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Experimental Studies |
-Hydroxylase1
Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Obstetrics-Gynecology and Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas 75235
Address all correspondence and requests for reprints to: Paul C. MacDonald, M.D., Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051. E-mail: macdonald{at}grnctr.swmed.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase of extrahepatic human tissues.
Progesterone and 5-dihydroprogesterone (5-DHP) are plasma-borne
precursors of 5-pregnan-3-ol-20-one, an anxiolytic/anesthetic
steroid, and 5-pregnan-3ß-ol-20-one in extrahepatic human tissues.
These two steroids are metabolized further by a saturated steroid
6-hydroxylase enzyme(s) that is distinct from the cytochrome P450
6-hydroxylase that catalyzes the 6-hydroxylation of
4-3-ketosteroids such as progesterone, cortisol, and
testosterone. Products of this saturated steroid 6-hydroxylase,
viz. 3ß/,6-dihydroxy-5-pregnan-20-ones, are
major radiolabeled urinary metabolites (excreted as glucuronosides) of
iv administered tritium-labeled 5-DHP in women and men. T47-D human
breast cancer cells, which are rich in saturated steroid
6-hydroxylase activity, were used as the enzyme source in this
study. The greatest total and the highest specific activity of
saturated steroid 6-hydroxylase were localized in microsome-enriched
preparations; enzyme activity was linear with incubation time up to 30
min and with microsome-enriched tissue protein concentrations between
0.05–0.5 mg/mL incubation mixture. The velocity of the reaction was
similar in incubations in which the pH was varied from 6.0–8.0, and
NADH and NADPH were equally effective in supporting the
6-hydroxylation of 5-pregnan-3ß-ol-20-one and
5-pregnan-3-ol-20-one. The more efficient substrates for this
enzyme were 5-pregnan-3ß-ol-20-one and
5-pregnan-3-ol-20-one, and the apparent Km
(
3.5 µmol/L) and maximum velocity (150 pmol/min·mg
microsome-enriched protein) for these two substrates were
indistinguishable. 5-Androstane-3ß,17ß-diol was less efficiently
6-hydroxylated, and 5-androstane-3,17ß-diol was an
inefficient substrate. The addition of a variety of inhibitors of
cytochrome P450 monooxygenases to the incubation mixtures did not
diminish significantly the 6-hydroxylation of
5-pregnan-3ß-ol-20-one, findings consistent with those of other
investigators who suggested that human saturated steroid
6-hydroxylase (of human prostate) is not a cytochrome P450. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydroprogesterone (5-DHP) is of considerable
physiological interest because these steroids are converted to
bioactive metabolites that act through progesterone
receptor-independent mechanisms. For example, plasma progesterone is
converted to deoxycorticosterone, a mineralocorticosteroid, in
extrahepatic, extraadrenal tissues (1, 2), and progesterone and
5-DHP are plasma-borne precursors of the bioactive steroid,
5-pregnan-3-ol-20-one, which is formed in a number of
extrahepatic tissues, including brain (3). 5-Pregnan-3-ol-20-one
acts to induce anxiolysis/anesthesia in all vertebrates studied,
including humans (4, 5, 6, 7), presumably through the stereospecific binding
of this steroid to the 
-aminobutyric acidA
(GABAA) receptor-chloride channel complex (8, 9, 10, 11, 12, 13, 14, 15, 16). This
steroid acts to increase the affinity of the receptor for GABA and
thereby facilitates the maintenance of the chloride channel in an open
state, a mechanism of action analogous to those of the barbiturates and
the benzodiazepine class of drugs (17, 18). These anxiolytic agents do
not act independently, but, rather, facilitate the neuroinhibitory
action of GABA.
In investigations conducted recently, it was established that
radioactive 3ß,6-dihydroxy-5-pregnan-20-one and
3,6-dihydroxy-5-pregnan-20-one (excreted as glucuronosides)
are major urinary metabolites of iv administered tritium- or
carbon-14-labeled 5-DHP in women and men (19). Fifteen years ago,
O’Hare and colleagues found that the major metabolite of progesterone
in six different human teratocarcinoma cell lines (of ovarian and
testicular origin) and in normal human breast epithelial and
mesenchymal cells was 3ß,6-dihydroxy-5-pregnan-20-one, and that
5-pregnan-3ß-ol-20-one was the substrate for 6-hydroxylation.
