Formation of delta 2- and delta 3-cholenoic acids from bile acid 3-sulfates by a human intestinal Fusobacterium strain.

We isolated two strains of an unnamed Fusobacterium species from human intestinal microflora, which stereospecifically transformed bile acid 3-sulfates into C-3-unsubstituted, ring A-unsaturated bile acids. Both 3 alpha- and 3 beta-sulfates of 5 beta-bile acids were metabolized to delta 3-5 beta-cholenoic acids; 3 beta-sulfates of 5 alpha-bile acids were converted into a mixture of delta 2-5 alpha-bile acids and 3 alpha-hydroxy-5 alpha-bile acids, whereas 3 alpha-sulfates of 5 alpha-bile acids were left intact. Unsulfated bile acids were not transformed into unsaturated derivatives. These strains differ from previously isolated intestinal bacteria, which desulfated bile acid sulfates without further transformation.     

Formation of delta 2- and delta 3-cholenoic acids from bile acid 3-sulfates by a human intestinal Fusobacterium strain

We isolated two strains of an unnamed Fusobacterium species from human intestinal microflora, which stereospecifically transformed bile acid 3-sulfates into C-3-unsubstituted, ring A-unsaturated bile acids. Both 3 alpha- and 3 beta-sulfates of 5 beta-bile acids were metabolized to delta 3-5 beta-cholenoic acids; 3 beta-sulfates of 5 alpha-bile acids were converted into a mixture of delta 2-5 alpha-bile acids and 3 alpha-hydroxy-5 alpha-bile acids, whereas 3 alpha-sulfates of 5 alpha-bile acids were left intact. Unsulfated bile acids were not transformed into unsaturated derivatives. These strains differ from previously isolated intestinal bacteria, which desulfated bile acid sulfates without further transformation.     

Isolation of a bile salt sulfatase-producing Clostridium strain from rat intestinal microflora.

Bile acid sulfates, formed in human and rat livers, are desulfated by the intestinal microflora. In our study we first isolated from conventional rat feces an unnamed bacterium, termed strain S1, which desulfated the 5 beta-bile salt 3 alpha-sulfates in vitro and in vivo after association with gnotobiotic rats. Strain S1 also possessed 12 alpha-hydroxysteroid dehydrogenase and bile salt-deconjugating activities. The strain was a strict anaerobic, CO2-requiring, gram-negative, sporeforming rod and was designated as belonging to the genus Clostridium. Growth was scarce in culture media, unless in the presence of 0.1% taurine, a sulfur-containing amino acid. Addition of this substance raised the number of bacteria in thioglycolate and peptone yeast media from 10(4) per ml to 10(6) to 10(7) per ml and increased the colony diameter on agar medium from 0.2 mm to 0.5 to 0.9 mm. Sulfatase activity was specific for the 5 beta-bile salt sulfates, leaving the 5 alpha-bile salt sulfates unchanged. In addition, the sulfatase activity was cell bound, and its production was dependent on the composition of the culture medium, although no minimal sulfur medium was required for sulfatase activity.     

Isolation of a rat intestinal Clostridium strain producing 5 alpha- and 5 beta-bile salt 3 alpha-sulfatase activity

An unnamed sporeforming microorganism, termed Clostridium sp. strain S2, possessing bile salt sulfatase activity was isolated from rat intestinal microflora. The microorganism was a strictly anaerobic, nonmotile, gram-negative, asaccharolytic, sporeforming rod requiring CO2, vitamin K, and taurine; the guanine-plus-cytosine content of the DNA was 40.8 mol% (Tm), and the strain was tentatively classified as an atypical Clostridium species. Sulfatase activity was specific for 3 alpha-sulfate esters of 5 alpha- and 5 beta-bile salts, leaving the 3 beta-, 7 alpha-, and 12 alpha-sulfates unchanged. Strain S2 also deconjugated tauro- and glyco-conjugated bile salts and partially reduced into the corresponding 6 alpha-hydroxy bile salts. By these reactions, alpha-muricholate and beta-muricholate were more than 80% converted into hyocholate and omega-muricholate, respectively. In addition, strain S2 produced 12 alpha-hydroxysteroid dehydrogenase converting deoxycholate into 3 alpha-hydroxy-12-oxo-5 beta-cholanoate. When strain S2 was associated with gnotobiotic rats, the fecal bile salts were more than 90% desulfated and the fecal excretion of allochenodeoxycholate was five times lower than in control rats.     

