutionary and biochemical implications of these findings are briefly considered.     

Formation of Hyodeoxycholic Acid from Muricholic Acid and Hyocholic Acid by an Unidentified Gram-Positive Rod Termed HDCA-1 Isolated from Rat Intestinal Microflora

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3α,6β,7β-trihydroxy bile acid β-muricholic acid into hyodeoxycholic acid (3α,6α-dihydroxy acid) by dehydroxylation of the 7β-hydroxy group and epimerization of the 6β-hydroxy group into a 6α-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3α,6α,7α-trihydroxy acid), α-muricholic acid (3α,6β,7α-trihydroxy acid), and ω-muricholic acid (3α,6α,7β-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted β-muricholic acid almost completely into hyodeoxycholic acid.     

Bile acids. LXIV. Synthesis of 5α-cholestane-3α,7α,25-triol and esters of new 5α-bile acids

Interest in the structural requirements of a sterol or bile acid for maximal activity by an hepatic microsomal steroid 12α-hydroxylase prompted the preparation of 5α-cholestane-3α, 7α, 25-triol and 5α-analogs of 3α, 7α-dihydroxy-5β-cholane-24-carboxylic acid. Methyl 3α, 7α-dihydroxy-5β-cholane-24-carboxylate derived from methyl chenodeoxycholate via the Arndt-Eistert reaction was allomerized with Raney nickel in boiling p-cymene to provide a number of products of which methyl 3,7-dioxo-5β- and 5α-cholane-24-carboxylates, methyl 3-oxo-7α-hydroxy-5β-and 5α-cholane-24-carboxylates, were identified. Reduction with K-Selectride of methyl 3-oxo-7α-hydroxy-5β-cholane-24-carboxylate, provided a high yield of methyl 3α, 7α-dihydroxy-5α-cholane-24-carboxylate. Treatment of this ester with an excess of methyl magnesium iodide afforded 5α-cholestane-3α, 7α, 25-triol. The products were characterized by thin-layer and gas liquid chromatography, proton resonance, infrared and mass spectrometry.     

The partial characterization of 1-oxygenated steroids from urine of a hypertensive newborn child

1. Four substances from the urine of a hypertensive newborn girl were partially characterized and shown to be 17α-hydroxy-5β-pregnane-1,3,20-trione, 3α,17α-dihydroxy-5β-pregnane-1,20-dione, 3α,17α,20α-trihydroxy-5β-pregnan-1-one and 5β-pregnane-1β,3α,17α,20α-tetrol. 2. The characterization rested mainly on R_M analysis of the substances and their derivatives by glycol fission, providing evidence for position and degree of substitution and for steroidal character. Supporting evidence was provided by chemically specific location reactions. 3. Certain problems in the manipulation of these β-disubstituted steroids are discussed.     

Substrate and inhibitor specificity of the cholesterol oxidase in bovine adrenal cortex

1. Cholesteryl 3β-sulphate is oxidized in vitro by preparations of bovine adrenal-cortex mitochondria to pregnenolone sulphate and isocaproic acid (4-methyl-pentanoic acid) without hydrolysis of the ester linkage. 2. Free cholesterol is the preferred substrate for adrenal-cortex cholesterol oxidase; the apparent K_m for cholesteryl sulphate is 500μm and for free cholesterol 50μm under the same conditions. 3. Cholesteryl 3β-acetate is hydrolysed by bovine adrenal-cortex mitochondria in vitro to free cholesterol, which is subsequently oxidized to more polar steroids and isocaproic acid. Evidence was obtained that other cholesterol esters behave similarly. Cholesterol esters may thus act as precursors of steroid hormones. 4. Cholest-4-en-3-one is only poorly oxidized to isocaproic acid and more polar steroids and thus is probably not a significant precursor of steroid hormones. 5. Cholesteryl esters inhibit the oxidation of cholesterol competitively (K_i for cholesteryl phosphate 28μm, for cholesteryl sulphate 110μm, for cholesteryl acetate 65μm) but pregnenolone esters do not inhibit this system. 6. Pregnenolone and 20α-hydroxycholesterol (both metabolites of cholesterol in this system) inhibit the oxidation of cholesterol non-competitively. K_i for pregnenolone is 130μm and K_i for 20α-hydroxycholesterol is 17μm. 7. 25-Oxo-27-norcholesterol inhibits cholesterol oxidation non-competitively (K_i16μm). A number of other Δ⁵-3β-hydroxy steroids inhibit cholesterol oxidation and evidence was obtained that the 3β-hydroxyl group was necessary for inhibitory activity. 8. Pregnenolone, 20α-hydroxycholesterol and 25-oxo-27-norcholesterol inhibit oxidation of cholesteryl sulphate by this system but their sulphates do not. 9. 3β-Hydroxychol-5-enoic acid, 3α-hydroxy-5β-cholanic acid and 3β-hydroxy-22,23-bisnorchol-5-enoic acid stimulated formation of isocaproic acid from cholesterol. 10. No evidence was obtained that phosphorylation or sulphation are obligatory steps in cholesterol oxidation by adrenal-cortex mitochondria. 11. The cholesteryl 3β-sulphate sulphatase of bovine adrenal cortex was found mostly in the microsomal fraction and was inhibited by inorganic phosphate.     