Contrarily, 6-hydroxyprogesterone was not a substrate for
5-reductase in these cells (20). Antila and co-workers confirmed
these findings in studies of one of the teratocarcinoma lines,
i.e. PA-1 (21). Ten years ago, Fennessey and colleagues
found that progesterone (10-6 mol/L) was metabolized
completely to a single product during 24-h incubations with T47-D
cells, which are human breast cancer cells characterized by the
presence of estrogen and progesterone receptors (22, 23). Horwitz and
associates also found that progesterone was metabolized to
3ß,6-dihydroxy-5-pregnan-20-one in MCF-7 human breast
carcinoma cells, but in these cells, a second product (in much smaller
quantities) also was identified, viz.
3,6-dihydroxy-5-pregnan-20-one. These investigators
established that the pathway of metabolism of progesterone to these
polar metabolites proceeded as follows:
progesterone
5-DHP5-pregnan-3ß/-ol-20-one3ß/,6-dihydroxy-5-pregnan-20-one.
They also demonstrated that 5-pregnan-3-ol-20-one was converted
quantitatively to two products, viz.
3,6-dihydroxy-5-pregnan-20-one and
3ß,6-dihydroxy-5-pregnan-20-one. Using 5ß-pregnan-3,20-dione
as substrate, there was complete metabolism to two products during an
18-h incubation, viz. 5ß-pregnan-3ß-ol-20-one (20%) and
5ß-pregnan-3-ol-20-one (80%). Incubation of
5ß-pregnan-3ß-ol-20-one gave a compound (31% of the total) that
appeared to be 3ß,6-dihydroxy-5ß-pregnane-20-one.
These findings are supportive of the view that the inactivation of
5-pregnanolones involves the 6-hydroxylation of the saturated
metabolites by an enzyme(s) distinct from the cytochrome P450
6-hydroxylase of human liver, which acts upon
4-3-keto-C19- and C21-steroids,
such as cortisol, progesterone, and testosterone, but does not act upon
5-reduced C21- or C19-steroids. This view is
supported further by the finding that 5-reductase does not act upon
6-hydroxysteroid substrates (20, 23). A similar metabolic pathway of
6-hydroxylation of saturated steroids exists in the human prostate.
Namely, testosterone is metabolized to DHT, thereafter to
5-androstane-3ß,17ß-diol, and thence to
5-androstane-3ß,6,17ß-triol in a reaction catalyzed by a
saturated steroid 6-hydroxylase. In human prostate, however, there
is a more dominant pathway for C19-steroid metabolism,
viz. the 7/ß-hydroxylation of
5-androstane-3ß,17ß-diol (24).
The purpose of this investigation was to characterize further the
6-hydroxylation of saturated steroids with specific emphasis on
defining the kinetics and substrate specificity of this saturated
steroid 6-hydroxylase reaction and in evaluating further the
possibility that this enzyme(s) is not a cytochrome P450
monooxygenase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[1,2-N-3H]5-Dihydrotestosterone (60
Ci/mmol) and
[1,2-N-3H]5-androstane-3,17ß-diol (51.1
Ci/mmol) were purchased from Amersham (Aylesbury, UK).
[1,2-N-3H]5-DHP (57 Ci/mmol),
[4-14C]5-DHP (56 mCi/mmol),
[9,11,12-N-3H]5-pregnan-3-ol-20-one (59.2 Ci/mmol),
and [4-14C]5-dihydrotestosterone (50 mCi/mmol) were
purchased from New England Nuclear (Boston, MA). These radiolabeled
steroids were purified by Celite-propylene glycol, Celite-ethylene
glycol, or t-butanol-based liquid partition chromatography
before use (25). Tritium and
[14C]5-pregnan-3ß-ol-20-ones were prepared by
reduction of radiolabeled 5-pregnane-3,20-dione with sodium
borohydride in ethanol. The 3ß-pregnanolones produced were purified
by Celite-propylene glycol and thin layer chromatography (TLC).
Tritium- and carbon-14-labeled 5-androstane-3ß,17ß-diol were
synthesized similarly by reduction of radiolabeled
5-dihydrotestosterone with sodium borohydride. The reduction
products were purified by TLC on alumina in the system benzene-ethanol
(93:7, vol/vol). [14C]5-Androstane-3,17ß-diol and
[14C]5-pregnan-3-ol-20-one were synthesized by
stereospecific reduction of [14C]5-DHT and
[14C]5-DHP, respectively, with potassium
trisiamylborohydride in tetrahydrofuran. Each product was purified by
TLC. [14C]3ß,6-Dihydroxy-5-pregnan-20-one and
[14C]3,6-dihydroxy-5-pregnan-20-one were
biosynthesized. [14C]5-DHP was added to the medium of
confluent cultures of either MCF-7 cells maintained in Eagle’s MEM
supplemented with FCS (10%), insulin, and pyruvate or to T47-D cells
maintained in RPMI 1640 supplemented with FCS (10%). After an
overnight incubation, radiolabeled steroids were extracted from the
medium with diethyl ether.