New insights on the specificity of heparin and heparan sulfate lyases from Flavobacterium heparinum revealed by the use of synthetic derivatives of K5 polysaccharide from E. coli. and 2-O-desulfated heparin

The capsular polysaccharide from E. Coli, strain K5 composed of ...-->4)beta-D-GlcA(1-->4)alpha-D-GlcNAc(1-->4)beta-D-GlcA (1-->..., chemically modified K5 polysaccharides, bearing sulfates at C-2 and C-6 of the hexosamine moiety and at the C-2 of the glucuronic acid residues as well as 2-O desulfated heparin were used as substrates to study the specificity of heparitinases I and II and heparinase from Flavobacterium heparinum. The natural K5 polysaccharide was susceptible only to heparitinase I forming deltaU-GlcNAc. N-deacetylated, N-sulfated K5 became susceptible to both heparitinases I and II producing deltaU-GlcNS. The K5 polysaccharides containing sulfate at the C-2 and C-6 positions of the hexosamine moiety and C-2 position of the glucuronic acid residues were susceptible only to heparitinase II producing deltaU-GlcNS,6S and deltaU,2S-GlcNS,6S respectively. These combined results led to the conclusion that the sulfate at C-6 position of the glucosamine is impeditive for the action of heparitinase I and that heparitinase II requires at least a C-2 or a C-6 sulfate in the glucosamine residues of the substrate for its activity. Iduronic acid-2-O-desulfated heparin was susceptible only to heparitinase II producing deltaU-GlcNS,6S. All the modified K5 polysaccharides as well as the desulfated heparin were not substrates for heparinase. This led to the conclusion that heparitinase II acts upon linkages containing non-sulfated iduronic acid residues and that heparinase requires C-2 sulfated iduronic acid residues for its activity.     

Steroid sulfatase activity in a Peptococcus niger strain from the human intestinal microflora.

A strictly anaerobic gram-positive coccus, identified as Peptococcus niger, that developed sulfatase activity towards steroid-3-sulfate esters was isolated from human fecal material. This strain desulfated the arylsulfate esters estrone-3-sulfate (100%) and beta-estradiol-3-sulfate (50%); only trace amounts of desulfated estriol-3-sulfate were found. In addition, alkylsulfatase activity was found towards the 3 alpha-sulfates of 5 alpha-androstane-17-one and 5 beta-androstane-17-one and towards the 3 beta-sulfates of 5 alpha-androstane-17-one, delta 5-androstene-17-one, 5 alpha-pregnane-20-one, and delta 5-pregnene-20-one, all of which were 100% desulfated. No sulfatase activity was found towards the 17-sulfate esters of beta-estradiol or delta 4-androstene-3-one-17 alpha-ol. The nonsteroid arylsulfate esters paranitrophenyl sulfate, paranitrocatechol sulfate, and phenolphthalein disulfate were desulfated 70, 40, and 40%, respectively. In addition to its sulfatase activity, this strain also developed C-17 oxidoreductase activity towards the estrogens and androsta(e)nes and C-3 oxidoreductase activity towards androsta(e)nes and pregna(e)nes.     

