J. Winter

Bacterial metabolism of natural and synthetic sex hormones undergoing enterohepatic circulation

Steroids undergoing enterohepatic circulation are exposed to bacterial metabolism particularly by obligate anaerobes which account for 99.99% of the fecal flora. The most common transformation is hydrolysis of conjugated steroids. The glucuronidases are synthesized by Escherichia coli and Bacteroides species. The bacterial catabolism of unconjugated steroids may be considered under several headings: 1. Reduction of ring-A due to clostridia species synthesizing specific enzymes; C. paraputrificum, 3 alpha,5 beta-reductase; C. innocuum, 3 beta,5 beta-reductase; and a new species C.J-1, 3 beta,5 alpha-reductase. 2. Reduction of the delta 5 bond by human fecal flora. The specific strain(s) synthesizing the enzyme have not yet been identified. 3. Reduction of 17-keto estrogens by the above mentioned ring-A reducing clostridia and by Eubacterium lentum. 4. Reduction of 17-keto androstenes by Bacteroides fragilis. 5. Desmolase mediated side chain cleavage at C17-C20 position of 17 alpha-hydroxysteroids by two new species Clostridium scindens and Eubacterium desmolans isolated from human and cat fecal flora respectively and by Clostridium cadavaris isolated from New York City sewage. 6. And 16 alpha- and 21-dehydroxylase by E. lentum a normal inhabitant of the human gut; it is the only organism known to synthesize 16 alpha- or 21-dehydroxylases. Due to the high specificity of the enzymes and the simplicity of extracting the metabolites, biosynthesis of reference compounds and radioimmunoassay reagents is practical and inexpensive. The enzymes can also be used for titration of specific bacterial strains in fecal flora and as markers for bacterial identification in particular for the strains difficult to be defined by regular biochemical reactions.

21-dehydroxylation of corticoids by anaerobic bacteria isolated from human fecal flora

Abstract Two human intestinal anaerobes, culture No. 116 and Eubacterium lentum , synthesize enzymes capable of metabolizing C-21 steroid hormones. 21-Dehydroxylase activity by culture No. 116 benefits from the presence of Escherichia coli or allied organisms in the medium; their effect appears to be a lowering of the Eh. The enzymes activity is independent of arginine and shows no sign of product inhibition. The enzyme, which is constitutive, functions at pH 6.5–7.3; its activity is unrelated to the 4-ene-3CO group but is inhibited by a C20-OH group. E. lentum (neotype), in addition to a 21-dehydroxylase, synthesizes a 3α-hydroxysteroid dehydrogenase active at Eh ± —150 mV, which explains the epimerization of 3α-pregnanolone observed in cultures of fecal flora. The metabolic alterations of deoxycorticosterone by cultures of fecal flora may be duplicated by Clostridium paraputrificum and E. lentum , whether acting in concert or sequentially.

REDUCTION OF ALDOSTERONE BY ANAEROBIC-BACTERIA - ORIGIN OF URINARY 21-DEOXY METABOLITES IN MAN

Abstract Aldosterone was incubated with pure cultures of human faecal micro-organisms and the products were analysed as methyloxime/trimethylsilyl ethers by gas chromatography/mass Spectrometry. Clostridium paraputrificum quantitatively reduced the A-ring of aldosterone to a 3α-hydroxy-5β group (tetrahydroaldosterone). In addition, aldosterone was quantitatively 21-dehydroxylated by culture no. 116 and Eubacterium lentum . Metabolism of aldosterone with mixed faecal flora yielded a single metabolite, 21-deoxy-tetrahydroaldosterone, reflecting the enzymatic action of both C. paraputrificum and culture no. 116. It is likely, therefore, that the 21-deoxymetabolites of aldosterone which have been identified in urine are formed by the action of these organisms during the passage of tetrahydroaldosterone through the gut following biliary excretion. The reduced metabolites are reabsorbed prior to urinary excretion. The specificity and high yield of the reactions carried out by these organisms suggest that they will prove useful for small scale production of reference compounds not yet commercially available, since almost all steroids with α-ketol side chain have been shown to be susceptible to transformation.

Formation of 20β-dihydrosteroids by anaerobic bacteria

Abstract Cortisol was metabolized to a variety of products, among them small amounts of cortol by fecal flora of humans and rats. A microorganism. Bifidobacterium adolescentis , isolated from both sources, synthesized a 20-hydroxysteroid dehydrogenase which reduced cortisol to 20β-dihydrocortisol. The metabolite was reduced to cortol by Clostridium paraputrificum . The 20-hydroxysteroid dehydrogenase showed a wide substrate specificity; it was independent of the 4-ene and the configuration at C-3, C-11, C-17 and C-21. Cortol was resistant to any further alteration by human fecal flora, i.e. it is a metabolic end product. As expected. B. adolescenris effectively prevented 21-dehydroxylation of cortisol by Eubacterium lentum .

Side-chain cleavage of cortisol by fecal flora

The side chain of certain C-21 steroids may be removed by an enzyme, desmolase, synthesized by intestinal bacteria. With a view to isolate these organisms we examined the conditions required for their multiplication and function. The model substrate, cortisol (11 beta, 17 alpha, 21-trihydroxy-4-pregnene-3,20-dione), was metabolized by mixed fecal flora of humans and rats to a number of C-21 and C-19 compounds. The major C-21 metabolites were 21-deoxycortisol, tetrahydro-21-deoxycortisol, and tetrahydrocortisol. The C-19 metabolites obtained were identified as 5 beta-androstane-3 alpha, 11 beta-triol and 5 xi-androstane-3 alpha, 11 beta-diol-17-one. The prevalence of converting microorganisms was approximately 10(6)/g feces in both humans and rats. Conversion required an Eh below -130 mV, and an initial pH of 7.0. Optimal yield of C-19 products occurred with a fecal dilution of 10(5), though C-19 metabolites were evident from 10(1) through 10(8) fecal dilutions. Preliminary investigation indicates that the ability of converting organisms to form colonies varied with the composition of the media and the gaseous environment.