Identification of bile alcohols in human bile

Human gallbladder bile was examined for bile alcohols. Following isolation and hydrolysis, the bile alcohols were analyzed by capillary gas-liquid chromatography-mass spectrometry. The following bile alcohols were identified with certainty by direct comparison with reference standards: 5 beta-cholane-3 alpha,-7 alpha,23,24-tetrol; 5 beta-cholane-3 alpha,7 alpha,12 alpha,24-tetrol; 24-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol; 27-nor-5 beta-cholest-25-ene-3 alpha,7 alpha,-12 alpha,24-tetrol; 3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one; 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol; 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol; 5 beta-cholestane-3 alpha,7 alpha,24-triol; 5 beta-cholestane-3 alpha,7 alpha,25-triol; 5 beta-cholestane-3 alpha,7 alpha,26-triol; 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol; (24R)- and (24S)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentols; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,-25,26-pentol; 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26,27-pentol; 26-methoxy-5 beta-cholestane-3 alpha,7 alpha,12 alpha,25-tetrol. There also existed two norcholestanetetrols and three cholestanetetrols with two hydroxyl substituents on the nucleus and two in the side chain. The human biliary bile alcohols occurred mainly as sulfate esters and in lesser amounts as glucuronoconjugated and unconjugated forms. The amount of total bile alcohols was about 0.9 mg (0.7-1.2 mg) in 1 g of bile solid, or 0.16 mumol (0.07-0.24 mumol) in 1 ml of gallbladder bile.     

Electrophysiological evidence for detection and discrimination of pheromonal bile acids by the olfactory epithelium of female sea lampreys (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

Electro-olfactograms were used to determine sensitivity and specificity of olfactory organs of female sea lampreys (Petromyzon marinus) to four bile acids: 3-keto petromyzonol sulfate and 3-keto allocholic acid from spermiating males and petromyzonol sulfate and allocholic acid from larvae. Spermiating male bile acids are thought to function as a mating pheromone and larval bile acids as a migratory pheromone. The response threshold was 10^–12 mol l^–1 for 3-keto petromyzonol sulfate and 10^–10 mol l^–1 for the other bile acids. At concentrations above 10^–9 mol l^–1, the sulfated bile acids showed almost identical potency, as did the non-sulfated bile acids. The two sulfated bile acids were more potent than the two non-sulfated ones. In addition, 3-keto petromyzonol sulfate and water conditioned with spermiating males induced similar concentration-response curves and response thresholds. Cross-adaptation experiments demonstrated that the sulfated and non-sulfated bile acids represent different odors to the olfactory epithelium of females. Further exploration revealed that 3-keto petromyzonol sulfate represents a different odor than petromyzonol sulfate, while 3-keto allocholic acid and allocholic acid represent the same odor. Results indicate that male-specific bile acids are potent and specific stimulants to the female olfactory organ, supporting the previous hypothesis that these bile acids function as a pheromone.Abbreviations 3kACA 3-keto allocholic acid - 3kPZS 3-keto petromyzonol sulfate - ACA allocholic acid - ANOVA analysis of variance - ELISA enzyme-linked immunosorbent assay - EOG electro-olfactogram - PIR percent initial response - PZS petromyzonol sulfate - SMW spermiating male washings     

The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C₅ acyl side chain of the C₂₄ steroid compound cholate involves the C₂₂ intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C₂₄ steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD⁺ as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.     

