![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Biological Engineering Division (M.H.C., P.L.S., J.S.W., S.R.T.), and Department of Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts
(Received December 15, 2004; accepted March 3, 2005)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
-/ß-, 6ß-, 15ß-, and 16ß-hydroxytestosterones, mainly catalyzed by cytochromes P450 2C9, 2C19, and 3A4. We now report the first determination that 11ß-hydroxytestosterone (11ß-OHT) can also be formed by human liver microsomal fractions. The structures of five hydroxylated metabolites of testosterone (2ß-, 6ß-, 11ß-, 15ß-, and 16ß-OHT) and the C-17 oxidative metabolite androstenedione were determined by liquid chromatography with UV detection at 240 nm and liquid chromatography-tandem mass spectrometry. Corresponding results were obtained by high-performance liquid chromatography-AMS analysis of incubations of [4-14C]testosterone with human liver microsomes. 6ß-Hydroxylation was always the dominant metabolic pathway, but 2ß-, 15ß-, and 16ß-OHT, and androstenedione were also formed. The previously undetected hydroxytestosterone, 11ß-OHT, was found to be a minor metabolite formed by human liver microsomal enzymes. It was formed more readily by CYP3A4 than by either CYP2C9 or CYP2C19. 11ß-Hydroxylation was inhibited by ketoconazole (IC50 = 30 nM) at concentrations similar to the IC50 (36 nM) for 6ß-hydroxylation Therefore, CYP3A4 could be mainly responsible for testosterone 11ß-hydroxylation in the human liver. These findings identify human hepatic biotransformation of testosterone to 11ß-OHT as a previously unrecognized extra-adrenal metabolic pathway.
). Endogenous and exogenous testosterone undergoes oxidative metabolism by the human liver cytochrome P450 enzymes (Wood et al., 1988; Yamazaki and Shimada, 1997; Rendic et al., 1999). ß-Hydroxylation at either the C6 or C16 position is the major route of testosterone oxidative metabolism, whereas 1ß-, 2/ß-, and 15ß-hydroxytestosterone are produced as minor metabolites. Human liver enzymes are also found to oxidize testosterone at the C17 position to form androstenedione (Fig. 1).
|
). An alteration of the steroid hydroxylation metabolism in liver microsomes therefore indicates altered expression of various P450 enzymes. Liver tumor microsomes, whether from chemically induced or spontaneous tumors, showed a radically modified steroid hydroxylation pattern (Feuer et al., 1986; Lange et al., 1989). The 11ß-hydroxylase is the most thoroughly studied mammalian enzyme involved in steroid hormone biosynthesis. The observation that 11-deoxycorticosterone and 11-deoxycortisol are hydroxylated at the 11ß-carbon position by the enzyme in the mitochondrial fraction of the adrenal suggests that a single reactive site on the enzyme is involved in both cases (Sharma et al., 1962). Biochemical abnormalities are completely linked to the congenital adrenal hyperplasia that causes altered excretion of the tetrahydro-metabolites of these compounds (White et al., 1994; Choi et al., 2002). 11ß-Hydroxylation was confirmed in human adrenal homogenate as the major metabolic pathway of testosterone (Chang et al., 1963) as well as of certain neutral and phenolic steroids (Knuppen and Breuer, 1962; Hudson and Killinger, 1972). On incubating testosterone with a testicular tumor, 11ß-hydroxytestosterone (11ß-OHT) also was observed (Savard et al., 1960). Although the liver is responsible for most of the metabolism of cholesterol and the steroid hormones, to our knowledge, no metabolic study showing the roles of 11ß-hydroxylase on the endogenous steroid metabolism in the human liver has been published.
