Potential bile acid metabolites. 16. Synthesis of stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids

New synthetic routes to the four possible stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids (and their methyl esters), one of which (3 alpha,6 alpha 7 beta,12 alpha) is new, and some related compounds are described. In addition, the 5 alpha-epimer of the new acid was obtained. The final products were obtained in high purity for use as reference compounds in the analysis of bile acids in human biologic samples. The results of analysis of the prepared stereoisomers by proton and carbon 13 nuclear magnetic resonance spectroscopies are briefly discussed along with the thin-layer and gas-liquid chromatographic properties.     

Catalytic properties and inhibition of hepatic cholesterol-epoxide hydrolase

Hepatic cholesterol-epoxide hydrolase is a microsomal enzyme which appears to be catalytically distinct from the epoxide hydrolase responsible for the catabolism of a wide variety of aromatic and aliphatic epoxides. The diastereomeric forms of cholesterol epoxide, cholesterol 5 alpha,6 alpha-, and cholesterol 5 beta,6 beta-epoxides are converted to cholestane-3 beta,5 alpha,6 beta-triol with equal facility. Kinetic analysis of cholesterol-epoxide hydrolase demonstrated that both diastereomers bind to a common catalytic site. Apparent Km values of 3.69 and 4.42 microM were derived for cholesterol 5 alpha,6 alpha- and cholesterol 5 beta,6 beta-epoxide, respectively. In addition, enzyme activity with both diastereomers was product-inhibited by cholestanetriol through a competitive mechanism with the apparent Ki for cholestanetriol being 10.8 and 6.8 microM against cholesterol alpha- and beta-epoxides, respectively. This inhibitory effect of cholestanetriol may account for the difference observed in the hydration rates for the cholesterol epoxide isomers when they are incubated together in the presence of liver microsomes. Inhibitors of epoxide hydrolase were studied, and three oxidation products were found to be particularly effective against cholesterol-epoxide hydrolase while producing no significant inhibition of styrene-epoxide hydrolase. These inhibitors were 7-ketocholesterol, 6-ketocholestanol, and 7-ketocholestanol, the latter displaying an apparent Ki lower than the Km for either cholesterol epoxide isomer. None of the xenobiotic epoxide hydrolase inhibitors or activators studied affected cholesterol-epoxide hydrolase activity.     

Identification of new bile alcohols, 5 beta-cholestane-3 alpha,7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha,7 alpha,26,27-tetrol in human gallbladder bile

The nature of cholestanetetrols present as the glucurono-conjugates in human gallbladder bile was studied. Glucurono-conjugated bile alcohols were isolated by ion exchange chromatography and, after enzymatic hydrolysis, were fractionated by reversed phase partition chromatography to give a fraction containing tetrahydroxy bile alcohols which was analyzed by gas-liquid chromatography and mass spectrometry. Along with the three previously identified bile alcohols, 5 alpha- and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,24-tetrols, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,26-tetrol, three new cholestanetetrols, possessing two hydroxyl groups in the ring system and two in the side chain, were detected in the tetrahydroxy bile alcohol fraction. These new bile alcohols were identified as 5 beta-cholestane-3 alpha, 7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha, 7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha,26,27-tetrol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of authentic standards prepared from chenodeoxycholic acid by partial synthesis.     

Metabolism of cholestane-3 beta,5 alpha,6 beta-triol. II. Identification of two major neutral metabolites in the rat

Rats were given a single oral dose of cholestane-3beta,5alpha,6beta-triol-4-(14)C, and their feces were collected. The two major neutral metabolites were separated and isolated by use of solvent fractionation and chromatographic methods. The metabolites were identified as cholestane-3beta,5alpha-diol-6-one and a mixture of long-chain fatty acid esters of cholestane-3beta,5alpha,-6beta-triol. Cholestane-3beta,5alpha-diol-6-one was identified using thin-layer and gas-liquid chromatography, infrared spectroscopy, and the spectrum produced by reaction with 65% sulfuric acid. The mixed esters of cholestane-3beta,5alpha,6beta-triol were subjected to basic hydrolysis, and the steroid moiety was identified using the same techniques employed for cholestane-3beta,5alpha-diol-6-one. The fatty acids were analyzed by gas-liquid chromatography of their methyl esters.     

