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Production of 19-hydroxy-11-deoxycorticosterone and 19-oxo-11-deoxycorticosterone from 11-deoxycorticosterone by cytochrome P-45011β
J: steroid Biochem. Vol. 26, No. 1, pp. 73-81,
Printed in Great Britain. Ali rights reserved
1987
0022-473t/87 $3.00 + 0.00
Copyright Q 1987Pergamon Journals Ltd
PRODUCTION OF 19-HYDROXY- llDEOXYCORTICOSTERONE AND 19-0X0-1 l-DEOXYCORTICOSTERONE FROM 1I-DEOXYCORTICOSTERONE BY CYTOCHROME P-450, 18 MIHO OHTA, SHIGERU FUJII, AKIRA WADA, TAIRA OHNISHI,TOSHIOYAMANO and MIT%JHIROOKAMOTO* Department of Biochemistry, Osaka University Medical School, 4-3-57 Nakanoshima, Osaka 530, Japan
Kita-ku,
(Received 25 June 1986)
Sag-Incubation of 1I-deoxyco~icosterone with a cytochrome P~5O,,~-r~onstitu~ system yielded, in addition to corticosterone and 18-hydroxy-I l~eoxycorti~osterone, a new steroid product. The retention time of the new product was identical with that of authentic 19-hydroxy-I 1-deoxycorticosterone on high performance liquid chromatography (HPLC). The turnover number of 19-hydroxy-1 l-deoxycorticosterone formation was 7.0 mol/min/mol P-450. When a large amount of cytochrome P-450,,! was used for the reaction and the products were analyzed by HPLC, the 19-hydroxy-1 I-deoxycorticosterone peak disappeared from the chromatogram and concomitantly new unidentified peaks appeared. These results suggest that 19-hydroxy-lI-deoxycorticosterone was further metabolized to other steroids by cytochrome P-450,,# Therefore, we next incubated 19-hydroxy-I ldeoxycorticosterone with cytochrome P-450,,, and analyzed the reaction products by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram. The retention time of one of the peaks coincided with that of authentic 19-0x0-1 I-deoxycorticosterone. This peak substance was purified by repeated HPLC and subjected to mass spectrometry and ‘H NMR analyses. Its field desorption mass spectrum (FlXMS) showed a M+ peak at m/e 344. The ‘H NMR spectrum showed the signal of an aldehyde proton instead of those of hydroxymethyl protons at the C-19 position. These results suggest that cytochrome P-450,,, can catalyze the t9-hydroxylation of 1I-deoxycorticosterone, and the 19-hydroxy-II-deoxycorticosterone produced is further oxidized at the C-19 position to 19-0x0-1 l-deoxycorticosterone.
INTRODUCTION It is known that removal of the 19-methyl group on a steroid nucleus affects both the biological and chemical properties of the corresponding methylated parent compound [l-3]. 19-Nor-l l-deoxycorticosterone is up to five times more potent than 1I-deoxycorticosterone in the sodium-retaining bioassay [ 1,4], and at molecular level has a higher affinity for the renal mineraloco~i~id receptor than aldosterone 151. This steroid has been shown to be markedly hypertensinogenic in the rat [6]. 19-Nor1I-deoxycorticosterone is not a product of adrenal steroid metabolism but appears to be formed extraadrenally from a precursor of adrenal gland origin [7]. Gomez-Sanchez et aL[7] suggested that 19-0x0-1 l-de19-hydroxy-1 I-deoxycorticosterone,
oxycorticosterone are the adrenal costerone. But processes which be elucidated. Cytochrome
and 19-oic- 11-deoxycorticosterone precursors of 1g-nor- 11-deoxycortithe enzymatic characteristics of the produce these metabolites remain to
mitoP-450] Is of adrenocortical chondria has been shown to catalyze both the llgand 18-hydroxylation of 11-deoxycorticosterone, the 1lo-hydroxylation of 18-hydroxy-1 l-deoxycorticosterone, the 18-hydroxylation of corticosterone, and the 1l/l- and 19-hydroxylation of 4-androstene-3,17dione [8-131. Recent investigations in our laboratory demonstrated that the formation of aldosterone from corticosterone is catalyzed by a cytochrome P-450,,,reconstituted system fortified with phospholipid [14-161, and that the 19-hydroxylation of 18-hydroxy-1 1-deoxycorticosterone is also catalyzed by cytochrome P-450,,, [17-191. Since cytochrome P*To whom correspondence should be addressed. 450,,, shows such versatile catalytic activity toward The trivial names used are: 19-hydroxy-I l-deoxycorticosterone, 19,21-dihydroxy-4-pregnene-3,20-dione; 19- steroid substrates, it is possible that the cytochrome can also catalyze the hydroxylation of 1I-deoxy0x0-1 I-deoxycorticosterone, 21-hydroxy-3,20-dioxo-4pregnene- 19-al; 19-061 ldeoxycorticosterone, 19- corticosterone at the C-19 position. Therefore, we 19-nor-l I-de- started to examine this possibility by using a reoic-2i-hydroxy-4-pre~ene-3,2O-dione; oxycorticosterone, 21-hydroxy-19-nor-4-pregneneconstituted system including cytochrome P-450,,, 18,19-dihydroxy-1 I-deoxycorticosterone, 3,20-dione; purified from bovine adrenal cortex. l&19,21-trihydroxy-4-pregnene-3,20-dione; l&hydroxyDuring this study, we found unknown metabolites 18,21-dihydroxy-4-pregnene11-deoxycorticosterone, 3,20-dione. of 11-deoxycorticosterone in the reaction mixture.
s,B.2611-F
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These metabolites were identified as 19-hydroxy-1 ldeoxycorticosterone and 19-0x0-1 l-deoxycorticosterone on the basis of the results of FD-MS analysis as well as those of NMR-spectroscopy. The results indicate that cytochrome P-450,,, catalyzes the hydroxylation of 1I-deoxycorticosterone at the C-19 position to 19-hydroxy-1 I-deoxycorticosterone, which is further oxidized to 19-0x0-1 l-deoxycorticosterone. EXPERIMENTAL
Chemicals
Most of the steroids used were purchased from Sigma (U.S.A.). 19-Hydroxy-1 I-deoxycorticosterone and 19-0x0-1 l-deoxycorticosterone were generous gifts from Dr T. Terasawa of the Shionogi Research Laboratory. NADPH, gluco~-6-phosphate and glucose-&phosphate dehydrogenase were obtained from Oriental Yeast Co. (Japan). Other chemicals were of the highest grade available from commercial sources. All solvents were of HPLC grade. Preparation of enzymes
Cytochrome P-450,,, was purified from bovine adrenocortical mitochondria according to the method described by Suhara et al.[ll] with the modifications of Wada et al.[ 161. The specific cytochrome P-450,,, content of the purified sample was 16 nmollmg protein. Aldrenodoxin and adrenodoxin reductase were purified from bovine adrenal mitochondria, by Dr Y. Nonaka of our laboratory, according to the methods described by Suhara et a/.[201 and Sugiyama et a/.(21], respectively.