Whether testosterone may undergo hydroxylation at the C11 position by human liver microsomal P450s has apparently been overlooked, perhaps due in part to techniques that were less specific and less sensitive than those now available. In many studies (van der Hoeven, 1984; Smith et al., 1992; Tachibana and Tanaka, 2001), testosterone metabolites were assayed by HPLC with UV detection (LC-UV) and/or gas chromatography-mass spectrometry or LC-mass spectrometry in which 11ß-OHT, which may have been present only at trace levels, might not have been detected. In the present study, we have re-examined hepatic microsomal metabolism of testosterone, using 14C labeling to ensure detection of all metabolites with equal sensitivity and LC-tandem mass spectrometry for structural identification. Individuals with congenital adrenal hyperplasia suffer from hypertension due to a complete deficiency of adrenal 11ß-hydroxylase (Eberlein and Bongiovanni, 1956). In most classic cases of this deficiency, there is overproduction of steroid precursors proximal to the blocked 11ß-hydroxylase, leading to pathological effects (Zachmann et al., 1983). In this paper, we demonstrate that the human liver is capable of generating 11ß-oxygenated steroids from precursors such as testosterone. This could potentially have implications for either diagnosis or therapeutic treatment of adrenal insufficiency diseases.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-methyltestosterone, were purchased from Steraloids (Newport, RI). [4-14C]Testosterone (specific activity, 50 mCi/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Pooled human liver microsomes and selectively expressed human cytochrome P450 enzymes (CYP2C9, CYP2C19, and CYP3A4) were purchased from BD Gentest (Woburn, MA). Liver microsome protein content was 20 mg/ml in 250 mM sucrose. Recombinant P450 isoforms were expressed in insect cells selectively transfected with a baculovirus expression system containing the cDNA for human CYP2C9 (total protein concentration, 4 mg/ml; P450 content, 500 pmol/mg), CYP2C19 (3 mg/ml, 360 pmol/mg), and CYP3A4 (5 mg/ml, 400 pmol/mg). The catalytic activities for CYP2C9 (according to diclofenac 4'-hydroxylase), CYP2C19 [(S)-mephenytoin 4'-hydroxylase), and CYP3A4 (testosterone 6ß-hydroxylation) were 8000, 900, and 2200 pmol/mg P450/min, respectively.
Single human liver samples were obtained from organ donors or patients undergoing liver resection (Tennessee Donor Service, Nashville, TN). Each liver was tested for pathogenicity using a polymerase chain reaction protocol. Liver samples (0.3 g) were homogenized in 10 ml of ice-cold 10 mM potassium phosphate buffer (pH 7.4) using a Polytron homogenizer. The cell debris, nuclei, and mitochondria were removed by centrifugation at 10,000g for 20 min at 4°C, and then the supernatant was ultracentrifuged at 120,000g for 2 h at 4°C. The pellet was resuspended in ice-cold phosphate buffer (pH 7.4) containing 1 mM EDTA and 20% glycerol (v/v; Guengerich, 1994). Aliquots of microsomal fractions were quickly stored at -80°C until used. Protein concentrations of the microsomal fractions were measured in triplicate, with bovine serum albumin as a calibration standard, using commercial protein assay kits (Bio-Rad, Hercules, CA). Cytochrome P450 was estimated by absorbance differences between 450 and 490 nm based on the method of Omura and Sato (1964).
The NADPH-regenerating system used for all NADPH-requiring oxidase assays was obtained from BD Gentest. The system consists of two solutions (solution A, 26.1 mM NADP+, 66 mM glucose 6-phosphate, and 66 mM MgCl2 in H2O; solution B, 40 U/ml glucose-6-phosphate dehydrogenase in 5 mM sodium acetate). A stock solution of steroids including 17-methyltestosterone was prepared at concentrations of 1 µmol/ml in methanol. The stock solution was used to prepare a working solution of varying concentrations (1–100 µmol/ml) in methanol.
Microsomal Assays. A 0.3-ml reaction mixture containing 0.33 mg/ml human liver microsomal protein or 120 pmol/ml human CYP2C9, CYP2C19, or CYP3A4, 50 µl of NADPH-regenerating solution A, 20 µl of solution B, and 0.2 µmol of testosterone in 100 mM potassium phosphate (pH 7.4) was incubated at 37°C for 20 min. After incubation, the reaction was terminated by the addition of 0.5 ml of acetonitrile, and 50 µl of methyltestosterone (1 mM) was added. The mixture was then centrifuged (12,000 rpm) for 10 min to precipitate protein. The supernatant, filtered with a VariSep Nylon Syringe Filter (0.2 µm x 4 mm; Varian, Inc., Palo Alto, CA), was dried under a stream of nitrogen and then reconstituted with 50 µl of 50% methanol.
Recovery Test and Calculations. Solutions containing testosterone and its six metabolites at varied concentrations were prepared for method validation. All quantitative calculations were based on the peak area ratios relative to that of the internal standard. To prepare the calibration curve, enzyme-free incubation solutions with increasing concentrations of added analyte (0.1–2 mM testosterone and 0.2–10 µM for all metabolites) and a fixed concentration of added 17-methyltestosterone (50 µl; 1 mM) were analyzed according to the procedure described above. The recovery of the procedure was measured by comparing the responses obtained from the extracted samples to those obtained from the corresponding unextracted reference standards prepared at the same concentrations.