3/3ß,6-Dihydroxy-5-pregnan-20-ones were purified by
Celite-ethylene glycol gradient-elution column chromatography (25). The
identity of the
[14C]3/3ß,6-dihydroxy-5-pregnan-20-ones was
verified by comigration on TLC with authentic
3ß,6-dihydroxy-5-pregnan-20-one and
3,6-dihydroxy-5-pregnan-20-one (supplied by the late Prof. D.
N. Kirk, Medical Research Council, London, England, United Kingdom). In
addition, identical mobilities on Celite-ethylene glycol chromatography
and in several TLC systems were found previously for these products and
urinary metabolites shown to be 3ß,6-dihydroxy-5-pregnan-20-one
and 3,6-dihydroxy-5-pregnan-20-one by gas chromatography/mass
spectrometry (19).
Other supplies
Nonradiolabeled steroids were obtained from Steraloids (Wilton, NH). TLC plates were purchased from Analtech (Newark, DE). Nicotinamide adenine dinucleotide (NAD+), NADH, nicotinamide adenine dinucleotide phosphate (NADP+), NADPH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase (from Torula yeast), BSA (fraction V), and chemical reagents and buffer additives were obtained from Sigma Chemical Co. (St. Louis, MO). Potassium trisiamylborohydride (KS-selectride) was purchased from Aldrich Chemical Co. (Milwaukee, WI). Culture media were obtained from Life Technologies (Grand Island, NY).
Cell culture and preparation of subcellular fractions
Monolayer cultures of T47-D cells were grown on plastic petri dishes. The cells were maintained in RPMI 1640 medium with FCS (10%) in a humidified atmosphere of air (95%)-CO2 (5%). Subcellular fractions were prepared from cells at confluence. Cells were initially disrupted by trituration through a 21-gauge needle and then were homogenized by 15–20 passes of a ground glass homogenizer (Duall 22, Kontes, Vineland, NJ). The homogenate volume was adjusted to 5 times the initial cell volume with an additional 0.25 mol/L sucrose, and this crude mixture was centrifuged (600 x g, 10 min). The pellet was rehomogenized by repeating the above steps. The supernatant fractions from the 600 x g centrifugations of the homogenates were combined and centrifuged at 15,000 x g for 20 min. Twice thereafter, the pellet was resuspended in fresh 0.25 mol/L sucrose and centrifuged for 10 min at 5,200 x g to obtain a mitochondrial-enriched fraction. The 5,200 x g supernate was centrifuged at 105,000 x g for 1 h to obtain a cytosol (supernate) and microsome-enriched fraction (pellet). This pellet was homogenized with a Dounce homogenizer and pelleted again at 105,000 x g. The particulate cell fractions were diluted with assay buffer [Tris hydroxyethyl aminomethane (50 mmol/L; pH 7.4), KCl (4.5 mmol/L), CaCl2 (2.5 mmol/L), and MgCl2 (3.0 mmol/L); 4 vol; 4 C] to the desired protein concentration. Protein concentrations were determined by the method of Lowry et al. (26), using BSA as the standard.
Enzyme assay conditions
All assays were conducted at 37 C. Routinely, the cell fraction (1 mL) was incubated with a solution that contained the cofactor [assay buffer (1 mL) that contained either NAD+, NADH, NADPH, or NADP+ (2 mmol/L) or an NADPH-generating system (i.e. 40 mmol/L glucose-6-phosphate, 5 mmol/L NADP+, and 2 U/mL glucose-6-phosphate dehydrogenase)]. The tritium-labeled steroid substrate and inhibitors (if used) were placed in a 16 x 100-mm glass tube, and the solvents therein were evaporated under nitrogen gas. The residue was dissolved in ethanol (10 µL). The cofactor-containing solution was then added, and the sample was warmed to 37 C for 5 min. In a separate container, the cellular fraction also was warmed. The reaction was initiated by the addition of the cellular fraction. After incubation for various times, the reaction was terminated by the addition of methanol (400 µL). Nonradiolabeled and 14C-labeled substrate and product steroids were quickly added followed immediately by the addition of diethyl ether (3 vol). Steroids were extracted from the reaction mixture with diethyl ether (twice, 3 vol).