For HPLC, a Gilson model 320 system equipped with a U.V.monitor was employed. For normal phase HPLC, a 4 x 250 mm silica column (M&S packs, 5 p) and a mobile phase of dichloromethane-ethanolwater = 98.1: 1.71:0.19 (by vol), at a flow rate of 1.2 mlimin, were used. For reverse phase HPLC, a 4.5 x 250 mm column (M&S packs, 5 p, ODS) was employed and the solvent system used comprised methanol-water = 60:40 (v/v), at a flow rate of 0.5 ml/min. For FD-MS, a JMS D-300 Mass Spectrometer equipped with an FD/FI/EI ion source was used. The ‘H NMR spectra were obtained at 200 MHz with a Varian XL-200 spectrometer. Chemical shifts (6) in CDCl, solution were measured in ppm with the residual CHCl, of the solvent system as the internal standard (S = 7.27). Enzyme assay and analysis
of products
For the usual enzyme assay, a steroid (100 PM) was incubated with the reconstituted cytochrome P-450,,B-system (a final volume of OSml) in the presence of a NADPH-regenerating system under aerobic conditions for 4 min according to the method described by Momoi et al.[18]. The products were
extracted with dichloromethane, and then the solvent was evaporated under nitrogen. The dried extract was analyzed by HPLC. The amounts of products were determined by monitoring the absorbance at 254 nm with cortisol as an internal standard. Preparation of an unidentified product [Product (ZI)]
19-Hydroxy-1 I-deoxycorticosterone (4 pmol) was incubated with cytochrome P-450,,, (2nmol) for 15min at 37°C in 20 ml of 10 mM Tris-HCl (pH 7.4) containing NADPH (100 pmol), glucose-6-phosphate (200 pmol), glucose-6-phosphate dehydrogenase (20 units), MgCl, (60pmol), adrenodoxin (600 nmol) and adrenodoxin reductase (20 nmol). The reaction was terminated by the addition of 10 ml ethanol. The reaction mixture was extracted three times with 100 ml of dichloromethane and then the solvent was evaporated under nitrogen. The residue was applied to the reverse phase HPLC system mentioned above and the fraction showing a retention time identical to that of 19-oxo-ll-deoxycorticosterone was collected and further applied to the same column repeatedly until the isolated product showed a single peak on chromatography. Analytical methods
Protein concentrations were determined according to the method of Lowry ef af.[22] with bovine serum albumin as the standard. The cytochrome P-450,,, content was estimated as described by Omura and Sato[23] using an extinction coefficient of 91 mM_’ cm-’ for the absorbance difference, at 450 nm and 490 nm, of the CO-binding reduced enzyme. The concentrations of adrenodoxin and adrenodoxin reductase were determined using t 414 = 10 mM-i.cm-’ and cdM= I1 mM-“cm-‘, respectively. RESULTS
HPLC-pro$ies of authentic steroids and identification of a new metabolite [Product (J)] as J9-hydroxy- JJdeoxy~orti~osterone
Figure 1 presents chromatograms of authentic steroids on normal phase HPLC and on reverse phase HPLC, respectively. These chromatographic systems well separated 19-hydroxy- 11-deoxycorticosterone from both corticosterone and 1X-hydroxy-I 1-deoxycorticosterone. The three steroids were eluted at 18, 12 and 16 min on normal phase HPLC, and at 22,44 and 38min on reverse phase HPLC, respectively. Using these HPLC systems, we examined the products of the reaction of 1I-deoxycorticosterone with a cytochrome P-450iIg-reconstituted system (Fig. 2). Three product peaks appeared in the chromatograms, two of which were identified as corticosterone (the product of 1lfi-hydroxylation) and 18-hydroxy-1 ldeoxycorticosterone (the product of 18-hydroxylation) on the basis of their retention times. The other steroid product (Product [I]) showed a retention time
19-Hydroxy-HOC and 1P0x0-DOC biosynthesis by P-45O,,B
(A) 0.04-
75
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10
0
20
0
3040
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2030
tmin)
3.00
_,..LL..L
40
50607080
(min)
t Fig. I. Analysis of authentic steroid mixtures by (A) normal phase HPLC and (B) reverse phase HPLC. The compounds tested were: (1) 1I-deoxycorticosterone, (2) corticosterone, (3) 18-hydroxy- 1l-deoxycorticosterone, (4) 19-hydroxy-1 I-deoxycorticosterone, (5) cortisol and (6) aldosterone. The HPLC conditions were as follows: (A) Column: M&S packs, silica 5 pm, 4 x 250 mm. Solvent system: CH,Cl,-EtOH-H,O (98.1:1.71 :O.19) (by vol.). Flow rate: 1.2 mi/min. (B) Column: M&S packs, ODS, 5 pm, 4.5 x 250 mm. FIow rate: OJml/min. Solvent system: MeOH:H,O=60:40 (v/v). The detailed conditions for these experiments are given under Experimental. Each peak number corresponds to the position of the corresponding authentic steroid.