Instrumentation and Chromatography. LC analyses were carried out with an Agilent 1100 binary pumping system (Agilent Technologies, Palo Alto, CA), using a 9725 Rheodyne injector (Rheodyne, Rohnert Park, CA) and a G1314A variable wavelength UV detector set at 240 nm. An aliquot of the sample (2 µl) from each incubation was injected onto a Capcell Pak C18 UG120 column (2.0 x 150 mm, 5-µm particle size; Shiseido Fine Chemicals, Tokyo, Japan) and eluted at a flow rate of 0.2 ml/min by gradients of solvent A (20 mM ammonium acetate in 10% methanol) and solvent B (90% methanol). The typical gradient (solvent B) was as follows: 0 min, 10%; 0 to 10 min, 10%; 10 to 20 min, 30%; 20 to 30 min, 55%; 30 to 38 min, 55%; 38 to 45 min, 100%; 45 to 50 min, 100%. The gradient was then returned to the initial condition (10% B) and held for 8 min before analysis of the next sample.
|
AMS analyses were conducted by the Biological Engineering Accelerator Mass Spectrometry Lab at Massachusetts Institute of Technology. The AMS instrument has previously been described in detail (Liberman et al., 2004a). The interface used to generate CO2 from liquid samples has also been described in detail (Liberman et al., 2004b), and an example of its application to the analysis of HPLC effluent in continuous fashion has been presented (Skipper et al., 2004).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
11ß-Hydroxylation. To determine whether the peak corresponding to 11ß-OHT was produced by P450-catalyzed hydroxylation of testosterone, incubations with pooled human liver microsomes but without added testosterone were conducted. Extracts were analyzed by LC-UV. No peak was detected at the retention time of authentic 11ß-OHT. To obtain further evidence that the peak observed at the retention time for 11ß-OHT was derived from metabolism of testosterone, we analyzed metabolites produced from 14C-labeled substrate. 14C was detected by AMS using a previously described system (Liberman et al., 2004b; Skipper et al., 2004) that facilitates continuous profiling of the isotope concentration in HPLC effluent. Results, shown in Fig. 3, depict the coincident detection of isotope and UV absorbance in a region of the chromatogram that ranges from 11ß-OHT to 16ß-OHT, and provide a good indication that the peak assumed to be 11ß-OHT is derived from testosterone.
|
Finally, multiple HPLC isolates of the peak corresponding to 11ß-OHT (37–38 min) were pooled and concentrated, and the contents were subjected to mass spectrometry. The CID mass spectrum was in good agreement with the mass spectrum of 11ß-OHT authentic standard (retention time 37.3 min) (Fig. 4). The other possible isomers, 2-, 6-, 7-, 11-, and 15-OHT, were ruled out on the basis of their LC retention times. Liver microsomal formation of 11ß-OHT was linear (r2 = 0.972) over the range of 1 to 50 mM substrate concentration.
|
; Yamazaki and Shimada, 1997), CYP3A4 was a major catalytic enzyme for formation of 15ß-, 6ß-, and 16ß-OHT, whereas it catalyzed formation of 2ß-OHT and androstenedione poorly. In contrast, CYP2C9 and CYP2C19 were found to form androstenedione at high rates. In addition, CYP2C9 and CYP2C19 were found in this study to hydroxylate testosterone less efficiently than CYP3A4 at all positions. In addition, CYP3A4 catalyzed the formation of 11ß-OHT at rates higher than those of CYP2C9 and CYP2C19. Catalytic ratios of 11ß-OHT versus 6ß-OHT in testosterone hydroxylation by recombinant P450 enzymes, including CYP2C9, CYP2C19, and CYP3A4, indicated 6ß-OHT as a major product. The ratios of 11ß-OHT/6ß-OHT were 0.56, 0.40, and 0.11 for CYP2C9, CYP2C19, and CYP3A4, respectively (Fig. 5). The 11ß-OHT/6ß-OHT ratio in CYP3A4 was 5- and 4-fold lower than those in CYP2C9 and CYP2C19, respectively, whereas the 11ß-OHT/testosterone (11ß-OHT/T) ratio in CYP3A4 (0.012) was 4 times higher than those in CYP2C9 (0.003) and CYP2C19 (0.003; Fig. 6).
