Hydrolysis of conjugated steroids by the combined use of β-glucuronidase preparations from helix pomatia and ampullaria: determination of urinary cortisol and its metabolites

Shibasaki, Hiromi; Tanabe, Chiharu; Furuta, Takahisa; Kasuya, Yasuji

doi:10.1016/s0039-128x(01)00118-0

Cited by 34 publications

(26 citation statements)

References 21 publications

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“…The supernatant was used for the LC-MS/MS analysis. The hydrolysis was performed according to the method described by Shibasaki et al (2001). Nonhydrolyzed urine samples were analyzed directly without any sample preparation.…”

Section: Finasteride Metabolites In Human Bile and Urinementioning

confidence: 99%

Identification of Finasteride Metabolites in Human Bile and Urine by High-Performance Liquid Chromatography/Tandem Mass Spectrometry

Lundahl¹,

Lennernäs²,

Knutson³

et al. 2009

Drug Metab Dispos

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ABSTRACT:The objective of this study was to further investigate the metabolism of the 5␣-reductase inhibitor, finasteride, and to identify previously unknown phase I and phase II metabolites in vitro and in vivo in human bile and urine. Healthy volunteers were given 5 mg of finasteride, directly to the intestine, and bile and urine were collected for 3 and 24 h, respectively. A single-pass perfusion technique, Loc-I-Gut, was used for drug administration and bile collection from the proximal jejunum, distal to papilla of Vater. Incubations with human liver microsomes/S9 fractions and different cofactors were performed with finasteride and the previously known metabolites, -hydroxy finasteride (M1) and finasteride--oic acid (M3). Liquid chromatography coupled to triple quadrupole mass spectrometry (MS) with positive/negative electrospray ionization and ion trap with MS n measurements were used for structural investigations and identification of metabolites. Two hydroxy metabolites of finasteride, other than M1, and one intact hydroxy finasteride glucuronide were identified in vitro and in bile and urine. The glucuronide and at least one of the hydroxy metabolites were previously unidentified. M1 and M3 were glucuronidated in vitro by specific human UDP-glucuronosyltransferases, UGT1A4 and UGT1A3, respectively. M1 glucuronide was not identified in vivo, and M3 glucuronide, an acyl glucuronide, was present in low amounts in bile from a few individuals. In conclusion, previously undescribed metabolites were identified, in vitro and in human urine and bile. Bile collection using the Loc-I-Gut technique followed by sensitive mass spectrometry analysis led to the discovery of novel, both phase I and phase II, finasteride metabolites in human bile.

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Section: Finasteride Metabolites In Human Bile and Urinementioning

confidence: 99%

Identification of Finasteride Metabolites in Human Bile and Urine by High-Performance Liquid Chromatography/Tandem Mass Spectrometry

Lundahl¹,

Lennernäs²,

Knutson³

et al. 2009

Drug Metab Dispos

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“…5. Of various methods for the reductive allomerization at C-5 of D 4 -3-ketosteroids reported previously, 24,25,29,30) it seemed to us that the most feasible route, starting from the readily available 1b through the inversion of the configuration at C-5, offered a better way to obtain the 3-ethoxy-3,5-diene intermediate (17). 24,25) The procedures employed were (1) the etherification of 1b with triethyl orthoformate in the presence of a catalytic amount of p-toluenesulfonic acid and (2) selective reduction of the D 3,5 -bond in the resulting 17 over 10% Pd/C catalyst, yielding enol ether (18), which after treatment with HCl gave the desired 19a.…”

Section: Resultsmentioning

confidence: 99%

“…The organic layer was washed with 5% NaHCO 3 and saturated brine, dried over anhydrous Na 2 SO 4 , and evaporated to dryness. Purification of the crude product by column chromatography on silica gel with hexane-EtOAc (7 : 3, v/v) as an eluent gave the intermediary 3-ethoxy-3,5-diene (17). This was dissolved in EtOAc-ethanol (12 ml; 1 : 1, v/v) and the solution was catalytically hydrogenated on 10% Pd/C (30 mg) overnight at room temperature.…”

Section: -Acetoxy-3amentioning

confidence: 99%

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Potential Corticoid Metabolites: Chemical Synthesis of 3- and 21-Monosulfates and Their Double-Conjugates of Tetrahydrocorticosteroids in the 5.ALPHA.- and 5.BETA.-Series