TLC
The extract residues were dissolved in small volumes of ethanol and applied to TLC plates. Steroid standards were applied to parallel lanes of the chromatograms. After development, the chromatograms were sprayed with primuline reagent, and standards were visualized under UV light. Zones of the chromatograms that contained radiolabeled compounds of interest were scraped from the plates and quantified by assay of an aliquot of the eluate for radioactivity and comparing this 3H/14C ratio to the quantity of carbon-14-labeled internal standard added.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylation
of 5-pregnan-3ß-ol-20-one
To examine the effect of cofactors, NADPH and NADH, in various
concentrations, on the velocity of 6-hydroxylation, a
microsome-enriched fraction prepared from T47-D cells was incubated
with [3H]5-pregnan-3ß-ol-20-one (10 µmol/L). The
concentration of added cofactor was varied from
10-9-10-3 mol/L. The reaction was initiated
by the addition of the microsome-enriched fraction (1 mL) to a solution
of the cofactor and substrate in the reaction buffer. The results of
this study are given in Fig. 1
. There was no difference
in the 6-hydroxylation of 5-pregnan-3ß-ol-20-one attributable
to the cofactor used.
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-reduced
steroid 6-hydroxylase activity with 5-pregnan-3ß-ol-20-one as
substrate in microsome-enriched preparations of T47-D cells
The subcellular localization of the saturated steroid
6-hydroxylase activity in T47-D cells was evaluated using
subcellular fractions isolated by differential centrifugation,
5-pregnan-3ß-ol-20-one (10 µmol/L) as substrate, and NADH (1
mmol/L) as cofactor. Both the specific activity and the total activity
were highest in microsome-enriched fractions prepared from T47-D cells
(Table 1). In all subsequent experiments,
microsome-enriched preparations of T47-D cells were used.
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-reduced steroid 6-hydroxylase activity in
microsome-enriched preparations of T47-D cells
[3H]5-Pregnan-3ß-ol-20-one, in various
concentrations, was incubated with microsome-enriched preparations of
T47-D cells with NADH (1 mmol/L) for 30 min. The apparent
Km and maximum velocity
(Vmax) for 6-hydroxylation of
5-pregnan-3ß-ol-20-one were 1.6 µmol/L and 124 pmol/min·mg
protein, respectively (Fig. 2, A and B). Product
formation with 5-pregnan-3ß-ol-20-one (10 µmol/L) and
microsome-enriched preparations of T47-D cells was linear with time of
incubation for 30 min (Fig. 2C). The velocity of 6-hydroxylation
also was assessed in assays in which the amount of microsome-enriched
protein of T47-D cells was varied, using 5-pregnan-3ß-ol-20-one
(10 µmol/L) as substrate. The reaction was linear with protein
concentrations between 0.05–0.055 mg (Fig. 2D).
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The effect of pH on the velocity of 6-hydroxylation of
5-pregnan-3ß-ol-20-one was evaluated using Tris-maleate buffers
that were prepared at various pH values from 6.0–8.0. The
microsome-enriched pellet was homogenized in a small volume of 0.25
mol/L sucrose, then diluted to a concentration of 0.27 mg/mL in
Tris-maleate buffers with NADH (2 mmol/L) at various pH levels. The
enzyme activity was similar at pH 6.0–8.0 (Fig. 3).
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-Hydroxylation of 5-pregnan-3-ol-20-one by
microsome-enriched fraction of T47-D cells
[3H]5-Pregnan-3-ol-20-one, in various
concentrations, was incubated (in duplicate) for 30 min in a total
reaction volume of 2 mL that contained either NADH (1 mmol/L) or NADPH
(1 mmol/L). The apparent Km of saturated steroid
6-hydoxylase was 3.5 µmol/L, and the Vmax
was 150 pmol/mg protein·min (Fig. 4A).
