Limits...
Metabolism of androstenone, 17β-estradiol and dihydrotestosterone in primary cultured pig hepatocytes and the role of 3β-hydroxysteroid dehydrogenase in this process.

Chen G, Bai Y, Ren L, Zhu D, Li Y, Fang M, Al-Kateb H, Doran O - PLoS ONE (2015)

Bottom Line: The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor.Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol.The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Agro-product Quality and Safety, Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.

ABSTRACT
Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

Show MeSH

Related in: MedlinePlus

Ion chromatograms and mass spectra (inset) of androstenone and its metabolites.(A) androstenone in the medium in absence of isolated hepatocytes; (B) androstenone in the medium in the presence of isolated hepatocytes. No androstenone was found after 24 h of cell culture; (C) identified androstenone metabolite (m/z+ = 257.2245). The samples were analyzed in ESI+ ionization mode; (D) identified androstenone metabolite 5α-androst-16-en-3β-glucuronide (m/z− = 449.2424) in ESI– ionization mode; (E) identified androstenone metabolite 5α-androst-16-en-3β-ol after enzyme hydrolysis by β-glucuronidase; (F) a mixture of authentic standards 5α-androst-16-en-3α-ol (3α-A) and 5α-androst-16-en-3β-ol (3β-A).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4295843&req=5

pone-0113194-g001: Ion chromatograms and mass spectra (inset) of androstenone and its metabolites.(A) androstenone in the medium in absence of isolated hepatocytes; (B) androstenone in the medium in the presence of isolated hepatocytes. No androstenone was found after 24 h of cell culture; (C) identified androstenone metabolite (m/z+ = 257.2245). The samples were analyzed in ESI+ ionization mode; (D) identified androstenone metabolite 5α-androst-16-en-3β-glucuronide (m/z− = 449.2424) in ESI– ionization mode; (E) identified androstenone metabolite 5α-androst-16-en-3β-ol after enzyme hydrolysis by β-glucuronidase; (F) a mixture of authentic standards 5α-androst-16-en-3α-ol (3α-A) and 5α-androst-16-en-3β-ol (3β-A).

Mentions: Figure 1A presents data on abundance of androstenone precursor ion (m/z+ = 273.2198) in the incubation medium before cell culturing. A time-course of androstenone metabolism showed that androstenone was non-detectable in the cell culture medium after 24 h of incubation (Figure 1B). LC-TOF-MS analysis of androstenone metabolites in ESI+ ionization mode identified a metabolite ion (m/z+ = 257.2245), which corresponds to a precursor ion of androstenone without a 3-keto group (Figure 1C). Analysis of androstenone metabolite in ESI– ionization mode demonstrated the presence of another ion with m/z− = 449.2424 (Figure 1D). A molecular formula of this ion was determined using MetID software. The analysis demonstrated that this ion corresponds to a glucuronide-conjugated androstenone (A–G). Further investigation was conducted to determine association between the results obtained with the two different ionization approaches. The fraction of m/z+ = 257.2245 from post-HPLC column was collected and analyzed in ESI– mode. The results demonstrated a sole mass spectrometric peak of m/z− = 449.2424, with the retention time corresponding to the peak of androstenone metabolite in the cultured medium samples analyzed in ESI– mode. These experiments confirmed that the ion m/z+ = 257.2245 is the glucuronide-conjugated androstenone which lost glucuronide structure (C6H8O6, MW = 176) in ESI+ ionization.


Metabolism of androstenone, 17β-estradiol and dihydrotestosterone in primary cultured pig hepatocytes and the role of 3β-hydroxysteroid dehydrogenase in this process.

Chen G, Bai Y, Ren L, Zhu D, Li Y, Fang M, Al-Kateb H, Doran O - PLoS ONE (2015)

Ion chromatograms and mass spectra (inset) of androstenone and its metabolites.(A) androstenone in the medium in absence of isolated hepatocytes; (B) androstenone in the medium in the presence of isolated hepatocytes. No androstenone was found after 24 h of cell culture; (C) identified androstenone metabolite (m/z+ = 257.2245). The samples were analyzed in ESI+ ionization mode; (D) identified androstenone metabolite 5α-androst-16-en-3β-glucuronide (m/z− = 449.2424) in ESI– ionization mode; (E) identified androstenone metabolite 5α-androst-16-en-3β-ol after enzyme hydrolysis by β-glucuronidase; (F) a mixture of authentic standards 5α-androst-16-en-3α-ol (3α-A) and 5α-androst-16-en-3β-ol (3β-A).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295843&req=5

pone-0113194-g001: Ion chromatograms and mass spectra (inset) of androstenone and its metabolites.(A) androstenone in the medium in absence of isolated hepatocytes; (B) androstenone in the medium in the presence of isolated hepatocytes. No androstenone was found after 24 h of cell culture; (C) identified androstenone metabolite (m/z+ = 257.2245). The samples were analyzed in ESI+ ionization mode; (D) identified androstenone metabolite 5α-androst-16-en-3β-glucuronide (m/z− = 449.2424) in ESI– ionization mode; (E) identified androstenone metabolite 5α-androst-16-en-3β-ol after enzyme hydrolysis by β-glucuronidase; (F) a mixture of authentic standards 5α-androst-16-en-3α-ol (3α-A) and 5α-androst-16-en-3β-ol (3β-A).
Mentions: Figure 1A presents data on abundance of androstenone precursor ion (m/z+ = 273.2198) in the incubation medium before cell culturing. A time-course of androstenone metabolism showed that androstenone was non-detectable in the cell culture medium after 24 h of incubation (Figure 1B). LC-TOF-MS analysis of androstenone metabolites in ESI+ ionization mode identified a metabolite ion (m/z+ = 257.2245), which corresponds to a precursor ion of androstenone without a 3-keto group (Figure 1C). Analysis of androstenone metabolite in ESI– ionization mode demonstrated the presence of another ion with m/z− = 449.2424 (Figure 1D). A molecular formula of this ion was determined using MetID software. The analysis demonstrated that this ion corresponds to a glucuronide-conjugated androstenone (A–G). Further investigation was conducted to determine association between the results obtained with the two different ionization approaches. The fraction of m/z+ = 257.2245 from post-HPLC column was collected and analyzed in ESI– mode. The results demonstrated a sole mass spectrometric peak of m/z− = 449.2424, with the retention time corresponding to the peak of androstenone metabolite in the cultured medium samples analyzed in ESI– mode. These experiments confirmed that the ion m/z+ = 257.2245 is the glucuronide-conjugated androstenone which lost glucuronide structure (C6H8O6, MW = 176) in ESI+ ionization.

Bottom Line: The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor.Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol.The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Agro-product Quality and Safety, Institute of Quality Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.

ABSTRACT
Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

Show MeSH
Related in: MedlinePlus