|
|
Inhibition of 11ß-Hydroxylation with Ketoconazole. KTZ is a selective inhibitor of CYP3A4 activity in human liver microsomes (IC50 = 40 nM), as evaluated by the 6ß-hydroxylation of testosterone (Eagling et al., 1998). To determine whether the CYP3A4 subfamily isoform also plays a predominant role in the 11ß-hydroxylation of testosterone in human liver microsomes, KTZ inhibition studies were conducted. The effects of coincubation with KTZ on testosterone 6ß- and 11ß-hydroxylation are shown in Fig. 7. As predicted, KTZ deactivated testosterone 6ß-hydroxylation, and the formation of 11ß-hydroxytestosterone was also inhibited with similar IC50 values, which were 36 nM and 30 nM for 6ß-hydroxylation and 11ß-hydroxylation, respectively.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
- and 21-hydroxylation) and then back to the mitochondria for 11ß-hydroxylation. Depending on whether the P450 hydroxylase is localized in the mitochondria or in the endoplasmic reticulum, there are two slightly different electron transport chains that function to transfer a pair of electrons from NADPH to the P450 enzyme. The most important component of the electron transport chain is the P450 protein, in that it determines the substrate specificity and dictates the precise site of hydroxylation. In principle, the three-dimensional structure of the substrate-binding domain of a P450 hydroxylase determines which substrates are able to acquire a new hydroxyl group.
A number of studies have shown that P450 enzymes in liver microsomes play an important role in the oxidative metabolism of steroids at positions other than C-11 (Waxman et al., 1991; Yamazaki and Shimada, 1997). Human liver microsomal CYP3A4 is responsible for the greatest portion of testosterone 6ß- and 16ß-hydroxylation (Kawano et al., 1987; Yamazaki and Shimada, 1997). CYP2C9 and CYP2C19 were also capable of oxidative catalysis of testosterone to form 2ß-, 6ß-, 15ß-, and 16ß-OHT, and androstenedione.
There is evidence for the occurrence of extra-adrenal hydroxylation of steroids at C-11. A virilizing testicular tumor, which exerts its effects by producing androgens, has been shown to form 11ß-OHT (Savard et al., 1960). 11ß-Hydroxylation of osaterone acetate (17-acetoxy-6-chloro-2-oxa-4,6-pregnandiene-3,20-dione), an exogenous steroid, by liver microsomal fraction (Minato et al., 1999) has also been demonstrated.
In the present study, hepatic hydroxylation at the 11ß-position of testosterone was observed, and the activity was higher with CYP3A4 than with CYP2C9 and CYP2C19. Chemical inhibition was used to further investigate the role of individual P450s. In many studies, KTZ mainly inhibits CYP3A4 activity at different concentrations (Newton et al., 1995; Yamazaki and Shimada, 1997; Eagling et al., 1998). Our data clearly indicate that KTZ selectively inhibited both testosterone 6ß- and 11ß-hydroxylation by CYP3A4 in human liver microsomes.
Human liver microsomal P450 enzymes are shown to be capable of metabolism of endogenous steroids as well as numerous therapeutic agents, and this metabolism seems to be differentially altered by a number of physiological and pathophysiological conditions. The oxidation of testosterone to 11ß-OHT by 11ß-hydroxylase in the adrenal cortex is well known. Although the physiological significance of the formation of 11ß-OHT by human liver remains unknown at present, our results suggest that this metabolite may participate in the generation of the androgens, which are formed in considerable quantities by the human liver catalyzed by CYP2C9, 2C19, and 3A4, as well as liver-associated, i.e., extra-adrenal, oxidation of testosterone to 11ß-OHT.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: OHT, hydroxytestosterone; LC, liquid chromatography; LC-UV, high-performance liquid chromatography with UV detection; HPLC, high-performance liquid chromatography; AMS, accelerator mass spectrometry; CID, collision-induced dissociation; KTZ, ketoconazole.
Address correspondence to: Dr. Steven R. Tannenbaum, Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 56-731A, Cambridge, MA 02139. E-mail: srt{at}mit.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxyoestrone by adrenal tissue. Biochim Biophys Acta 58: 147-148.[Medline]
This article has been cited by other articles:
|
|
 |
|
  S. K. Balani, N. V. Nagaraja, M. G. Qian, A. O. Costa, J. S. Daniels, H. Yang, P. R. Shimoga, J.-T. Wu, L.-S. Gan, F. W. Lee, et al. EVALUATION OF MICRODOSING TO ASSESS PHARMACOKINETIC LINEARITY IN RATS USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY Drug Metab. Dispos., March 1, 2006; 34(3): 384 - 388. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, 6ß-hydroxylation; 
, 11ß-hydroxylation. Data are the mean values from three experiments.