Androstadienedione Research Articles

Physiological and biochemical improvement of the enzyme side-chain degradation of cholesterol by Fusarium solani

The conversion of cholesterol (C) to androstenedione (AD) and androstadienedione (ADD) was influenced significantly by the composition of the cultivation medium. A medium composed of (g/l): glucose, 50; peptone, 20; corn steep solid, 3; supported relatively high AD and ADD outputs. C convertibility was further accelerated when the cultivation medium was adjusted with phosphate buffer with the pH range 5.5–7.38. Supplementation of the medium with 8-hydroxyquinoline or methanol prevented the degradation of the C nucleus with concomitant formation of good AD and ADD yields.

Study of key operational parameters for the side-chain cleavage of sitosterol by free mycobacterial cells in Bis-(2-ethylhexyl) phthalate

The selective side-chain cleavage of β-sitosterol by free cells of Mycobacterium sp. NRRL B-3805 is a well-established multi-enzymatic process for the production of the pharmaceutical steroid precursors androstenedione (AD) and androstadienedione (ADD). In this study, bis(2-ethylhexyl) phthalate (BEHP) was used as a reaction medium for carrying out the process with freely suspended cells. The work aimed to show that microbial sitosterol side-chain cleavage is possible in this essentially mono-phasic organic medium, provided that some important parameters are adequately controlled. The effects of the biocatalyst/substrate mass ratio, system aeration rate and minimum buffer addition to the organic medium on the product yield and the reaction rate were thus evaluated.

Microbial transformation of hydrophobic compound in cloud point system

Microbial transformations of hydrophobic compounds are often constrained by practical difficulties, such as substrate solubility and product inhibition. A novel approach, microbial transformation in cloud point system (CPS), was developed. The system could provide microbial cells with aqueous environment and could dissolve substrate and products in surfactant phase. A microbial transformation of cholesterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (4-AD) has been carried out in the system consisting of nonionic surfactants Triton X-100 and Triton X-114, which is more effective than the microbial transformation in conventional media. The biocompatibility and bioavailability in the cloud point system were investigated by determination of solubilization of surfactant phase and observation of dilute phase and coacervate phase under microscopy. The system parameters of microbial transformation were optimized. It indicated that CPS has the potential capability to be utilized as an effective microbial transformation medium.

Confirmatory analysis of 17β-boldenone, 17α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine, faeces, feed and skin swab samples by liquid chromatography–electrospray ionisation tandem mass spectrometry

The origin, i.e. natural occurrence or illegal treatment, of findings of 17α-boldenone (α-Bol) and 17β-boldenone (β-Bol) in urine and faeces of cattle is under debate within the European Union. A liquid chromatographic positive ion electrospray tandem mass spectrometric method is presented for the confirmatory analysis of 17β-boldenone, 17α-boldenone and an important metabolite/precursor androsta-1,4-diene-3,17-dione (ADD), using deuterium-labelled 17β-boldenone (β-Bol-d3) as internal standard. Detailed sample preparation procedures were developed for a variety of sample matrices such as bovine urine, faeces, feed and skin swab samples. The method was validated as a quantitative confirmatory method according to the latest EU guidelines and shows good precision, linearity and accuracy data, and CCα and CCβ values of 0.1–0.3 and 0.4–1.0 ng/ml, respectively. Currently, the method has been successfully applied to suspect urine samples for more than a year, and occasionally to faeces, feed and swab samples as well. Results obtained from untreated and treated animals are given and their impact on the debate about the origin of residues of 17β-boldenone is critically discussed. Finally, preliminary data about the degree of conjugation of boldenone residues are presented and a simple procedure for discrimination between residues from abuse versus natural origin is proposed.

Model of bioconversion of cholesterol in cloud point system

Bioconversion of cholesterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (4-AD) in cloud point system (CPS) was studied. A mathematical model was presented based on the mass transfer rate and microbial growth rate. The calculations for cell concentration, substrate concentration, and product concentration as functions of time were made. Sensitive analysis of model was made. Some parameters, such as substrate concentration, mass transfer coefficient, and maximum product inhibition concentration, indicated that the maximum product concentration is the most significant parameter in batch process. The prediction was validated by adjusting substrate concentration, stirring speed, and surfactant concentration. It showed the batch process of cholesterol bioconversion in CPS was controlled by inhibition of product. Adjusting surfactant concentration can diminish inhibition of product and increase final product concentration. The results also indicated that CPS can increase the substrate concentration of aqueous phase in batch process.