of 18 min on normal phase HPLC and one of 22 min on reverse phase HPLC. These retention times are identical to those of authentic 19-hydroxy-I l-deoxycorticosterone. Moreover, the ‘H NMR spectrum of Product (I) was essentially identical to that of authentic 19-hydroxy- 1I-deoxycorticosterone (data not shown). To confirm that 19-hydroxy-1 I-deoxycortico-
sterone is a real product generated from ll-deoxycorticosterone by cytochrome P-450,,,, we performed the following experiments. The time-course of the formation of IPhydroxy- 11-deoxycorticosterone (Product [Ij) was examined. The formation of 19hydroxy- 11-deoxycorticosterone proceeded almost linearly with time up to 4min under the standard assay conditions (Fig. 3A). The turnover number of
T
(B)
(A) 1
2
0.04
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i=i
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10
20
30
(mini
40
I,~~
50
0
20
d
II-*
40
60
80
i J,.,,
fmin)
Fig. 2. HPLC profiles of reaction products of 1I-deoxycorticosterone and cytochrome P-450,,8. (A) Normal phase HPLC and (B) reverse phase HPLC. The peak numbers correspond to the positions of the authentic steroids on HPLC, (1) 1I-Deoxycorticosterone, (2) corticosterone, (3) IS-hydroxy-1 l-deoxy~rti~sterone, (4) 19-hydroxy-1 l-d~xycorti~s~rone, and (5) cortisol (the internal standard). The chromatogtaphic conditions were as in Figs l(A) and (B). 11-Deoxycorticosterone (1OOpM) was incubated with cytochrome P-450,,, (39 pmol) in the presence of adrenodoxin, adrenodoxin reductase and a NADPH-generating system (a final volume of 0.5 ml). The extract of the reaction mixture was subjected to (A) normal phase HPLC and (B) reverse phase HPLC, respectively, and the eluate was monitored as to the absorbance at 254 nm, The detailed conditions for this experiment are given under Experimental.
MIHO OHTA et
76
al.
a- (El)
-1.6
7-
-1.4
-c
-1.2
T ‘E \
-0.8
“0
0 -
(A)
E
1 0.4
L 0’
10.2
L 1
2
Incubation
3
4
5
time (min)
0
I 20
40
P-450,,~
60
80
100
2 a
0.0
(pmol)
Fig. 3. (A) Time-course of the formation of 19-hydroxy-1 I-deoxycorticosterone (Product [I]). 1I-Deoxycorticosterone (100 NM) was incubated with the reconstituted cytochrome P-450,,, (44 pmol)-system in a final volume of 0.5 ml at 37°C for the indicated periods. For the detailed conditions and the analytical method, see under Experimental. (B) Effect of the concentration of cytochrome P-450,,, on the formation of 19-hydroxy-1 I-deoxycorticosterone (Product [I]). 1I-Deoxycorticosterone (100 FM) was incubated with the indicated amounts of cytochrome P-450,,8-system in a final volume of 0.5ml. The products were analyzed as described under Experimental. Closed and open circles show the rate of production of 19-hydroxy-1 I-deoxycorticosterone (Product [I]) and that of corticosterone, respectively. The value obtained on incubation without cytochrome P-450,,, is due to a noise on HPLC.
19-hydroxy- 1 1-deoxycorticosterone formation under these conditions was determined to be 7.0 mol/ min/mol P-450. The dose dependency of the formation of 19-hydroxy- 11-deoxycorticosterone (Product [I]) as to the cytochrome P-450,,, concentrations was studied. As shown in Fig. 3(B), a linear relationship was observed between the 19-hydroxy1I-deoxycorticosterone formation and the amount of cytochrome P-450,,, in a range below 100 PM. After heat-treatment, cytochrome P-450,,, (treated for 5 min at 100°C) did not produce any corticosterone, 1%hydroxy-1 l-deoxycorticosterone or 19-hydroxyI I-deoxycorticosterone (data not shown). Metabolism of 19-hydroxy- 11 -deoxycorticosterone through the cytochrome P-450,,p-catalyzed reaction
When a large amount of cytochrome P-450,,, was used for the reaction and the reaction products were analyzed by HPLC, the peak of 19-hydroxy-lldeoxycorticosterone (Product [I]) unexpectedly decreased in the chromatogram and new unidentified peaks appeared instead (one of the new peaks is denoted as Product (II) in Fig. 4). Figure 5 shows the amounts of corticosterone, 1%hydroxy-1 l-deoxycorticosterone and 19-hydroxy- 11-deoxycorticosterone (Product [I]) produced on the incubation of 11-deoxycorticosterone with a high concentration of cytochrome P-450, iD. The addition of more than 200 pmol of cytochrome P-450,,, to the incubation medium resulted in a decrease in the production of 19-hydroxy- 11-deoxycorticosterone, while the amounts of corticosterone and 1%hydroxy- ll-
deoxycorticosterone produced reached plateaus and never decreased in the cytochrome P-450,,, concentration range investigated. We considered that the substrate was exhausted within 4min in the incubation mixture with more than 200 pmol of cytochrome P-450,,,. These results suggest that the new unidentified peak (Product [II] in Fig. 4) was probably derived from 19-hydroxy-1 l-deoxycorticosterone. Figure 6 shows the time courses of the production of corti19costerone, 1%hydroxy-1 1-deoxycorticosterone, hydroxy-1 I-deoxycorticosterone (Product [I]) and the new unidentified product (Product [II]) in the presence of 318 pmol of cytochrome P-450,,,. Under these conditions, the amounts of corticosterone and 18-hydroxy-1 I-deoxycorticosterone accumulated in the reaction mixture reached plateaus after 2 min because of exhaustion of the substrate, and thereafter they did not significantly decrease up to 15 min. On the other hand, 19-hydroxy-1 I-deoxycorticosterone (Product [I]) linearly accumulated in the reaction mixture up to 2min, and then decreased. In accord with the beginning of the decrease in 1Phydroxy-1 ldeoxycorticosterone, Product (II) started to appear in the reaction mixture and increased progressively. Stoichiometry between the decrease in 19-hydroxy1 1-deoxycorticosterone and the increase in Product (II) could not be established, because Product (II) was one of several unidentified peaks seen in the chromatogram (see Fig. 4B). These results suggest that the 19-hydroxy-1 I-deoxycorticosterone produced was further metabolized to other steroids by
19-Hydroxy-DOC and IP0x0-DOC biosynthesis by P-450,,,
77 7
(AlI
(A)(2) 1
l
2
Iminl
I
_ (mini
-!