16α-Dehydration of corticoids by bacteria isolated from rat fecal flora

Abstract Two strains (No. 144 and No. 146) of rat intestinal anaerobic bacteria, phenotypically similar to Eubacterium lentum, were isolated and found capable of 16α-dehydrating corticoids. The initial step in the 16α-dehydration of 16α-hydroxyprogesterone was dehydration at the C-16 and C-17 position with the accumulation of 16-dehydroprogesterone. This step required the side chain at C-17. In bacterial cultures the 16-dehydroprogesterone was then slowly reduced to iso-progesterone. 16α-Hydroxypregnanolone was also converted to iso-pregnanolone by these bacteria. 16α-Dehydratase was easily demonstrated in cell fractions of strain No. 144 incubated either aerobically or anaerobically. The same extracts did not convert 16-dehydroprogesterone to iso-progesterone under similar assay conditions. 16α-Dehydration occurred at all substrate concentrations tested (20 to 200 μg/ml) provided the pH of the growth medium was between 6 and 8 and the Eh below -130mV. Strain No. 146 had both 16α-dehydration and 21-dehydroxylation activities. The two enzymes functioned independently. A role for intestinal bacteria in the biotransformation of biliary 16α-hydroxylated steroids and subsequent excretion in the urine is proposed.

Urinary metabolites of 18-hydroxylated corticosteroids: Microbial preparation of reference compounds

Abstract Clostridium paraputrificum quantitatively reduced 18-hydroxy-11-deoxycorticosterone, 18-hydroxy-11-dehydrocorticosterone and 18-hydroxycorticosterone to their 3α-hydroxy-5β metabolites. This microbial transformation is eminently suited for the preparation of rare or expensive reference steroids. Mass spectra of the methyloxime-trimethylsilyl ethers of the metabolites are illustrated.

Characterization of a C21 neutral steroid hormone transforming enzyme, 21-dehydroxylase, in crude cell extracts of Eubacterium lentum.

A strain of the obligate anaerobe, Eubacterium lentum, isolated from human feces, catalyzes the 21-dehydroxylation of 11-deoxycorticosterone to progesterone. A quantitative radiochromatographic assay was developed to measure 21-dehydroxylase activity in cell extracts. Maximum enzyme activity in cell extracts required both a reduced pyridine nucleotide and an oxidized flavin coenzyme. However, photochemically reduced flavin (FMNH2) could replace the requirement for NAD(P)H plus oxidized flavin. NAD(P)H : flavin (either FMN or FAD) oxidoreductase activity was detected spectrophotometrically in cell extracts assayed under anaerobic conditions. 21-Dehydroxylase was active from pH 5.4 to 8.5 with an apparent optimum between 6.4 and 6.8 using mixtures of NADH plus FMN as coenzymes. The substrate concentration at half-maximal reaction velocity was 8.0 microM and a specific acitivity of 5.8 nmol [3H]progesterone formed . h-1 . mg-1 protein was determined using [3th]deoxycorticosterone as substrate. Atabrine, rotenone, acriflavin, and 2,4-dinitrophenol (all at 1 mM) inhibited 21-dehydroxylase activity in cell extracts by 25, 24, 35 and 84%, respectively. These results suggest that 21-dehydrogenase may be coupled to a NAD(P)H : flavin oxidoreductase system in E. lentum.

Cofactor requirements of steroid- 17-20-desmolase and 20α-hydroxysteroid dehydrogenase activities in cell extracts of Clostridium scindens

Two neutral steroid-transforming activities were demonstrated in cell extracts of Clostridium scindens. Steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase were found to be inducible in cells cultured in the presence of cortisol. Both activities required manganese ions and NAD+ or NADH for activity. Cortisol, cortisone and 11-desoxycortisol were substrates as well as inducers of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. 17 alpha-Hydroxyprogesterone was an effective inducer but did not serve as a substrate for either enzyme activity. C. scindens is the first bacterial species of the normal human intestinal flora reported to elaborate inducible steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. The results of cofactor, substrate specificity and induction studies suggest that these two activities may reside in the same enzyme complex.

Bacterial formation of aldosterone metabolites

The experiments described in this paper demonstrate that most of the metabolic alterations of the aldosterone molecule, hitherto attributed to hepatic enzymes, equally well may be carried out by enzymes synthesized by anaerobic bacteria from the human gut. The steroid reductases synthesized by Clostridium paraputrificum, Clostridium J-1, and Clostridium innocuum convert aldosterone to the 3 alpha, 5 beta tetrahydroaldosterone (THA), 3 beta, 5 alpha-THA, and 3 alpha, 5 alpha-THA, respectively. All three enzymes metabolize 5 alpha.dihydroaldosterone to a single compound: 3 beta, 5 alpha-THA. Bifidobacterium adolescentis reduces aldosterone to 20 beta-dihydroaldosterone. In mixed cultures of B. adolescentis and clostridia, the individual enzymes operate independently of each other; however, about half of the aldosterone metabolites are in the free form and half in the acetal form. By appropriate selection of substrate and bacterial strains, therefore, it is possible to biosynthesize not only three of the THA isomers but also the hexahydroisomers in free form as well as in the acetal form.