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-hydroxylation of 5-reduced
C19- and
C21-steroids
This experiment was conducted to evaluate the substrate
specificity of the saturated steroid 6-hydroxylase activity in T47-D
cells. Substrates consisted of
[3H]5-pregnan-3ß-ol-20-one,
[3H]5-androstane-3,17ß-diol, and
[3H]5-androstane-3ß,17ß-diol in various
concentrations. Incubations were conducted for 30 min at 37 C. In the
case of the C21-steroid substrates,
[14C]5-pregnan-3ß-ol-20-one,
[14C]5-pregnan-3ß,6-diol-20-one, and
nonradiolabeled 5-pregnan-3ß-ol-20-one were quickly added to
monitor recovery and facilitate identification of compounds of interest
during extraction and TLC analysis. Nonmetabolized substrate and the
5-pregnan-3ß,6-diol-20-one product were quantified by reference
to the 3H/14C ratio of the purified product and
the amount of carbon-14-labeled compound added. The results are given
in Fig. 5.
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-triol recovery standard was available.
After termination of the assay,
[14C]5-androstane-3/ß,17ß-diol was added. The
amount of substrate remaining was quantified in a fashion identical to
that described. The amount of putative
5-androstane-3/ß,6,17ß-triol formed was quantified
assuming similar recoveries of the substrate and product.
Effects of inhibitors of cytochrome P450 enzymes on the velocity of
6-hydroxylation of 5-pregnan-3ß-ol-20-one
This experiment was conducted to assess the effects of various
potential inhibitors of cytochrome P450 enzymes on the velocity of
6-hydroxylation of 5-pregnan-3ß-ol-20-one. The inhibitors used
were ketoconazole, antipyrine, ellipticine, propanone, and
napthoflavone. These agents are nonspecific inhibitors of cytochrome
P450 enzymes.
A microsome-enriched fraction of a homogenate of T47-D cells was
prepared as described. Tritium-labeled 5-pregnan-3ß-ol-20-one (in
various concentrations or at 10 µmol/L) was incubated at 37 C with
the microsome-enriched cell fraction for 30 min in a final volume of 2
mL. An NADPH-generating system was used. Enzyme inhibitors were present
in a final concentration of 1 µmol/L. The findings of this study are
given in Fig. 6.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydroxy-5-pregnane-20-one in selected extrahepatic
cells, and the reaction sequence involved is as follows:
5-reductase3-ketosteroid-3ß-reductase6-hydroxylase. In
addition, 5-pregnan-3-ol-20-one is an equally efficient substrate
for this saturated steroid 6-hydroxylase.
The apparent Km of the saturated steroid
6-hydroxylase(s) for 5-pregnan-3ß-ol-20-one or
5-pregnan-3-ol-20-one was 3.5 µmol/L, and the
Vmax was approximately 150 pmol/mg protein·min
in microsome-enriched preparations of T47-D cells. NADH and NADPH were
equally effective as cofactors for this enzymatic reaction. This latter
finding is consistent with those of Gemzik and associates (24), who
presented evidence that the saturated steroid 6-hydroxylase enzyme
of human prostate tissue is not a cytochrome P450 (24). They found that
neither carbon monoxide nor antibody against NADPH-cytochrome P450
reductase inhibited the 6-hydroxylation of
5-androstane-3ß,17ß-diol in human prostate microsomes. They also
demonstrated that the reaction was not inhibited by a variety of
imidazole-type antimycotic agents, viz. ketoconazole,
clortimazole, and miconazole (10 µmol/L). In this study, there was no
reduction in 6-hydroxylase activity of microsome-enriched
preparations of T47-D cells treated with yet other inhibitors of
cytochrome P450s.
A saturated steroid-hydroxylating enzyme also has been characterized in
rat tissues, viz. prostate, seminal vesicles, brain,
hypothalamus, and anterior pituitary. This enzyme, however, is
distinctly different from the C-6 and C-7/ß-hydroxylases of
human prostate. The saturated steroid-hydroxylating enzyme of rat
tissues, which catalyzes the C-6/ß and C-7/ß hydroxylation of
5-androstane-3ß,17ß-diol, is a single cytochrome P-450 that
gives rise to C-6/7-hydroxylations in a fixed ratio of products (27),
namely 6/7/7ß = 7:3:1 (C-6ß hydroxylation is a minor
reaction). In human prostate, however, 6-hydroxylation is not the
quantitatively more important pathway of C19-steroid
metabolism; rather, the C-7/ß-hydroxylation of
5-androstane-3ß,17ß-diol is dominant (24). The rat enzyme also
catalyzes the 7-hydroxylation of 5-pregnane-3ß-ol-20-one, but
not 5-pregnan-3-ol-20-one (28).