Fig. 4. Conversion of IQ-hydroxy-1I-deoxycorticosterone (Product [I] to new metabolites catalyzed by the reconstituted cytochrome P-450,,8 system. 11-Deoxycorticosterone (100 p M) was incubated with (A) a small amount (106pmol) and (B) a large amount (530pmol) of the reconstituted cytochrome P-450,,B-systemin a final volume of 0.5 ml at 37°C for 4 min. The products were analyzed as described under Ex~rimental. (1) Normal phase HPLC. (2) Reverse phase HPLC. Each peak number corresponds to the position of the corresponding authentic steroid. (1) 1I-Deoxycorticosterone, (2) corticosterone, (3) 18-hydroxy-1 I-deoxycorticosterone, (4) IQ-hydroxy-1 I-deoxycorticosterone, and (5) cortisol (the internal standard).
the cytochrome P-450,,,. Therefore we performed the following experiments. 19-Hydroxy- 1I-deoxycorticosterone was incubated as a substrate with the cytochrome F-45O,~~-reconstituted system and the products were analyzed by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram (data not shown). One of the peaks (Product [II]) showed a retention time of 4min on normal phase HPLC and one of 30min on reverse phase HPLC, respectively. These retention times coincide with those of authentic lPoxo-1 l-deoxycorticosterone. As the amount of
P- 450,,B I pm011 Fig. 5. Effect of the cytoehrome P-450,,, concentration on 19-hydroxy- i 1~eoxyco~icosterone formation. I I-Deoxycorticosterone (100 PM) was incubated with various amounts of the reconstituted cytochrome P-450,,8-system in a final volume of 0.5ml at 37°C for 4min. The amounts of 19-hydroxy-ll-deoxycorticosterone (Product [I]) (a), corticosterone (0) and It-hydroxy-I l~eoxycorticosterone [IS-(OH)DOC] (A) accumulated in the media were determined by HPLC.
cytochrome P-450,,, increased, the production of Product (II) increased (Fig. 7). Heat-inactivated cytochrome P-450,,, (treated for 5 min at 100°C) did not produce any new metabolites from 19-hydroxy-lldeoxycorticosterone (data not shown). Identification of Product (II) as 19-0~0 - 11 -deoxy corticosterone
To further confirm the chemical identity of Product (II), large scale of purification of it was carried out as described under Experimental. The purified sample was subjected to mass analysis involving a field desorption technique. Figure 8 shows the FD-MS spectra of authentic 19-0x0-1 I-deoxycorticosterone and Product (II). Molecular ion peaks of Product (II) as well as of authentic 19-0x0-1 l-deoxyco~icosterone were detected at mie 344. To obtain better insight into the chemical structure of Product (II), ‘H NMR spectra of authentic 19-0x0-1 I-deoxycorticosterone and Product (II) were obtained. Figure 9(A) shows the ‘H NMR spectrum of authentic 19-0x0- 11-deoxycorticosterone in CDCI,. The signal of an aldehyde proton appeared at S = 9.93 ppm. According to the results of Genard et aE.[23] and Fujii et aI.[lS], the resonance peaks around 6 = 3.23 and at zi = 4.19 and 5.99 ppm were assigned as due to 21-OH-, 21-CH,- and 4H-proton, respectively. The resonance peak that appeared at around 6 = 1.4 ppm (indicated by an asterisk) was considered to be due to a contaminant in the solvent, because further purification of this material by HPLC caused a decrease in this signal. Figure 9(B) shows the spectrum of Product (II), which is essentially identical to that of authentic 19-0x0-1 I-deoxycorticosterone. The combined characteristics of the HPLC elution profiles, the FD-MS
MIHO OHTA et al.
78
o
z
6 8 10 12 INCUBATION TIME I mini
1L
16
Fig. 6. Metabolism of 1I-deoxycorticosterone by the reconstituted cytochrome P-450,,8-system.1I-Deoxycorticosterone (100 PM) was incubated with the reconstituted cytochrome P-450,,, (318pmol)-system in a final volume of 0.5 ml at 37°C for the indicated periods. The products were analyzed as described under Experimental. The amounts of 19-hydroxy-1l-deoxycorticosterone (Product [r]) (a), corticosterone (0) 18-hydroxy-11-deoxycorticosterone (18(0H)DOC) (A) and Product (II) (0) were estimated from the absorbance at 254nm.
spectrum and the ‘H NMR spectrum provided strong evidence of the production of 19-oxo-ll-deoxycorticosterone by cytochrome P-450, i8. These results together indicate that cytochrome P-45O1,8 can catalyze the 19-hydroxylation of 1l-de19IOH)DOC~
$ Producf
(III
P_45O,,rJ
(1) 148
I
LI
0
pm01
10
20
30
40
50
60
(min) Fig. 7. HPLC profiles of reaction products of 19hydroxy-1 I-deoxycorticosterone with the reconstituted cytochrome P-450,,B-system. 19-Hydroxy-1 l-deoxycorticosterone (100 PM) was incubated with the indicated amounts of the reconstituted cytochrome P-450,,8-system at 37°C for 4min. The products were analyzed by reverse phase HPLC. The detailed conditions for this experiment are given under Experimental. The amounts of cytochrome P-450,,8 used were (1) 148 pmol, (2) 44 pmol and (3) 15 pmol in 0.5 ml of the reaction mixture. The striped peak corresponds to Product (II), and its retention time is identical with that of authentic 19-0x0-1 I-deoxycorticosterone. 19(0H)DOC, 19-hydroxyl-deoxycorticosterone. F, cortisol (the internal standard).
oxycorticosterone and the 19-hydroxy- 1l-deoxycorticosterone thus produced is further metabolized to 19-0x0- 11-deoxycorticosterone. DISCUSSION
In this study, we demonstrated that 1l-deoxycorticosterone can be hydroxylated at the C-19 position as well as the 11/I- and l&positions by cytochrome P-450,,,. By means of FD-MS and ‘H NMR spectrometric analyses, we confirmed that the structures of the products are 19-hydroxy- 1l-deoxycorticosterone and 19-0x0- 1 1-deoxycorticosterone, and found that the rate of the formation of 19hydroxy-1 I-deoxycorticosterone is comparable to that of 18-hydroxy-11-deoxycorticosterone. To our knowledge, this is the first report of the production of 1Phydroxy-1 1-deoxycorticosterone and 19-0x01I-deoxycorticosterone by a cytochrome P-450,,, system, although Sato et a1.[9] reported that this cytochrome can catalyze the 19-hydroxylation of androstenedione, and more recently Momoi et a/.[ 181 reported that this cytochrome can also catalyze the 19-hydroxylation of 18-hydroxy-1 l-deoxycorticosterone. 19-Hydroxy-1 I-deoxycorticosterone was first isolated from bovine and hog adrenal glands [25-271. Recently, 19-hydroxy-1 I-deoxycorticosterone was found in incubates of regenerating adrenal capsules 3-4 days after enucleation [28], and 19-nor-l l-deoxycorticosterone was identified in the urine of rats with regenerating adrenal glands [29]. That enucleation in the adrenal glands of rats causes adrenal regeneration hypertension has been well demonstrated [30,3 11, and the possibility that a mineralocorticoid other than aldosterone, perhaps 19-nor-l l-deoxycorti-
I9-Hydroxy-DOC
and 19-0x0-DOC biosynthesis by P-450,,,
19
CH,OH
I c=o
19-KHO)-DOC 1
H\c//o
hl+ 344
/
0
M+l
E
& ’
M+
1, x4.0
344
Product
(II
I
I---
0 300
350
400
450
500
m/e Fig. 8. The field desorption mass spectra of authentic 19-0x0-1 I-deoxycorticosterone and the Product (II). Product (II) was purified and analyzed by FD-MS as described under Experimental. 19(CHO)DOC, 19-0x0-1 I-deoxycorticosterone.