Confirmatory analysis of 17β-boldenone, 17α-boldenone and androsta-1,4-diene-3,17-dione in bovine urine by liquid chromatography–tandem mass spectrometry

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for confirmatory analysis of 17β-boldenone (17β-BOL), 17α-boldenone (17α-BOL) and androsta-1,4-diene-3,17-dione (ADD) in bovine urine was developed. [ 2H 2]17β-Testosterone (17β-T-d 2) was used as the internal standard. Sample preparation involved enzymatic hydrolysis and purification on a C 18 solid-phase extraction column. Chromatographic separation of the analytes was obtained using an RP-C 18 HPLC column. LC–MS–MS detection was carried out with an atmospheric pressure chemical ionisation (APCI) source equipped with a heated nebulizer (HN) interface operating in the positive ion mode. For unambiguous hormone confirmation, three analyte precursor–product ion combinations were monitored during multiple-reaction monitoring (MRM) LC–MS–MS analysis. Overall recovery (%), repeatability (relative standard deviations, RSD, %) and within-laboratory reproducibility (RSD, %) ranged from 92.2 to 97.7%, from 6.50 to 2.94% and from 13.50 to 5.04%, respectively, for all analytes. The limit of quantification in bovine urine was 0.20 ng ml −1 for 17β-BOL and ADD and 0.50 ng ml −1 for 17α-BOL. The validated method was successfully applied for determination of 17β-BOL, 17α-BOL and ADD in a large number of bovine urine samples collected within the national Official Residue Control Program.

Lecithin‐enhanced biotransformation of cholesterol to androsta‐1,4‐diene‐3,17‐dione and androsta‐4‐ene‐3,17‐dione

AbstractA biotransformation process using Mycobacterium sp was studied for androsta‐1, 4‐diene‐3,17‐dione (ADD) and androsta‐4‐ene‐3,17‐dione (AD) production from cholesterol. Cholesterol has a poor solubility in water (∼1.8 mg dm−3 at 25 °C), which makes it difficult to use as the substrate for biotransformation. Lecithin is a mixture of phospholipids of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which behave like surfactants and can form planar bi‐layer structures in an aqueous medium. Therefore, a small amount of lecithin (<1 g dm−3) can be used to form stable colloids with cholesterol at a relatively high concentration (20 g dm−3) in water. In this work, an energy density of 1000 J cm−3 from sonication was provided to overcome the self‐association of cholesterol and to generate a stable lecithin–cholesterol suspension that could be used for enhanced biotransformation. The lecithin–cholesterol suspension was stable and could withstand typical autoclaving conditions (121 °C, 15 psig, 20 min). In contrast to conventional surfactants, such as Tween 80, that are commonly used to help solubilize cholesterol, lecithin did not change the surface tension of the aqueous solution nor cause any significant foaming problem. Lecithin was also biocompatible and showed no adverse effect on cell growth. Compared with the medium with Tween 80 as the cholesterol‐solubilizing agent, lecithin greatly improved the biotransformation process in regard to its final product yield (∼59% w/w), productivity (0.127–0.346 g dm−3 day−1), ADD/AD ratio (6.7–8), as well as the long‐term process stability. Cells can be reused in repeated batch fermentations for up to seven consecutive batches, but then lose their bioactivity due to aging problems, possibly caused by product inhibition and nutrient depletion.© 2002 Society of Chemical Industry

Studies on the catalytic function of aromatase: aromatization of 6-alkoxy-substituted androgens

To gain insight into the catalytic function of aromatase, we studied aromatization of a series of 6α- and 6β-ether-substituted (methoxy, ethoxy, and n-butoxy) androst-4-ene-3,17-dione (AD) steroids ( 1 and 2) and their androsta-1,4-diene-3,17-dione (ADD) derivatives ( 3 and 4) with human placental aromatase by gas chromatography–mass spectrometry (GC–MS). Among the steroids examined, 6β-methoxy and 6β-ethoxyADDs ( 4a and 4b) are suicide substrates of aromatase. All of the steroids were found to be converted into the corresponding 6-alkoxy estrogens. Introduction of the alkoxy groups at C-6 of AD or ADD decreased the ability of these to serve as a substrate of aromatase. In 6α-alkoxy steroid series, compounds 1 and 3, the aromatization rate increased by elongating the 6-methoxy group up to the n-butoxy group whereas, in the 6β-isomers series, 2 and 4, the rate decreased due to this structural modification. 6β-Alkoxy steroids, 2 and 4, including the suicide substrates, were extremely poor substrates for the aromatization reaction. Apparent K m values obtained for 6α-alkoxy compounds 1 and 3 were similar to each other, ranging from 92 to 111 nM, as shown by their previously-obtained K i values. The findings indicate that the stereochemistry as well as the bulkiness of the 6-ether-substituent play an important role in the ability to serve as a substrate. It is also predicted that the aromatization reaction and the mechanism-based inactivation reaction would be related and have a definite partition number which is characteristic to the compound in a series of suicide substrates.