Steroid sulfatase activity in a Peptococcus niger strain from the human intestinal microflora

A strictly anaerobic gram-positive coccus, identified as Peptococcus niger, that developed sulfatase activity towards steroid-3-sulfate esters was isolated from human fecal material. This strain desulfated the arylsulfate esters estrone-3-sulfate (100%) and beta-estradiol-3-sulfate (50%); only trace amounts of desulfated estriol-3-sulfate were found. In addition, alkylsulfatase activity was found towards the 3 alpha-sulfates of 5 alpha-androstane-17-one and 5 beta-androstane-17-one and towards the 3 beta-sulfates of 5 alpha-androstane-17-one, delta 5-androstene-17-one, 5 alpha-pregnane-20-one, and delta 5-pregnene-20-one, all of which were 100% desulfated. No sulfatase activity was found towards the 17-sulfate esters of beta-estradiol or delta 4-androstene-3-one-17 alpha-ol. The nonsteroid arylsulfate esters paranitrophenyl sulfate, paranitrocatechol sulfate, and phenolphthalein disulfate were desulfated 70, 40, and 40%, respectively. In addition to its sulfatase activity, this strain also developed C-17 oxidoreductase activity towards the estrogens and androsta(e)nes and C-3 oxidoreductase activity towards androsta(e)nes and pregna(e)nes.     

Partial characterization of the steroidsulfatases in Peptococcus niger H4.

The strictly anaerobic intestinal Peptococcus niger H4 synthesizes three different steroidsulfatase enzymes: a constitutive arylsulfatase and two inducible alkylsteroidsulfatases. The arylsulfatase desulfates estrogen-3-sulfates and phenylsulfates. The two alkylsteroidsulfatases desulfate, respectively, 3 alpha-sulfates and 3 beta-sulfates of delta 5, 5 alpha, and 5 beta androstanes, pregnanes, and bile acids. Cholesterol-3 beta-sulfate was not desulfated by the alkylsteroidsulfatases nor were steroids or bile acids that were sulfated in positions other than the 3 position. The alkylsteroidsulfatases were induced by their substrates; bile acid sulfates, however, were poor inducers of the 3 beta-sulfatase and did not induce the 3 alpha-sulfatase activity. In intact bacterial cells, taurine and sulfite suppressed the induction of the alkylsteroidsulfatases and inhibited the activity of the arylsulfatase and alkylsteroidsulfatases. In cell homogenates, the arylsulfatase and alkylsteroidsulfatases activities were inhibited by sulfite and sulfate but not by taurine. Our results support the hypothesis that the main function of the steroidsulfatases in P. niger H4 is to provide the bacteria with sulfur for dissimilatory purposes.     

Partial characterization of the steroidsulfatases in Peptococcus niger H4

The strictly anaerobic intestinal Peptococcus niger H4 synthesizes three different steroidsulfatase enzymes: a constitutive arylsulfatase and two inducible alkylsteroidsulfatases. The arylsulfatase desulfates estrogen-3-sulfates and phenylsulfates. The two alkylsteroidsulfatases desulfate, respectively, 3 alpha-sulfates and 3 beta-sulfates of delta 5, 5 alpha, and 5 beta androstanes, pregnanes, and bile acids. Cholesterol-3 beta-sulfate was not desulfated by the alkylsteroidsulfatases nor were steroids or bile acids that were sulfated in positions other than the 3 position. The alkylsteroidsulfatases were induced by their substrates; bile acid sulfates, however, were poor inducers of the 3 beta-sulfatase and did not induce the 3 alpha-sulfatase activity. In intact bacterial cells, taurine and sulfite suppressed the induction of the alkylsteroidsulfatases and inhibited the activity of the arylsulfatase and alkylsteroidsulfatases. In cell homogenates, the arylsulfatase and alkylsteroidsulfatases activities were inhibited by sulfite and sulfate but not by taurine. Our results support the hypothesis that the main function of the steroidsulfatases in P. niger H4 is to provide the bacteria with sulfur for dissimilatory purposes.     

12 beta-dehydrogenation of bile acids by Clostridium paraputrificum, C. tertium, and C. difficile and epimerization at carbon-12 of deoxycholic acid by cocultivation with 12 alpha-dehydrogenating Eubacterium lentum.