(A) CDCL,
(B) CDCI,
ISCHOPOC
PRODUCTUI)
Fig. 9. ‘H NMR spectra of authentic 19-0x0-1 I-deoxycorticosterone and Product (II). (A) 19-0x0-1 Ideoxycorticosterone [19(CHO)DOC] in CDCl,; (B) Product (II) in CDCl,. The peak indicated by an asterisk is due to the solvent.
80
MIHO OHTA et al.
costerone, may be important in the pathogenesis of adrenal regeneration hypertension was suggested [7,29]. 1g-Nor- 11-deoxycorticosterone has been shown to be a potent mineralocorticoid in the rat bioassay for sodium retention [l] and in sodium transport across the toad bladder [32], whereas 19-hydroxy-11-deoxycorticosterone does not exhibit significant mineralocorticoid activity. From these results it was suggested that 19-nor-l l-deoxycorticosterone may play a significant role in the pathogenesis of sodium retention following adrenal enucleation. Although the biosynthetic pathway for 19-nor- 11-deoxycorticosterone remains unknown, several intermediate products of 1l-deoxycorticosterone oxygenated at the C-19 position were found in an adrenal homogenate by Gomez-Sanchez et a1.[7]. They reported the formation of 19hydroxy-1 l-deoxycorticosterone, 19-0x0-1 l-deoxycorticosterone and 19-oic- 11-deoxycorticosterone from 1 1-deoxycorticosterone in adrenal glands of rats undergoing adrenal regeneration and of intact rats [7]. On the other hand, as 19-nor-ll-deoxycorticosterone itself was not found in the adrenal homogenate, it was proposed that 19-nor-l l-deoxycorticosterone may be generated at the target site of its mineralocorticoid action from one of these putative precursors [31]. Therefore, the existence of 19-hydroxy- 11-deoxycorticosterone and 19-0x0- 1ldeoxycorticosterone might reflect their biological significance as precursors of 1g-nor-1 1-deoxycorticosterone. P-450, 18 was reported Recently, cytochrome to catalyze the formation of aldosterone from corticosterone or 18-hydroxycorticosterone [14-161. Aldosterone is well known as the most potent physiologically-occurring mineralocorticoid. The recent description of the formation of 18,19dihydroxy- 11-deoxycorticosterone by a cytochrome P-450,,8-reconstituted system suggests the possible existence of 1g-nor- 18-hydroxy-1 l-deoxycorticosterone [17]. Okamoto et a/.[331 reported that synthetic 19-nor-18-hydroxy-l l-deoxycorticosterone showed an affinity to the aldosterone receptor of the rat renal cytosol similar to that of 18-hydroxy-1 ldeoxycorticosterone, and that its sodium retaining activity was also similar to that of 18-hydroxy-1 ldeoxycorticosterone. These reports [14-191 and our present results suggest that cytochrome P-450,,, is deeply involved in the biosynthesis of mineralocorticoids in adrenal glands. Further studies are necessary to find further metabolites of 1l-deoxycorticosterone in the cytochrome P-450,,B-catalyzed reaction and to determine if 19-hydroxy-1 l-deoxycorticosterone and 19-0x0-1 I-deoxycorticosterone are precursors of 19-nor-l l-deoxycorticosterone. Acknowledgements-A part of this study was supported by research grants from the Ministry of Education and Culture of Japan, and the Science and Technology Agency of I
REFERENCES 1. Kagawa C. M. and Van Arman G. G.: Sodium re-
taining activity of 19-nor-steroids in adrenalectomized rats. Proc. Sot. exp. Biol. Med. 94 (1957) 444-447. 2. Wynne K. N., Mercer J., Stockigt J. R. and Funder J. W.: 19-nor analogs of adrenal steroids: mineralocorticoid and glucocorticoid receptor activity. Endocrinology 107 (1980) 1278-1280. G., A., Ringold H. J., Rosenkranz 3. Zaffar& Sondheimer F.. Thomas G. H. and Djerassi C.: Steroids. Synthesis of 19-nor-A4-pregneie- 11,17,21triol-3,20-dione (19-norhydrocortisone) and related 19-nor-adrenal hormones. J. Am. them. Sec. 80 (1958) 611&6114. 4. Axelrad B. J., Cates J. E., Johnson B. B. and Luetscher J. A. Jr: Bioassay of mineralocorticoids: Relationship of structure to physiological activity. Endocrinology 55 (1954) 568-574. 5. Funder J. W., Mercer J., Ingram
B., Feldman D., Wynne K. and Adam W. R.: 1PNor deoxycorticosterone (19-nor DOC): Mineralocorticoid receptor affinity higher than aldosterone, electrolyte activity lower. Endocrinology 103 (1978) 151k-1517. 6. Hall C. E., Gornez-Sanchez C. E., Holland 0. B. and Nasseth D.: Influence of 19-nor-deoxycorticosterone on blood pressure, saline consumption, and serum electrolytes, corticosterone, and renin activity. Endocrinology 105 (1979) 6OCMO4. I. Gomez-Sanchez C. E., Gomez-Sanchez
E. P., Shackleton C. H. L. and Milewich L.: Identification of 19-hydroxydeoxycorticosterone, 19-oxo-deoxycorticosterone, and 19-oic-deoxycorticosterone as products of deoxycorticosterone metabolism by rat adrenals. Endocrinology 110 (1982) 386389. 8. Takemori S., Sato H., Gomi T., Suhara K. and Katagiri M.: Purification and properties of cytochrome P-450,,, from adrenocortical mitochondria. Biochem. biophys. Res. Commun. 67 (1975) 1151-1157. 9. Sato H., Ashida N., Suhara K., Itagaki E., Takemori S. and Katagiri M.: Properties of an adrenal cytochrome P-450 (P->50,,8) for- the hydroxylations of corticosteroids. Archs Biochem. Biophys. 190 (1978) 307-314. 10. Watanuki M., Tilley B. E. and Hall P. F.: Cytochrome P-450 for 1lb- and 18-hydroxylase activities of bovine adrenocortical mitochondria: one enzyme or two? Biochemistry 17 (1978) 127-130. Il. Suhara K., Gomi T., Sato H., Itagaki E., Takemori S. and Katagiri M.: Purification and immunochemical characterization of the two adrenal cortex mitochondrial cvtochrome P-450-Proteins. Archs Biochpm .- . Biophys. ldo (1978) 29&299. 12. Kim C. Y., Sugiyama T., Okamoto M. and Yamano T.: Regulation of 18-hydroxycorticosterone formation in bovine adrenocortical mitochondria. J. steroid Biochem. 18 (1983) 593-599. 13. Suhara K., Takeda K. and Katagiri M.: P-450,,,denendent conversion of cortisol to cortisone. 2nd -.._ 19-hydroxyandrostenedione to 19-oxoandrostenedione. Biochem. biophys. Res. Commun. 136 (1986) 369-375. 14. Wada A., Okamoto M., Nonaka Y. and Yamano T.: Aldosterone biosynthesis by a reconstituted cytochrome P-450,,, system. Biochem. biophys. Res. Commun. 119 1
(1984) 365-371.