Bioconversion of 3 beta-acetoxypregna-5,16-diene-20-one to androsta-1,4-diene-3,17-dione by mixed bacterial culture.

To isolate a bacterium capable of degrading 3 beta-acetoxypregna-5,16-diene-20-one (16-DPA) to androsta-1,4-diene-3,17-dione (ADD) and to decipher the biodegradation pathway. Isolation on mineral salt agar containing 16-DPA as sole carbon source yielded two bacteria identified as Pseudomonas diminuta and Comamonas acidovorons. These bacteria failed to degrade 16-DPA individually in pure cultures but converted 16-DPA to ADD in a mixed culture. The intermediates accumulated during the bioconversion were identified as pregna-4,16-diene-3,20-dione and pregna-1,4,16-triene-3,20-dione. The degradation pattern of 16-DPA by mixed bacterial culture revealed the reaction sequence as (i) cleavage of C-3 acetyl function, (ii) dehydrogenation at C-1 and C-2 positions and (iii) cleavage of C-17 side-chain. The present work opens a new approach towards the production of a female sex hormone precursor and elucidates the biodegradation pathway of 16-DPA by mixed bacterial culture.

Enzymic aromatization of 6-alkyl-substituted androgens, potent competitive and mechanism-based inhibitors of aromatase.

To gain insight into the relationships between the aromatase inhibitory activity of 6-alkyl-substituted androgens, potent competitive inhibitors, and their ability to serve as a substrate of aromatase, we studied the aromatization of a series of 6alpha- and 6beta-alkyl (methyl, ethyl, n-propyl, n-pentyl and n-heptyl)-substituted androst-4-ene-3,17-diones (ADs) and their androsta-1,4-diene-3,17-dione (ADD) derivatives with human placental aromatase, by gas chromatography-mass spectrometry. Among the inhibitors examined, ADD and its 6alpha-alkyl derivatives with alkyl functions less than three carbons long, together with 6beta-methyl ADD, are suicide substrates of aromatase. All of the steroids, except for 6beta-n-pentyl ADD and its n-heptyl analogue as well as 6beta-n-heptyl AD, were found to be converted into the corresponding 6-alkyl oestrogens. The 6-methyl steroids were aromatized most efficiently in each series, and the aromatization rate essentially decreased in proportion to the length of the 6-alkyl chains in each series, where the 6alpha-alkyl androgens were more efficient substrates than the corresponding 6beta isomers. The Vmax of 6alpha-methyl ADD was approx. 2.5-fold that of the natural substrate AD and approx. 3-fold that of the parent ADD. On the basis of this, along with the facts that the rates of a mechanism-based inactivation of aromatase by ADD and its 6alpha-methyl derivative are similar, it is implied that alignment of 6alpha-methyl ADD in the active site could favour the pathway leading to oestrogen over the inactivation pathway, compared with that of ADD. The relative apparent Km values for the androgens obtained in this study are different from the relative Ki values obtained previously, indicating that there is a difference between the ability to serve as an inhibitor and the ability to serve as a substrate in the 6-alkyl androgen series.

Effect of inhibitors of cell envelope synthesis on β‐sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683

The role of the lipid bilayer and the peptidoglycan of the mycobacterial cell wall in the permeation of beta-sitosterol into the cell and its transformation to androst-1-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) was studied. Specific inhibitors were used at concentrations affecting the biosynthesis of the assumed target structures, but causing only partial cell growth inhibition or exerting no effect on growth. m-Fluorophenylalanine and DL-norleucine which are known to disorganize the biosynthesis of amphipatic components of the outer layer of the lipid bilayer, used at concentrations 250 micrograms/ml and 400 micrograms/ml, respectively, increased the formation rate of AD+ADD from 0.3 (control) to 0.7 and 0.8 mg products/g dry weight/h. The disorganization of the underlying mycolyl-arabinogalactan structure by the action of the ethambutol at the concentration 40 micrograms/ml, at which the cell growth was apparently not affected, caused the decrease of the product formation from 135 mg/l to 70 mg/l. In the presence of isoniazid (350 micrograms/ml) only trace amounts of AD accumulated during 48 hours of transformation indicating much lower activity than that of the intact cells. The most effective among the tested inhibitors of peptidoglycan synthesis were glycine (15 mg/ml) and vancomycin (150 micrograms/ml) which enhanced the transformation activity of the treated cells nearly three times. Increased transformation rate was also obtained by the action of colistin at concentrations ranging from 10 micrograms/ml to 15 micrograms/ml.