Biosynthesis of 5,6-dihydroxyprostaglandin E1 and F1 alpha from 5,6-dihydroxyeicosatrienoic acid by ram seminal vesicles

cis-5(6)Epoxy-cis-8,11,14-eicosatrienoic acid was recently found to be metabolized by ram seminal vesicles to 5-hydroxyprostaglandin I 1 alpha and 5-hydroxyprostaglandin I 1 beta, 5(6)epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1. The epoxide can be hydrolyzed by epoxide hydrolases to 5,6-dihydroxy-8,11,14-eicosatrienoic acid. The latter was incubated with microsomes of ram seminal vesicles for 2 min at 37 degrees C and the polar metabolites were purified by reversed phase HPLC and analyzed by capillary column gas chromatography-mass spectrometry. The major metabolite was identified as 5,6-dihydroxyprostaglandin F 1 alpha. In the presence of glutathione (1 mM), 5,6-dihydroxyprostaglandin E1 was also formed. The 3H-labelled vicinal diol and the 3H-labelled epoxide were metabolized to polar products to a similar extent, but the formation of prostaglandin E compounds in the presence of glutathione was lower from the diol than from the epoxide or from arachidonic acid. The likely prostaglandin endoperoxide intermediates in the metabolism of the diol (5,6-dihydroxyprostaglandin G1 and 5,6-dihydroxyprostaglandin H1) thus appear to be less prone to be isomerized to prostaglandin E compounds than prostaglandins G2 and H2 and their 5(6)epoxy counterparts. 5(6)Epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1 can be chemically transformed into 5,6-dihydroxyprostaglandin B1. The latter can be analyzed by HPLC or by mass fragmentography, and a simple chemical synthesis of 5,6-dihydroxyprostaglandin B1 from prostaglandin E2 is described.     

Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of alpha 6 beta 4 and alpha 2 beta 1 integrins.

In this study, we used clone A, a human colon carcinoma cell line, to characterize those integrins that mediate colon carcinoma adhesion to laminin. Monoclonal antibodies specific for the human beta 1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of alpha 2 beta 1 and alpha 3 beta 1. A monoclonal antibody specific for alpha 2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen I. An alpha 3-specific antibody (P1B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the alpha 2 beta 1 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express alpha 3 beta 1, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the alpha 6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen I. This antibody precipitated the alpha 6 subunit in association with the beta 4 subunit. There was no evidence of alpha 6 beta 1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin alpha subunits, in association with two distinct beta subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of alpha 3 beta 1 and alpha 2 beta 1, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.     

Colon cancer cells adhesion and spreading on autocrine laminin-10 is mediated by multiple integrin receptors and modulated by EGF receptor stimulation

Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-alpha (TGF-alpha) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-alpha and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS-PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin alpha5, beta1, and gamma1 chains, respectively, indicating that LIM1215 cells secrete laminin-10 (alpha5 beta1 gamma1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via alpha2 beta1 and alpha3 beta1 integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin alpha3 or beta1 but not alpha2, alpha6, or beta4 subunits. Spreading is almost completely inhibited by blocking alpha3 + alpha2, alpha3 + alpha6, or beta1 + beta4 integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of alpha6 beta4 integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for alpha3 beta1 integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-alpha and laminin-10 and autocrine induction of appropriate integrins.     

Synthesis of 3 alpha,6 beta,7 alpha,12 beta- and 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholanoic acids.

Chemical synthesis of 3 alpha,6 beta,7 alpha,12 beta- and 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholan-24-oic acids is described. 3 alpha,12 beta-Dihydroxy-5 beta-chol-6-en-24-oic acid used as the starting material in the synthesis was prepared via oxidation of 3 alpha,12 alpha-dihydroxy-5 beta-chol-6-en-24-oic acid 3-hemisuccinate at C-12 followed by reduction with potassium/tertiary amyl alcohol. alpha-Epoxidation of the ester diacetate of 3 alpha,12 beta-dihydroxy-5 beta-chol-6-en-24-oic acid with m-chloroperbenzoic acid followed by cleavage of the epoxide with acetic acid and alkaline hydrolysis yielded 3 alpha,6 beta,7 alpha,12 beta-tetrahydroxy-5 beta-cholan-24-oic acid (overall yield 25%). N-Methylmorpholine-N-oxide-catalyzed osmium tetroxide oxidation of the ester diacetate of 3 alpha,12 beta-dihydroxy-5 beta-chol-6-en-24-oic acid followed by alkaline hydrolysis yielded 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholan-24-oic acid (overall yield 33%). The structures of the synthesized bile acids were confirmed from their proto nuclear magnetic resonance and mass spectral fragmentation patterns.     