15 Ohnishi T., Wada A., Nonaka Y., Okamoto M. and Yamano T.: Effect of phospholipid on aldosterone biosynthesis by a cyt&hrome P1450,,8-reconstituted svstem. Biochem. In?. 9 (1984) 715-723. 16 Wada A., Ohnishi T., Nonaka Y., Okamoto M. and Yamano T.: Synthesis of aldosterone by a reconstituted system of cytochrome P-450,,, from bovine adrenocortical mitochondria. J. Biochem. 98 (1985) 245-256.
I9-Hydroxy-DOC
and I9-oxo-DOC biosynthesis by P-450,,#
17. Okamoto M., Momoi K., Fujii S. and Yamano T.: 18,19~ihydroxydeoxyco~icosterone; a novel product of cytochrome P-45O,,,+atalyzed reaction. Biochem. biophys. Res. Commun. 109 (1982) 236-241. 18. Momoi K., Okamoto M., Fujii S., Kim C. Y., Miyake Y. and Yamano T.: 19-Hvdroxvlation of 18-hydroxy1I -deoxycorticosterone catalyzed by cytoehrome P-450,,, of bovine adrenoeortex. J. bid. Chem. 258 (1983) 8855-8860. 19. Fujii S., Momoi K., Okamoto M., Yamano T., Okada T. and Terasawa, T.: 18,19-dihydroxydeoxycorticosterone, a new metabolite produced from 18-hydroxydeoxycorticosterone by cytochrome P-450,,,. Chemical synthesis and structural analysis by ‘H NMR. Biochemistry 23 (1984) 2558-2564. 20. Suhara K., Takemori S. and Katagiri M.: Improved purification of bovine adrenal iron-sulfur protein. Biochim. bjuphys. Acta 263 (1972) 272-278. 21 Sugiyama T. and Yamano T.: Purification and crystalization of NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria. FEES Letr. 52 (1975) 145-148. 22 Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193 (1951) 265-275. 23. Omura T. and Sato R.: The carbon monooxide-binding pigment of liver microsomes. J. bid. Chem. 239 (1964) 2370-2378. 24. Genard P., Palem-Vliers M., Denoel J., Van Cauwenberge H. and Eechaute W.: Molecular configuration and conformation of aldosterone, 1S-hydroxy-11deoxycorticosterone and a new urinary 18-hydroxysteroid-an N.M.R. study. J. steroid Biochem. 6 (1975) 201-210. 25. Mattox V. R.: Isolation of 19-hydroxy-1 I-desoxycorti-
26.
27.
28.
29.
30.
31.
32.
33.
81
costerone from beef adrenal extract. Proc. Stafi Mrg Mayo Clin. 30 (1955) 18&182. Neher R. and Wettstein A.: Isolierung and konstitutionsermittlung weiterer pregnanverbindungen aus nebennieren. Helu. chim. Acra 39 (1956) 2062-2088. Levy H. and Kushinsky S.: The isolation of 19-hydroxy-1 I-deoxy~orti~osterone and an unknown, active mineralocorticoid from bovine adrenal perfusions of progesterone. Archs Biochem. Biophys. 59 (1955) 290-293. Dale S. L., Holbrook M. M. and Melbey J. C.: Identification of 19-hydroxydeoxyco~icosterone in regenerating rat adrenal incubations. Steroid 36 (1980) 601-609. Gomez-Sanchez C. E., Holland 0. B., Murry B. A., Lloyd H. A. and Milewich L.: 19-Nor-deoxycorticosterone: A potent mineralocorticoid isolated from the urine of rats with regenerating adrenals. ~n~oc~j~~f~gy 105 (1979) 708-711. Skelton F. R.: Adrenal regeneration and adrenalregeneration hypertension. Physiol. Review 39 (1959) 162-182. Melby J. C., Dale S. L. and Griffing G. T.: Hyperrension. McGraw-Hill Book Company, New York (1983) pp. 349-359. Perrone R. D., Schwartz J. H., Bengele H. H., Dale S. L., Melby J. C. and Alexander, E. A.: Mineralocorticoid activity of 19-nor-DOC and IP-OH-DOC in toad bladder. Am. J. Physiot. 241 (1981) 406409. Okamoto M., Momoi K., Yamano T., Nakamura M., Odaguchi K., Shimizu T., Okada T. and Terasawa T.: Affinity of 18,19-dihydroxydeoxyco~icosterone and 18-hydroxy-19-nor-deoxycorticosterone to aldosterone receptor and their mineralocorticoid activity. Biochem. Int. 7 (1983) 687694.