Microbial transformation of sterols to C19-steroids by Rhodococcus equi

A wild-type strain of Rhodococcus equi, isolated from soil, degraded cholesterol, β-sitosterol, stigmasterol and mixed sterois to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). A definite preference for a relatively simply structured cholesterol side chain was observed. Highest specific cholesterol side-chain cleavage was associated with active growth of the culture. Maximum yield of ADD was obtained when sodium acetate and cholesterol were incorporated together in the medium. Specific side-chain cleavage required the presence of 2,2'-dipyridyl, an inhibitor of ring cleavage.

Untersuchungen zur Regioselektivität von Wittig‐Horner‐ und Reformatsky‐Reaktionen an ausgewählten 3,17‐Dioxosteroiden

Investigations on Regioselectivity of Wittig‐Horner and Reformatsky‐Reactions on Selected 3,17‐Dioxo SteroidsThe Reformatsky synthesis and the Wittig‐Horner olefination of 3,17‐dioxo steroids like androsta‐1,4‐diene‐3,17‐dione (ADD) 1 and androst‐4‐ene‐3,17‐dione (AD) 2 is described. There are differences in the regioselective introduction of identical substructures with these two methods resulting from different nucleophilicity and stereoelectronical relations of these two reagents. In Reformatsky synthesis the oxo group in 3‐position predominantly reacts, whereas in the Wittig‐Horner olefination (in the case of 1) the reaction occurs mainly in the 17‐position.The structures of all new compounds has been confirmed by spectroscopical data.

Steroid transformation in aqueous two-phase systems: side-chain degradation of cholesterol by Mycobacterium sp.

We report on the use of aqueous two-phase systems for the side-chain degradation of cholesterol into androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) by Mycobacterium sp. A problem encountered at the start of this investigation, extensive aggregation of the cells, was solved by careful choice of the polymer and detergent composition of the phase system. The systems showing the best properties were composed of polyethylene glycol, dextran and Brij 35 or polyvinylpyrrolidone, dextran and Brij 35 (or Tween 40). The bacterium maintained in the upper polyethylene glycol-rich (or polyvinylpyrrolidone-rich) phase was found to have high activity. The substrated partitioned to the upper phase or the interface (if present as crystals), while the products had partition coefficients around 2.

Biotransformation of sugar‐cane sterols into Androsta 1,4‐dione‐3,17‐dione (ADD) by Arthrobacter globiformis Str. Oxydans

Arthrobacter globiformis isolated from garden soil was cultured on phosphate salt medium containing phytosterols extracted from sulphitation pressmud, a sugar industry waste, as sole source of carbon. The bacterium grew well on sterols and transformed them into precursors of steroidal drugs and hormones. Addition of α,α′‐dipyridyl and sodium arsenite as metabolic inhibitors in the culture medium enhanced the accumulation of a 17‐ketosteroid which acts as precursor of steroidal drugs. The optimum temperature and pH conditions for its maximum accumulation were found to be 32 ± 0.5d̀C and 7.2. Spectroscopic analyses by u.v., i.r., p.m.r. and mass spectra of the 17‐ketosteroid formed by microbial activity in the medium confirmed it to be androsta‐1,4‐diene‐3,17‐dione (ADD). The bioconversion of sugar‐cane sterols into 17‐ketosteroid in varying culture condition is discussed.

Microbial Transformation of Sterols

Cholesterol decomposing ability of 1589 microbial strains was examined. Two hundreds and thirty six strains from actinomycetes, bacteria, molds, and yeasts were found capable of oxidizing cholesterol into cholestenone. Cholesta-1,4-dien-3-one was produced by 5 strains of Streptomyces. The complete decomposition of cholesterol molecule was observed in the genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. α,α′-Dipyridyl and arsenite inhibited decomposing enzymes giving rise to cholestenone, cholesta-1,4-dien-3-one, and an intermediate probably devoid of the sterol side chain.Selective cleavage of the side chains of various sterols at C-17, giving rise to androsta-1,4-diene-3,17-dione (ADD), occurred in the presence of α,α′-dipyridyl by microorganisms of the following genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Strept...

Microbial Transformation of Sterols

Cholesterol decomposing ability of 1589 microbial strains was examined. Two hundreds and thirty six strains from actinomycetes, bacteria, molds, and yeasts were found capable of oxidizing cholesterol into cholestenone. Cholesta-1,4-dien-3-one was produced by 5 strains of Streptomyces. The complete decomposition of cholesterol molecule was observed in the genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. α,α′-Dipyridyl and arsenite inhibited decomposing enzymes giving rise to cholestenone, cholesta-1,4-dien-3-one, and an intermediate probably devoid of the sterol side chain.Selective cleavage of the side chains of various sterols at C-17, giving rise to androsta-1,4-diene-3,17-dione (ADD), occurred in the presence of α,α′-dipyridyl by microorganisms of the following genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Strept...