Synthesis of biological precursors of cholic acid

This paper describes a new and convenient procedure for the synthesis of 5β-cholestane-3α,7α,12α,24-tetrol(24R and 24 S) and 5β-cholestane-3α, 7α, 12α, 26-tetrol starting from 5β-cholestane-3α,7α,12α,25-tetrol. Dehydration of the 25-hydroxytetrol with glacial acetic acid and acetic anhydride yielded a mixture of 5β-cholest-24-ene-3α,7α,12α-triol and the corresponding Δ²⁵ compound. Hydroboration and oxidation of the mixture of Δ²⁴ and Δ²⁵ unsaturated bile alcohols resulted in the formation of 5β-cholestane-3α,7α,12α,24ξ-tetrol and 5β-cholestane-3α,7α,12α,26-tetrol. In addition, smaller amounts of 5β-cholestane-3α, 7α, 12α, 23ξ-tetrol and 5β-cholestane-3α, 7α, 12α-triol were also obtained.The bile alcohols epimeric at C-24 were resolved by analytical and preparative TLC, characterized by gas-liquid chromatography and mass-spectrometry. Tentative assignments of the 24R and 24S configuration was made on the basis of molecular rotation differences. These compounds will be useful for biological studies of cholic acid biosynthesis.     

Identification of genes involved in inversion of stereochemistry of a C-12 hydroxyl group in the catabolism of cholic acid by Comamonas testosteroni TA441

Comamonas testosteroni TA441 degrades steroids such as testosterone via aromatization of the A ring, followed by meta-cleavage of the ring. In the DNA region upstream of the meta-cleavage enzyme gene tesB, two genes required during cholic acid degradation for the inversion of an α-oriented hydroxyl group on C-12 were identified. A dehydrogenase, SteA, converts 7α,12α-dihydroxyandrosta-1,4-diene-3,17-dione to 7α-hydroxyandrosta-1,4-diene-3,12,17-trione, and a hydrogenase, SteB, converts the latter to 7α,12β-dihydroxyandrosta-1,4-diene-3,17-dione. Both enzymes are members of the short-chain dehydrogenase/reductase superfamily. The transformation of 7α,12α-dihydroxyandrosta-1,4-diene-3,17-dione to 7α,12β-dihydroxyandrosta-1,4-diene-3,17-dione is carried out far more effectively when both SteA and SteB are involved together. These two enzymes are encoded by two adjacent genes and are presumed to be expressed together. Inversion of the hydroxyl group at C-12 is indispensable for the subsequent effective B-ring cleavage of the androstane compound. In addition to the compounds already mentioned, 12α-hydroxyandrosta-1,4,6-triene-3,17-dione and 12β-hydroxyandrosta-1,4,6-triene-3,17-dione were identified as minor intermediate compounds in cholic acid degradation by C. testosteroni TA441.     

New evidence for the structure of myxinol

1. Preliminary spectroscopic examination of a second component of hagfish bile salts suggested that it might be 3β,7α,26(27)-trihydroxy-5α-cholestane. 2. Impure reduction products of the 3β,26(27)-dihydroxycholestane-7,16-dione previously made from myxinol disulphate appeared also to have the 5α-configuration. 3. Infrared, nuclear-magnetic-resonance and mass-spectrographic as well as optical-rotatory-dispersion measurements on 3β,26(27)-dihydroxycholestane-7,16-dione showed that it was a 5α-compound. 4. Myxinol is thus 3β,7α,16α,26(27)-tetrahydroxy-5α-cholestane; new nuclear-magnetic-resonance measurements on myxinol tetra-acetate at higher resolution confirm this structure.     

Isoform-selective Oligomer Formation of Saccharomyces cerevisiae p24 Family Proteins