Printed in Great Britain. Ali rights reserved
1987
0022-473t/87 $3.00 + 0.00
Copyright Q 1987Pergamon Journals Ltd
PRODUCTION OF 19-HYDROXY- llDEOXYCORTICOSTERONE AND 19-0X0-1 l-DEOXYCORTICOSTERONE FROM 1I-DEOXYCORTICOSTERONE BY CYTOCHROME P-450, 18 MIHO OHTA, SHIGERU FUJII, AKIRA WADA, TAIRA OHNISHI,TOSHIOYAMANO and MIT%JHIROOKAMOTO* Department of Biochemistry, Osaka University Medical School, 4-3-57 Nakanoshima, Osaka 530, Japan
Kita-ku,
(Received 25 June 1986)
Sag-Incubation of 1I-deoxyco~icosterone with a cytochrome P~5O,,~-r~onstitu~ system yielded, in addition to corticosterone and 18-hydroxy-I l~eoxycorti~osterone, a new steroid product. The retention time of the new product was identical with that of authentic 19-hydroxy-I 1-deoxycorticosterone on high performance liquid chromatography (HPLC). The turnover number of 19-hydroxy-1 l-deoxycorticosterone formation was 7.0 mol/min/mol P-450. When a large amount of cytochrome P-450,,! was used for the reaction and the products were analyzed by HPLC, the 19-hydroxy-1 I-deoxycorticosterone peak disappeared from the chromatogram and concomitantly new unidentified peaks appeared. These results suggest that 19-hydroxy-lI-deoxycorticosterone was further metabolized to other steroids by cytochrome P-450,,# Therefore, we next incubated 19-hydroxy-I ldeoxycorticosterone with cytochrome P-450,,, and analyzed the reaction products by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram. The retention time of one of the peaks coincided with that of authentic 19-0x0-1 I-deoxycorticosterone. This peak substance was purified by repeated HPLC and subjected to mass spectrometry and ‘H NMR analyses. Its field desorption mass spectrum (FlXMS) showed a M+ peak at m/e 344. The ‘H NMR spectrum showed the signal of an aldehyde proton instead of those of hydroxymethyl protons at the C-19 position. These results suggest that cytochrome P-450,,, can catalyze the t9-hydroxylation of 1I-deoxycorticosterone, and the 19-hydroxy-II-deoxycorticosterone produced is further oxidized at the C-19 position to 19-0x0-1 l-deoxycorticosterone.
INTRODUCTION It is known that removal of the 19-methyl group on a steroid nucleus affects both the biological and chemical properties of the corresponding methylated parent compound [l-3]. 19-Nor-l l-deoxycorticosterone is up to five times more potent than 1I-deoxycorticosterone in the sodium-retaining bioassay [ 1,4], and at molecular level has a higher affinity for the renal mineraloco~i~id receptor than aldosterone 151. This steroid has been shown to be markedly hypertensinogenic in the rat [6]. 19-Nor1I-deoxycorticosterone is not a product of adrenal steroid metabolism but appears to be formed extraadrenally from a precursor of adrenal gland origin [7]. Gomez-Sanchez et aL[7] suggested that 19-0x0-1 l-de19-hydroxy-1 I-deoxycorticosterone,
oxycorticosterone are the adrenal costerone. But processes which be elucidated. Cytochrome
and 19-oic- 11-deoxycorticosterone precursors of 1g-nor- 11-deoxycortithe enzymatic characteristics of the produce these metabolites remain to
mitoP-450] Is of adrenocortical chondria has been shown to catalyze both the llgand 18-hydroxylation of 11-deoxycorticosterone, the 1lo-hydroxylation of 18-hydroxy-1 l-deoxycorticosterone, the 18-hydroxylation of corticosterone, and the 1l/l- and 19-hydroxylation of 4-androstene-3,17dione [8-131. Recent investigations in our laboratory demonstrated that the formation of aldosterone from corticosterone is catalyzed by a cytochrome P-450,,,reconstituted system fortified with phospholipid [14-161, and that the 19-hydroxylation of 18-hydroxy-1 1-deoxycorticosterone is also catalyzed by cytochrome P-450,,, [17-191. Since cytochrome P*To whom correspondence should be addressed. 450,,, shows such versatile catalytic activity toward The trivial names used are: 19-hydroxy-I l-deoxycorticosterone, 19,21-dihydroxy-4-pregnene-3,20-dione; 19- steroid substrates, it is possible that the cytochrome can also catalyze the hydroxylation of 1I-deoxy0x0-1 I-deoxycorticosterone, 21-hydroxy-3,20-dioxo-4pregnene- 19-al; 19-061 ldeoxycorticosterone, 19- corticosterone at the C-19 position. Therefore, we 19-nor-l I-de- started to examine this possibility by using a reoic-2i-hydroxy-4-pre~ene-3,2O-dione; oxycorticosterone, 21-hydroxy-19-nor-4-pregneneconstituted system including cytochrome P-450,,, 18,19-dihydroxy-1 I-deoxycorticosterone, 3,20-dione; purified from bovine adrenal cortex. l&19,21-trihydroxy-4-pregnene-3,20-dione; l&hydroxyDuring this study, we found unknown metabolites 18,21-dihydroxy-4-pregnene11-deoxycorticosterone, 3,20-dione. of 11-deoxycorticosterone in the reaction mixture.
s,B.2611-F
73
These metabolites were identified as 19-hydroxy-1 ldeoxycorticosterone and 19-0x0-1 l-deoxycorticosterone on the basis of the results of FD-MS analysis as well as those of NMR-spectroscopy. The results indicate that cytochrome P-450,,, catalyzes the hydroxylation of 1I-deoxycorticosterone at the C-19 position to 19-hydroxy-1 I-deoxycorticosterone, which is further oxidized to 19-0x0-1 l-deoxycorticosterone. EXPERIMENTAL
Chemicals
Most of the steroids used were purchased from Sigma (U.S.A.). 19-Hydroxy-1 I-deoxycorticosterone and 19-0x0-1 l-deoxycorticosterone were generous gifts from Dr T. Terasawa of the Shionogi Research Laboratory. NADPH, gluco~-6-phosphate and glucose-&phosphate dehydrogenase were obtained from Oriental Yeast Co. (Japan). Other chemicals were of the highest grade available from commercial sources. All solvents were of HPLC grade. Preparation of enzymes
Cytochrome P-450,,, was purified from bovine adrenocortical mitochondria according to the method described by Suhara et al.[ll] with the modifications of Wada et al.[ 161. The specific cytochrome P-450,,, content of the purified sample was 16 nmollmg protein. Aldrenodoxin and adrenodoxin reductase were purified from bovine adrenal mitochondria, by Dr Y. Nonaka of our laboratory, according to the methods described by Suhara et a/.[201 and Sugiyama et a/.(21], respectively.