Assay of unesterified cholesterol-5,6-epoxide in human serum by isotope dilution mass spectrometry. Levels in the healthy state and in hyperlipoproteinemia

Accurate methods based on isotope dilution-mass spectrometry were developed for assay of free cholesterol-5,6-epoxide (sum of 5 alpha,6 alpha- and 5 beta,6 beta-epimer) and cholestane-3 beta,5 alpha,6 beta-triol in human serum. In all serum samples tested, the level of cholesterol epoxides was well above the detection limit (about 10 ng/ml) whereas the level of cholestane-3 beta,5 alpha,6 beta-triol was below or near the detection limit in most cases. Immediate addition of antioxidant was found to be necessary in order to obtain reproducible results in the serum analyses, and prolonged storage of frozen samples had to be avoided. The level of cholesterol epoxide in healthy subjects 23-35 years of age ranged from 67 ng/ml to 293 ng/ml (mean 131 ng/ml, n = 9). There was a tendency to higher levels with increasing age, but there was no correlation to serum cholesterol. In marked contrast to results previously reported with a less accurate method, patients with various forms of hyperlipoproteinemia did not have increased levels of cholesterol epoxide. On the contrary, many of these patients had levels lower than normal.     

Synthesis of epoxide and vicinal diol regioisomers from docosahexaenoate methyl esters

Docosahexaenoic acid (22:6(n-3)) was recently shown to be metabolized by liver microsomes to vicinal diol regioisomers. To identify the diols, and to compare their biological actions with those of epoxide precursors, we developed a chemical method to synthesize microgram to milligram amounts of epoxides and corresponding diols. In brief, methylated docosahexaenoate was reacted for 15 min with 0.1 eq m-chloroperoxybenzoic acid. After normal- and reverse-phase high performance liquid chromatography, six products were isolated. The underivatized or hydrogenated products were characterized and identified using capillary gas-liquid chromatography and mass spectrometry. The products were identified as 19,20-, 16,17-, 13,14-, 10,11-, 7,8-, and 4,5-epoxy-docosapentaenoate. Per incubation, the total epoxide yield from 22:6(n-3) was 8.6%. By reincubating unused substrate 10-20 times (cycling), the total epoxide could be increased to 55-70%. As found for epoxides of arachidonic and eicosapentaenoic acids, the yield of individual regioisomers increased as the distance between the targeted double bond and carbomethoxy group increased. Each epoxide regioisomer was hydrolyzed to its corresponding vicinal diol. The gas-liquid chromatographic retention times and mass spectra of the diol products were found to match those of metabolites produced by cytochrome P-450 monooxygenases.     

Identification of 3,6,7,12-tetrahydroxy-5 beta-cholan-24-oic acids in human biologic fluids

Unusual bile acids, 3 alpha, 6 alpha, 7 alpha, 12 alpha-, and 3 alpha, 6 beta, 7 beta, 12 alpha-tetrahyroxy-5 beta-cholan-24-oic acids, were identified in all amniotic fluid (four samples) and urine (six samples) from adult patients with cholestatic liver disease by gas-liquid chromatography/mass spectrometry. For the certain identification of these bile acids in the biologic samples, the chemical syntheses of 3 alpha, 6 beta, 7 alpha, 12 alpha- and 3 alpha, 6 beta, 7 beta, 12 alpha-tetrahydroxy-5 beta-cholan-24-oic acids were conducted.     

Identification of bile alcohols in rat bile

Bile alcohols in rat bile were analyzed by gas-liquid chromatography-mass spectrometry. Six bile alcohols were newly identified as minor constituents in addition to 5 beta-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, major bile alcohol of rat bile. The bile alcohols newly identified were 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol, 5 alpha-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol, and 5 beta-cholestane-3 alpha,6 beta,7 beta,25,26-pentol. The biliary bile alcohols of the rat occurred mainly as the sulfuric acid esters and, in lesser amounts, as glucuronoconjugated and unconjugated forms. The amount of total bile alcohols was about 27.9 nmol in 1 ml of bile.     

Synthesis of 11 alpha-hydroxyprogesterone haptens

C-4 and C-6 bridged haptens of 11 alpha-hydroxyprogesterone, an anti-androgen, were respectively synthesized via 4, 5 and 5 alpha, 6 alpha epoxide ring openings using 3-mercaptopropanoic acid. Substitution of 6-bromo derivative of progesterone using the same reagent unexpectedly afforded a C-4 substituted product instead of the normal C-6 substituted product previously reported in the literature.     