p24 family proteins are evolutionarily conserved transmembrane proteins involved in the early secretory pathway. Saccharomyces cerevisiae has 8 known p24 proteins that are classified into four subfamilies (p24α, -β, -γ, and -δ). Emp24 and Erv25 are the sole members of p24β and -δ, respectively, and deletion of either destabilizes the remaining p24 proteins, resulting in p24 null phenotype (p24Δ). We studied genetic and physical interactions of p24α (Erp1, -5, and -6) and γ (Erp2, -3, and -4). Deletion of the major p24α (Erp1) partially inhibited p24 activity as reported previously. A second mutation in either Erp5 or Erp6 aggravated the erp1Δ phenotype, and the triple mutation gave a full p24Δ phenotype. Similar genetic interactions were observed among the major p24γ (Erp2) and the other two γ members. All the p24α/γ isoforms interacted with both p24β and -δ. Interaction between p24β and -δ was isoform-selective, and five major α/γ pairs were detected. These results suggest that the yeast p24 proteins form functionally redundant αβγδ complexes. We also identified Rrt6 as a novel p24δ isoform. Rrt6 shows only limited sequence identity (∼15%) to known p24 proteins but was found to have structural properties characteristic of p24. Rrt6 was induced when cells were grown on glycerol and form an additional αβγδ complex with Erp3, Erp5, and Emp24. This complex was mainly localized to the Golgi, whereas the p24 complex containing Erv25, instead of Rrt6 but otherwise with the same isoform composition, was found mostly in the ER.     

Altered metabolism of bile acids in cholestasis: Determination of 1β- and 6α-hydroxylated metabolites

Trihydroxy and tetrahydroxy bile acid metabolites substituted at the C-1 or C-6 position were studied using the urine, serum and liver tissue from sixteen patients with cholestatic liver diseases. Following extraction, isolation and hydrolysis, bile acids were converted into the dimethylethylsilyl derivatives and assayed by capillary gas chromatography—mass spectrometry. Five 1β-hydroxylated bile acids, viz. 1β,3α,12α-trihydroxy-, 1β,3α,7β-trihydroxy-1, 1β,3α,7α,12α-tetrahydroxy-5β-cholanoic acids and an epimer of the first compound, and two 6α-hydroxylated bile acids, viz. 3α,6α,7α-trihydroxy-, 3α,6α,7α,12α-tetrahydroxy-5β-cholanoic acids, were completely or partially identified. Large amounts of 1β-hydroxylated and 6α-hydroxylated bile acids were found in the urine, whereas only trace amounts were detected in the serum and liver tissue. These findings indicate that altered metabolism, such as 1β- or 6α-hydroxylation of bile acids, is enhanced in cholestasis, and that the resulting hydroxylated metabolites are eliminated in the urine.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Endogenous Gibberellins from Sporophytes of Two Tree Ferns, Cibotium glaucum and Dicksonia antarctica

Gibberellin A₁ (GA₁), 3-epi-GA₁, GA₄, GA₉, 11α-hydroxyGA₁₂, 12α-hydroxyGA₁₂, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₅, GA₃₇, GA₄₀, GA₅₈, GA₆₉, GA₇₀, and GA₇₁ have been identified from Kovats retention indices and full scan mass spectra by capillary GC-MS analyses of purified extracts from sporophytes of the tree fern, Cibotium glaucum. Abscisic acid, dihydrophaseic acid, an epimer of 4′-dihydrophaseic acid, and the epimeric ent-6α, 7α, 16α, 17-(OH)₄ and ent-6α, 7α, 16β, 17-(OH)₄ derivatives of ent16, 17-dihydrokaurenoic acid, in addition to the epimeric 16α, 17- and 16β, 17-dihydroxy-16, 17-dihydro derivatives of GA₁₂, were also identified in extracts of C. glaucum. An oxodihydrophaseic acid and a hydroxydihydrophaseic acid were also detected. In extracts of sporophytes of Dicksonia antarctica, GA₄, GA₉, 12α- and 12β-hydroxyGA₁₂, GA₁₅, GA₂₅, and GA₃₇ were identified by the same criteria, as well as abscisic acid, phaseic acid, 8′-hydroxymethylabscisic acid and dihydrophaseic acid. This is the first time that GA₄₀ has been identified in a higher plant; it is also the first report of the natural occurrence of the two gibberellins, 11α- and 12β-hydroxyGA₁₂. The total gibberellin (GA) content in C. glaucum (tall) was at least one order of magnitude greater than that of D. antarctica (dwarf) based on total ion current response in GC-MS and bioassay data. Abscisic acid was a major component of D. antarctica and the oxodihydrophaseic acid was a major component of C. glaucum.     

Anti-inflammatory components of the Vietnamese starfish Protoreaster nodosus

Background

In the present study, we examined the inhibitory effects of a methanolic extract, dichloromethane fraction, water layer, and polyhydroxylated sterols (1–4) isolated from the Vietnamese starfish Protoreaster nodosus on pro-inflammatory cytokine (IL-12 p40, IL-6, and TNF-α) production in LPS-stimulated bone marrow-derived dendritic cells (BMDCs) using enzyme-linked immunosorbent assays (ELISA).