Gemzik and associates demonstrated convincingly that the
6-hydroxylation of 5-androstane-3ß,17ß-diol in human prostate
is catalyzed by an enzyme distinctly different from that which
catalyzes the C-7/ß-hydroxylation of this substrate. The
C-7-hydroxylating enzyme is a cytochrome P450-monooxygenase, whereas
the saturated steroid 6-hydroxylase of prostate and other
extrahepatic tissues seemingly is not. It appears that the
C-7/ß-hydroxylation of the saturated steroid,
5-androstane-3ß,17ß-diol, is the preferred pathway for
inactivation of bioactive C19-steroids in the human
prostate and other tissue sites of androgen action, whereas
C-6-hydroxylation is the preferred pathway of inactivation of
5-reduced C21-steroids such as
5-pregnan-3-ol-20-one and 5-pregnan-3ß-ol-20-one in tissue
sites of progesterone metabolite action. In this study, we found no
evidence for 7-hydroxylation of 5-pregnan-3/3ß-ol-20-one by
chromatographic or gas chromatographic-mass spectrometric criteria.
The human saturated steroid 6-hydroxylase enzyme is widely
distributed in extrahepatic tissues, but with highly variable levels of
enzyme activity among cells. In addition to teratocarcinoma cell lines
of ovarian and testicular origin (20, 21), T47-D and MCF-7 breast
carcinoma cells (22, 23), and normal breast epithelial and mesenchymal
cells (20), we found (unpublished studies) saturated steroid
6-hydroxylase enzyme activity in human skin fibroblasts, peripheral
blood monocytes, endometrial stromal and epithelial cells, myometrial
smooth muscle cells, and leukemic cell lines (HL-60, THP-1, and
U-937).
In young women during the luteal phase of the ovarian cycle and
throughout pregnancy, progesterone is secreted into blood in very large
quantities. The levels of plasma progesterone may reach
10-7 mol/L during the midluteal phase, and during
pregnancy near term, the plasma levels of progesterone are about 5
x 10-7 mol/L (2). The levels of progesterone attained
during human pregnancy are much greater than those in most other
mammalian species (by 5- to 10-fold), including subhuman primates (29),
and the levels of 5-DHP in plasma also are very high, about 10%
those of progesterone after ovulation and during early pregnancy
(4–6 ng/mL), but increasing to about 40% near term (30–60
ng/mL) (30, 31, 32). Because of the very high metabolic clearance rate of
5-DHP (3600 L/24 h) (33), the daily production rate of plasma
5-DHP may exceed 100 mg during the third trimester of pregnancy.
This rate of 5-DHP production is the greatest known for any steroid
other than progesterone. The very high rate of 5-DHP formation is
partially accounted for as follows: 1) approximately 50% of the
irreversible clearance of plasma progesterone is attributable to
metabolism in tissue sites other than the liver (2), and about 80% of
the total extrahepatic clearance proceeds by way of initial
5-reduction of progesterone to give 5-DHP (2, 33); 2)
5-reductase enzyme activity is widely distributed in extrahepatic
tissues, and progesterone is rapidly taken up by cells because
progesterone is highly lipophilic and is not bound with high affinity
to plasma proteins; and 3) the apparent Km of
5-reductase for progesterone is low, appreciably lower, for example,
than the Km for testosterone. While half of the
plasma progesterone metabolism is attributable to extrahepatic
clearance, even more (two thirds) of plasma 5-DHP is metabolized
outside of the liver (33). Therefore, the extrahepatic metabolism of
progesterone appears to be accounted for by three primary pathways: 1)
extraadrenal 21-hydroxylase (1%), 2) 20-hydroxysteroid
dehydrogenase(s) (20%), and 3) the 5-reductase pathway
(80%), which includes 3-hydroxysteroid dehydrogenase(s),
3-ketosteroid-3ß-reductase (and possibly other isoforms of the
3ß-hydroxysteroid dehydrogenase family of enzymes), and saturated
steroid 6-hydroxylase(s). Several of these enzyme reactions are
demonstrable in many extrahepatic cells. Thus, the 6-hydroxylation
of saturated C21- and C19-steroids in
extrahepatic tissues appears to be a mechanism for the inactivation of
bioactive metabolites of progesterone and testosterone in several
species, including humans.
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Acknowledgments |
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Footnotes |
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Received November 7, 1996.
Revised January 22, 1997.
Accepted February 3, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-pregnane-3,20-dione and progesterone in peripheral blood of
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-pregnane-3,20-dione
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