Bile salts of the coelacanth, Latimeria chalumnae

Bile salts of the coelacanth, Latimeria chalumnae, Smith, have been analyzed and shown to have three bile alcohols, latimerol, 5 alpha-cyprinol, and 5 alpha-cholestane-3 beta, 7 alpha,-12 alpha,25,26-pentol, two C24 bile acids, chenodeoxycholic acid and cholic acid, one C26 bile acid, probably 3 beta, 7 alpha, 12 alpha-trihydroxy-27-nor-5 alpha-cholestan-26-oic acid, and two C27 bile acids, 3 alpha,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid and 3 beta,7 alpha,12 alpha-trihydroxy-5 alpha-cholestan-26-oic acid as determined by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry.     

Structures of the major oligosaccharides from a human rectal adenocarcinoma glycoprotein

Four oligosaccharides in the reduced form were isolated from RMG (a mucin-type glycoprotein from a human rectal adenocarcinoma). They were 1) Sia alpha s2 leads to 6GalNAc-ol; 2) Sia alpha 2 leads to 6(Gal beta 1 leads to 3)GalNAc-ol; 3) Sia alpha 2 leads to 6(GlcNAc beta 1 leads to 3)GalNAc-ol; and 4) Sia alpha 2 leads to 6(GalNAc alpha 1 leads to 3)GalNAc-ol. The amounts of oligosaccharides 1, 2, 3, and 4 corresponded to 27, 5, 11, and 8% of the total N-acetylgalactosaminitol produced on alkaline borohydride treatment of RMG. To determine the structures of oligosaccharides 2, 3, and 4, a mixture of the three was subjected to methylation analysis which revealed that the N-acetylgalactosaminitol was substituted at both C-3 and C-6 and other sugars at the nonreducing ends. Desialized oligosaccharides were prepared, and the structures were deduced by analysis of the permethylated sugars on gas-liquid chromatography/mass spectrometry. Anomeric configurations were determined by exoglycosidase digestions except for galactose which was analyzed by chromium trioxide oxidation.     

Bioconversion of arachidonic acid to prostaglandins and related compounds in human myometrium and uterine cervix

Arachidonic Acid metabolites in human myometrium and uterine cervix were studied using silicic acid column chromatography, thin layer chromatography, reversed phase partition chromatography and gas-liquid chromatography. Myometrium produced 6-ketoPGF1 alpha, PGF2 alpha, PGE2, thromboxane B2. Uterine cervix produced 6-ketoPGF1 alpha, PGF2 alpha, PGE2, thromboxane B2, and one hydroxyacid. There was no difference between the rate of conversion of prostaglandins in myometrium and cervix. But only cervix could convert arachidonic acid to hydroxyacid.     

Mutagenic characterization of cholesterol epoxides in Chinese hamster V79 cells

The uptake, metabolism and alkylating properties of the diastereomeric cholesterol epoxides were studied using Chinese hamster lung fibroblasts (V79 cells). Specific emphasis is given to the comparative cyto- and geno-toxic effects of cholesterol 5 beta,6 beta-epoxide (beta CE) and cholesterol 5 alpha,6 alpha-epoxide (alpha CE) and data are provided for the first time indicating that beta CE can induce more 6-thioguanine-resistant cells than alpha CE. Cholesterol 5 beta,6 beta-epoxide induced colonies of cells resistant to 6-thioguanine at 2-3-fold the frequencies observed with the alpha-isomer, but neither compound produced ouabain-resistant colonies. The cytotoxicity (LD50) of alpha CE was estimated to be 45-50 microM whereas beta CE displayed an LD50 of 25-29 microM. Inhibition of DNA synthesis (IC50) was observed over the same dose ranges as the LD50 for each epoxide isomer. The epoxides were assimilated by cells to an equal extent, however, beta CE was metabolized to cholestane 3 beta,5 alpha-6 beta-triol twice as rapidly as the alpha-isomer. Both epoxides reacted with 4-(4'-nitrobenzyl)-pyridine to a similar extent, and with identical nucleophilic selectivity at pH 7.4, but their alkylating activity was estimated on this basis to be two orders of magnitude less than methyl methanesulfonate. Binding experiments with the DNA or cultured V79 cells or with calf-thymus DNA indicated that interactions were noncovalent and DNA binding did not correlate with the potency of the epoxides to induce the 6-thioguanine-resistant phenotype. Our results could be interpreted as indicating that both cholesterol epoxide isomers are weak mutagens or that they might induce some epigenetic event repressing the hypoxanthine guanine-phosphoribosyltransferase gene. The similarity of the epoxides' alkylating activity and their DNA-binding properties are inconsistent with their different potencies in inducing the 6-thioguanine-resistant phenotype, suggesting that the mechanism leading to this phenotype is not necessarily the result of DNA alkylation.