Results

The methanolic extract and dichloromethane fraction exerted potent inhibitory effects on the production of all three pro-inflammatory cytokines, with IC₅₀ values ranging from 0.60 ± 0.01 to 26.19 ± 0.64 μg/mL. Four highly pure steroid derivatives (1–4) were isolated from the dichloromethane fraction and water layer of P. nodosus. Potent inhibitory activities were also observed for (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) on the production of IL-12 p40 and IL-6 (IC_50s = 3.11 ± 0.08 and 1.35 ± 0.03 μM), and for (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) and (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) on the production of IL-12 p40 (IC_50s = 0.01 ± 0.00 and 1.02 ± 0.01 μM). Moreover, nodososide (4) exhibited moderate inhibitory effects on IL-12 p40 and IL-6 production.

Conclusion

This is the first report of the anti-inflammatory activity from the starfish P. nodosus. The main finding of this study is the identification oxygenated steroid derivatives from P. nodosus with potent anti-inflammatory activities that may be developed as therapeutic agents for inflammatory diseases.     

Cerebrospinal Fluid Steroidomics: Are Bioactive Bile Acids Present in Brain?

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C₂₇ and C₂₄ intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C₂₆ sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.     

Bile Salt Degradation by Nonfermentative Clostridia

Eight strains of nonfermentative clostridia were characterized on the basis of their intracellular nicotine adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent hydroxysteroid dehydrogenase (HSDH) content, ability to deconjugate taurocholate, growth characteristics, and metabolic products, including utilization of lactate and pyruvate. Two cultures of Clostridium sporosphaeroides (representing one strain obtained from two different sources), one strain of Clostridium irregularis, four strains of an unnamed species (Clostridium group SPH-1), and one strain of an unnamed species (Clostridium group P) were studied. Both cultures of C. sporosphaeroides contained low amounts of 7α-HSDH; C. irregularis contained only a low amount of 3α-HSDH. All four strains of Clostridium SPH-1 contained both 12α- and 7α-HSDH in the ratio of approximately 10:1. The strain of Clostridium group P contained only 12α-HSDH and was devoid of any other bile salt oxidoreductases. The enzyme preparation from Clostridium group P was useful in spectrophotometric quantitative studies of 12α-OH groups. Correlation of bile salt degradative activities with other phenotypic tests for characterization of and differentiation among such organisms is discussed.     

Bile salts of the West Indian manatee, Trichechus manatus latirostris: novel bile alcohol sulfates and absence of bile acids

The bile salts present in gallbladder bile of the West Indian manatee, Trichechus manatus latirostris, an herbivorous marine mammal of the tropical and subtropical margins of the Atlantic Ocean, were found to consist of a mixture of bile alcohol sulfates. Bile acids, previously believed to be present in all mammals, were not detected. Using chromatography, mass spectrometry, and 1H- and 13C-nuclear magnetic resonance spectroscopy, the major bile alcohol was identified as 5 beta-cholestane-3 alpha,6 beta,7 alpha-25,26-pentol; that is, it had the nuclear structure of alpha-muricholic acid and the side chain structure of bufol. This compound has not been described previously and the trivial name "alpha-trichechol" is proposed. The second most abundant compound was 5 beta-cholestane-3 alpha,7 alpha,25,26-tetrol. Other bile alcohols were tentatively identified as 5 beta-cholestane-3 alpha,6 beta,7 beta,25,26-pentol (named beta-trichechol), 3 alpha,6 alpha,7 beta, 25-26-pentol (named omega-trichechol) and 5 beta-cholestane-3 alpha,6 beta,7 alpha,26-tetrol. The 1H and 13C NMR spectra of the four 6,7 epimers of 3,6,7 trihydroxy bile acids are described and discussed. All bile alcohols were present as ester sulfates, the sulfate group being tentatively assigned to the 26-hydroxy group. 12-Hydroxy compounds were not detected. The manatee is the first mammal found to lack bile acids, presumably because it lacks the enzymes required for oxidation of the 26-hydroxy group to a carboxylic acid. Trichechols, like other bile salts, are water-soluble end products of cholesterol metabolism; whether they also function as biological surfactants in promoting biliary cholesterol secretion or lipid digestion is unknown.