Okihara

Mitamura

Hasegawa

et al. 2010

CHEMICAL & PHARMACEUTICAL BULLETIN

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As the principal glucocorticoid in humans, cortisol plays a crucial role in modulating the metabolic and homeostatic process that protects against stress, shock, inflammation, etc.3-7) It is synthesized primarily in the zona fasciculata with a small contribution from the zona reticularis.3) While cortisol is essentially secreted by the adrenal glands, cortisone is mainly produced using 11b-hydroxysteroid dehydrogenase isoenzymes, which interconvert cortisol to hormonally inactive cortisone.3) Cortisol and cortisone are extensively metabolized to tetrahydro-reduced derivatives: tetrahydrocortisol (THF) and tetrahydrocortisone (THE), and their 5a-stereoisomer (allo-THF and allo-THE, respectively). 3,[7][8][9] Metabolism decreases the biological activity of hormones and increases their water solubility by converting them to hydrophilic compounds that can be excreted in urine. Unmetabolized cortisol and cortisone comprise only ca. 0.1% of the total urinary cortisol metabolites. At least 90% of the tetrahydro-derivatives of cortisol and cortisone metabolites are excreted into the urine as sulfate or glucuronide conjugates.10-13) Tetrahydro-11-deoxycortisol (THS) and its 5a-stereoisomer (allo-THS) which are tetrahydro-reduced metabolites of 11-deoxycortisol (11-DOC), a biosynthetic precursor of cortisol, are also excreted in urine as conjugated forms. 8,10,12) In order to obtain information regarding the related biochemistry, physiology, and pathophysiology of endocrine disorders, it is necessary to identify all the corticosteroid metabolites found in urine, and determine the exact structure of their conjugates, their concentration, and the dynamics of their formation and disposal.At present, analytical methods for the detection of conjugated tetrahydrocorticosteroids are based on gas chromatography and mass spectrometric determination of hydrolyzed and derivatized compounds.14-18) Although these methods are robust and sensitive, sample preparation in these indirect methods is time consuming, and the GC-MS sample throughput is relatively low. Thus the development or more straightforward methods based on the direct analysis of steroid conjugates without the need for a deconjugation process is of great interest. The application of liquid chromatographic separation interfaced by soft ionization techniques, such as electrospray ionization (ESI) with tandem mass spectrometry and/or ion trap mass spectrometry, offers an effective analytical tool for the direct monitoring of the urinary conjugates of tetrahydrocorticosteroids. 19,20) Recently, we reported a highly sensitive and specific liquid chromatography/ESI-linear ion trap mass spectrometry method for the direct measurement of the glucuronide conjugates of THF, THE, THS, and their 5a-stereoisomer in human urine.21) The authentic glucuronides had been chemically synthesized by Hosoda et al. [22][23][24][25] One significant drawback of sulfate conjugate analysis, however, is still the lack of reference materials, which are essential for the development and application of ...

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“…The assay is ideal for use in large projects involving huge number of samples. In humans, aldosterone is mainly excreted as a glucuronide in urine (Mohring et al, Tait et al) and thus estimation of total aldosterone is usually done after hydrolysis of the conjugate [25,26]. We have used the enzymatic hydrolysis method (β-glucuronidase, Helix pomatia, Sigma) as optimised by Ferchaud et al [25,35] to pre-treat mouse urine.…”

Section: Discussionmentioning

confidence: 99%

Development of a highly sensitive ELISA for aldosterone in mouse urine: Validation in physiological and pathophysiological states of aldosterone excess and depletion

et al. 2009

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Repository Use PolicyThe full-text may be used and/or reproduced, and given to third parties for personal research or study, educational or not-for-profit purposes providing that:• The full-text is not changed in any way AbstractClinical studies have established aldosterone as a critical physiological and pathophysiological factor in salt and water homeostasis, blood pressure control and in heart failure. Genetic and physiological studies of mice are used to model these processes. A sensitive and specific assay for aldosterone is therefore needed to monitor adrenocortical activity in murine studies of renal function and cardiovascular diseases. Methods: Antibodies against aldosterone were raised in sheep as previously described. HRP-Donkey-anti-sheep IgG enzyme tracer was produced in our laboratory using the Lightning-Link HRP technique. Aldosterone ELISA protocol was validated and optimised to achieve the best sensitivity. Cross reactivity with likely competing steroids and specificity before and after chromatography were checked.. The assay was validated by analysing the urine of mice collected under various experimental conditions designed to stimulate or suppress aldosterone in the presence of other potentially interfering steroid hormones. Results: Cross-reactivity with major interfering steroids was minimal: corticosterone=0.0028%, cortisol=0.0006%, DOC=0.0048% except for 5α-dihydroaldosterone=1.65%. Minimum detection limit of this ELISA was 5.7 pmol/L (1.6 pg/mL). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between the results obtained before and after solvent extraction and HPLC separation step (Y=1.048X+0.006, R 2 =0.998, n=42). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels were (i) approximately 60 fold higher in females than males; (ii) increased 6 fold by dietary sodium restriction; (iii) increased x fold by ACTH infusion; (iv) reduced by >60% in Cyp11b1 null mice. Conclusion We describe an ELISA for urinary aldosterone which is suitable for repeated non-invasive measurements in mice. Female aldosterone levels are higher than males. Unlike humans, most aldosterone in mouse urine is not conjugated. Increased levels were noted in response to dietary sodium restriction and ACTH treatment. The sensitivity of the assay is sufficient to detect suppressed levels in mouse models of congenital adrenal hyperplasia..

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Hydrolysis of conjugated steroids by the combined use of β-glucuronidase preparations from helix pomatia and ampullaria: determination of urinary cortisol and its metabolites

Cited by 34 publications

References 21 publications

Identification of Finasteride Metabolites in Human Bile and Urine by High-Performance Liquid Chromatography/Tandem Mass Spectrometry

Identification of Finasteride Metabolites in Human Bile and Urine by High-Performance Liquid Chromatography/Tandem Mass Spectrometry

Potential Corticoid Metabolites: Chemical Synthesis of 3- and 21-Monosulfates and Their Double-Conjugates of Tetrahydrocorticosteroids in the 5.ALPHA.- and 5.BETA.-Series

Development of a highly sensitive ELISA for aldosterone in mouse urine: Validation in physiological and pathophysiological states of aldosterone excess and depletion

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