12 beta-Hydroxysteroid dehydrogenating activities were detected in 13 strains of Clostridium paraputrificum, 1 strain of C. tertium, and 1 strain of C. difficile, together with a 3 alpha- and 3 beta-hydroxysteroid dehydrogenase system in many strains. Redox reactions a C-12 of disubstituted and trisubstituted bile acids were performed unspecifically by representative strains of C. paraputrificum. 3 alpha,12 beta-, 3 beta,12 beta-Dihydroxy-, 3 alpha, 7 alpha, 12 beta-trihydroxy-, and 3-keto,12 beta-hydroxy-5 beta-cholanoic acids, so far not known as bacterial bile acid metabolites, were identified. Epimerization of the 12 alpha-hydroxyl group of deoxycholate via the 12-keto intermediate was achieved by cocultivation of C. paraputrificum and Eubacterium lentum, elaborating a 12 alpha-hydroxysteroid dehydrogenase only. In addition, epimerization at C-12 was demonstrated with mixed human fecal cultures.     

LC/ESI-tandem mass spectrometric determination of bile acid 3-sulfates in human urine 3beta-Sulfooxy-12alpha-hydroxy-5beta-cholanoic acid is an abundant nonamidated sulfate

We developed a highly sensitive and quantitative method to detect bile acid 3-sulfates in human urine employing liquid chromatography/electrospray ionization-tandem mass spectrometry. This method allows simultaneous analysis of bile acid 3-sulfates, including nonamidated, glycine-, and taurine-conjugated bile acids, cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and lithocholic acid (LCA), using selected reaction monitoring (SRM) analysis. The method was applied to analyze bile acid 3-sulfates in human urine from healthy volunteers. The results indicated an unknown compound with the nonamidated common bile acid 3-sulfates on the chromatogram obtained by the selected reaction monitoring analysis. By comparison of the retention behavior and MS/MS spectrum of the unknown peak with the authentic specimen, the unknown compound was identified as 3beta,12alpha-dihydroxy-5beta-cholanoic acid 3-sulfate.     

12 beta-dehydrogenation of bile acids by Clostridium paraputrificum, C. tertium, and C. difficile and epimerization at carbon-12 of deoxycholic acid by cocultivation with 12 alpha-dehydrogenating Eubacterium lentum

12 beta-Hydroxysteroid dehydrogenating activities were detected in 13 strains of Clostridium paraputrificum, 1 strain of C. tertium, and 1 strain of C. difficile, together with a 3 alpha- and 3 beta-hydroxysteroid dehydrogenase system in many strains. Redox reactions a C-12 of disubstituted and trisubstituted bile acids were performed unspecifically by representative strains of C. paraputrificum. 3 alpha,12 beta-, 3 beta,12 beta-Dihydroxy-, 3 alpha, 7 alpha, 12 beta-trihydroxy-, and 3-keto,12 beta-hydroxy-5 beta-cholanoic acids, so far not known as bacterial bile acid metabolites, were identified. Epimerization of the 12 alpha-hydroxyl group of deoxycholate via the 12-keto intermediate was achieved by cocultivation of C. paraputrificum and Eubacterium lentum, elaborating a 12 alpha-hydroxysteroid dehydrogenase only. In addition, epimerization at C-12 was demonstrated with mixed human fecal cultures.     

Studies on steroid conjugates: IX. Urinary excretion of sulfate conjugated metabolites of cortisol in man.

Following i.v. administration of [4-14C]cortisol, various sulfate conjugated metabolites of cortisol in urine were identified and their respective excretion rates measured. The results obtained demonstrated the following: 1) sulfate conjugates as a group are excreted considerably slower than glucuronide conjugates; 2) sulfate conjugates of steroids with non-reduced ring-A (C-21 sulfates) are excreted (and presumably formed) much faster than steroid-3-sulfates, which require reduction of the ring-A prior to the conjugation; 3) the excretion of C-3 sulfates of ring-A reduced steroids with glycerol side-chain (cortols and cortolones) is significantly faster than those of the corresponding steroids with dihydroxyacetone side-chain (THF, THE and their 5alpha-isomers); 4) the relative concentrations of C-21 sulfates of steroids with ring-A intact (FK, EK, ER, epiER and 6beta-hydroxycortisol) are much higher than the concentrations of C-21 glucuronides of these steroids.     

Structural features and some binding properties of proteoheparan sulfate enzymatically labeled by calf brain microsomes

Previous studies established that brain microsomes catalyze the transfer of [35S]sulfate from 3'-phosphoadenosine 5'-phospho[35S]sulfate to an O-linked oligosaccharide chain of a membrane glycoprotein and sulfamino groups of a membrane-associated proteoheparan sulfate (R. R. Miller and C. J. Waechter (1979) Arch. Biochem. Biophys. 198, 31-41). A large fraction of the proteoheparan [35S]sulfate can be released by treating the enzymatically labeled membranes from calf brain with 1 M NaCl. The salt-extracted 35S-labeled proteoglycan has been partially purified by a combination of ion-exchange and gel filtration chromatography. Based on chromatographic analyses, the 35S-labeled proteoglycan labeled in vitro is proposed to be a family of proteoheparan [35S]sulfates having an average molecular weight estimated to be 55,000. Variation in the length of the 35S-labeled polysaccharide chains partially accounts for the differences in molecular size of the proteoheparan [35S]sulfates. Binding studies reveal that the intact proteoheparan [35S]sulfates, as well as the free 35S-labeled polysaccharides released by mild alkali treatment, rapidly reassociate with calf brain membrane preparations. The association with calf brain membranes is saturable and reversible. Consistent with the binding being a specific interaction, only iduronic acid-containing glycosaminoglycans inhibit the association of the 35S-labeled proteoglycan with calf brain membranes and facilitate the disassociation. Neither the binding of the 35S-labeled proteoglycan to membranes nor the displacement was affected by hyaluronic acid, chondroitin 4-sulfate, or chondroitin 6-sulfate. The binding of the enzymatically labeled proteoheparan sulfate is reduced by preincubating membranes with either trypsin or chymotrypsin, but not with neuraminidase or phospholipase D. These results suggest that at least one class of proteoheparan sulfates could be specifically bound to one or more brain membrane proteins. The results also suggest a role for iduronosyl residues, and perhaps the stereochemical relationship of the carboxyl group to the O-sulfate moiety at C-2, in the recognition process.     

The synthesis of diazo, halo, and sulfoxy bile acid derivatives: potential affinity labels.

Bile acid derivatives, with and without C-3 sulfate groups, and having either the diazo- or halomethylketone moieties, have been synthesized in good yield and purity. The synthetic sequence, COOH leads to COC1 leads to COCHN2 leads to COCH2X, was used with deoxycholic and cholic acids, which requires carefully controlled quench, work-up, and purification procedures, especially for the 3-sulfate esters (made from deoxycholic acid derivatives only). The pure title compounds are anticipated to be useful chemical probes (affinity labels), especially the completely water soluble sulfates, toward our studies of ileal active transport of bile salts. A new use for Sephadex LH-20 as a sulfate ester protecting group is reported. Also developed were the use of acetamide hydrochloride complex as a mild hydrochlorination reagent and a neutral desalting method for sulfate esters of deoxycholic acid derivatives.     

Isolation and identification of intestinal steroid-desulfating bacteria from rats and humans.

We isolated 12 strictly anaerobic steroid-3-sulfate-desulfating strains from the intestinal floras of rats and humans. Two strains (S1 and S2) of the same atypical Clostridium species and an atypical Lactobacillus strain (termed R9) were obtained from rats. The human isolates were identified as Eubacterium cylindroides (two strains, H1 and H2), Peptococcus niger (two strains, H4 and H89), and Clostridium clostridiiforme. We also isolated, from different human fecal samples, four strains of phenotypically similar asaccharolytic Bacteroides strains, H6.2a, H6.2b, H65, and H175. Aryl steroid sulfatase activity for estrogen sulfates was present in all isolates. Alkyl steroid sulfatase activity for both 3 alpha- and 3 beta-sulfates was found only in P. niger H4. The same P. niger strain and Clostridium strains S1 and S2 also possessed bile acid sulfatase activity.     

Bile acid sulfates in serum bile acids determination.

Some bile acid sulfates were synthesized and characterized. The configuration of sulfate groups at C-3, C-7 and C-12 positions was confirmed by Nuclear Magnetic Resonance analysis. These sulfates were utilized in a study of their chemical behaviour in different analytical procedures currently used for serum bile acids determination. Procedures for bile acids extraction from serum with ethanol or Amberlite XAD-2 result in an important loss of the most polar sulfated bile acids. Complete separation of unsulfated from sulfated bile acids on Sephadex LH-20 is not achieved when deconjugation of the most polar bile acid sulfate is slow but does not produce artifacts. Enzymatic determination of bile acids gives positive response with some bile acid sulfates. The current procedures of serum bile acids determination are discussed in consideration of these results.     

Importance of estrogen sulfates in breast cancer

Estrogen sulfates are quantitatively the most important form of circulating estrogens during the menstrual cycle and in the post-menopausal period. Huge quantities of estrone sulfate and estradiol sulfate are found in the breast tissues of patients with mammary carcinoma. It has been demonstrated that different estrogen-3-sulfates (estrone-3-sulfate, estradiol-3-sulfate, estriol-3-sulfate) can provoke important biological responses in different mammary cancer cell lines: there is a significant increase in progesterone receptor. On the other hand, no significant effect was observed with estrogen-17-sulfates. The reason for the biological response of estrogen-3-sulfates is that these sulfates are hydrolyzed, and no sulfatase activity for C17-sulfates is present in these cell lines. [3H]Estrone sulfate is converted in a very high percentage to estradiol (E2) in different hormone-dependent mammary cancer cell lines (MCF-7, R-27, T-47D), but very little or no conversion was found in the hormone-independent mammary cancer cell lines (MDA-MB-231, MDA-MB-436). Different anti-estrogens (tamoxifen and derivatives) and another potent anti-estrogen: ICI 164,384, decrease the concentration of estradiol very significantly after incubation of estrone sulfate with the different hormone-dependent mammary cancer cell lines. No significant effect was observed for the uptake and conversion of estrone sulfate in the hormone-independent mammary cancer cell lines. Progesterone provokes an important decrease in the uptake and in estradiol levels after incubation of [3H]estrone sulfate with the MCF-7 cells. It is concluded that in breast cancer: (1) Estrogen sulfates can play an important role in the biological response of estrogens; (2) Anti-estrogens and progesterone significantly decrease the uptake and estradiol levels in hormone-dependent mammary cancer cell lines; (3) The control of the sulfatase and 17 beta-hydroxysteroid dehydrogenase activities, which are key steps in the formation of estradiol in the breast, can open new possibilities in the treatment of hormone-dependent mammary cancer.     

The effect of chondroitin sulfate molecular weight and degree of sulfation on the activity of a sulfotransferase from chicken embryo epiphyseal cartilages

The transfer of [35S] sulfate from [35S]PAPS, by means of PAPS: chondroitin sulfate sulfotransferase, to various chondroitin sulfates, with different degrees of sulfation and molecular weights is reported. Analyses by digestion with chondroitin AC and specific 4- or 6-sulfatases indicate that the sulfation occurs only in position 6 of the non-sulfated N-acetyl galactosamine moiety. The 50-70% desulfated chondroitin 4/6-sulfates are two times better sulfate acceptors than totally desulfated chondroitin, and the affinity of the sulfotransferase increases markedly from the octa-to the deca-saccharide. These results suggest that sulfation increases sharply only after the growing polysaccharide contains about 10 sugar residues, in the early stages of polymerization, and that the sulfation of chondroitin sulfate may be a process in which the addition of some sulfate groups facilitates further sulfation.