For HPLC, a Gilson model 320 system equipped with a U.V.monitor was employed. For normal phase HPLC, a 4 x 250 mm silica column (M&S packs, 5 p) and a mobile phase of dichloromethane-ethanolwater = 98.1: 1.71:0.19 (by vol), at a flow rate of 1.2 mlimin, were used. For reverse phase HPLC, a 4.5 x 250 mm column (M&S packs, 5 p, ODS) was employed and the solvent system used comprised methanol-water = 60:40 (v/v), at a flow rate of 0.5 ml/min. For FD-MS, a JMS D-300 Mass Spectrometer equipped with an FD/FI/EI ion source was used. The ‘H NMR spectra were obtained at 200 MHz with a Varian XL-200 spectrometer. Chemical shifts (6) in CDCl, solution were measured in ppm with the residual CHCl, of the solvent system as the internal standard (S = 7.27). Enzyme assay and analysis
of products
For the usual enzyme assay, a steroid (100 PM) was incubated with the reconstituted cytochrome P-450,,B-system (a final volume of OSml) in the presence of a NADPH-regenerating system under aerobic conditions for 4 min according to the method described by Momoi et al.[18]. The products were
extracted with dichloromethane, and then the solvent was evaporated under nitrogen. The dried extract was analyzed by HPLC. The amounts of products were determined by monitoring the absorbance at 254 nm with cortisol as an internal standard. Preparation of an unidentified product [Product (ZI)]
19-Hydroxy-1 I-deoxycorticosterone (4 pmol) was incubated with cytochrome P-450,,, (2nmol) for 15min at 37°C in 20 ml of 10 mM Tris-HCl (pH 7.4) containing NADPH (100 pmol), glucose-6-phosphate (200 pmol), glucose-6-phosphate dehydrogenase (20 units), MgCl, (60pmol), adrenodoxin (600 nmol) and adrenodoxin reductase (20 nmol). The reaction was terminated by the addition of 10 ml ethanol. The reaction mixture was extracted three times with 100 ml of dichloromethane and then the solvent was evaporated under nitrogen. The residue was applied to the reverse phase HPLC system mentioned above and the fraction showing a retention time identical to that of 19-oxo-ll-deoxycorticosterone was collected and further applied to the same column repeatedly until the isolated product showed a single peak on chromatography. Analytical methods
Protein concentrations were determined according to the method of Lowry ef af.[22] with bovine serum albumin as the standard. The cytochrome P-450,,, content was estimated as described by Omura and Sato[23] using an extinction coefficient of 91 mM_’ cm-’ for the absorbance difference, at 450 nm and 490 nm, of the CO-binding reduced enzyme. The concentrations of adrenodoxin and adrenodoxin reductase were determined using t 414 = 10 mM-i.cm-’ and cdM= I1 mM-“cm-‘, respectively. RESULTS
HPLC-pro$ies of authentic steroids and identification of a new metabolite [Product (J)] as J9-hydroxy- JJdeoxy~orti~osterone
Figure 1 presents chromatograms of authentic steroids on normal phase HPLC and on reverse phase HPLC, respectively. These chromatographic systems well separated 19-hydroxy- 11-deoxycorticosterone from both corticosterone and 1X-hydroxy-I 1-deoxycorticosterone. The three steroids were eluted at 18, 12 and 16 min on normal phase HPLC, and at 22,44 and 38min on reverse phase HPLC, respectively. Using these HPLC systems, we examined the products of the reaction of 1I-deoxycorticosterone with a cytochrome P-450iIg-reconstituted system (Fig. 2). Three product peaks appeared in the chromatograms, two of which were identified as corticosterone (the product of 1lfi-hydroxylation) and 18-hydroxy-1 ldeoxycorticosterone (the product of 18-hydroxylation) on the basis of their retention times. The other steroid product (Product [I]) showed a retention time
19-Hydroxy-HOC and 1P0x0-DOC biosynthesis by P-45O,,B
(A) 0.04-
75
(8) ’
3.04
x
/ f
“d ; 3.02 u I
o.oc I I
10
0
20
0
3040
10
2030
tmin)
3.00
_,..LL..L
40
50607080
(min)
t Fig. I. Analysis of authentic steroid mixtures by (A) normal phase HPLC and (B) reverse phase HPLC. The compounds tested were: (1) 1I-deoxycorticosterone, (2) corticosterone, (3) 18-hydroxy- 1l-deoxycorticosterone, (4) 19-hydroxy-1 I-deoxycorticosterone, (5) cortisol and (6) aldosterone. The HPLC conditions were as follows: (A) Column: M&S packs, silica 5 pm, 4 x 250 mm. Solvent system: CH,Cl,-EtOH-H,O (98.1:1.71 :O.19) (by vol.). Flow rate: 1.2 mi/min. (B) Column: M&S packs, ODS, 5 pm, 4.5 x 250 mm. FIow rate: OJml/min. Solvent system: MeOH:H,O=60:40 (v/v). The detailed conditions for these experiments are given under Experimental. Each peak number corresponds to the position of the corresponding authentic steroid.
of 18 min on normal phase HPLC and one of 22 min on reverse phase HPLC. These retention times are identical to those of authentic 19-hydroxy-I l-deoxycorticosterone. Moreover, the ‘H NMR spectrum of Product (I) was essentially identical to that of authentic 19-hydroxy- 1I-deoxycorticosterone (data not shown). To confirm that 19-hydroxy-1 I-deoxycortico-
sterone is a real product generated from ll-deoxycorticosterone by cytochrome P-450,,,, we performed the following experiments. The time-course of the formation of IPhydroxy- 11-deoxycorticosterone (Product [Ij) was examined. The formation of 19hydroxy- 11-deoxycorticosterone proceeded almost linearly with time up to 4min under the standard assay conditions (Fig. 3A). The turnover number of
T
(B)
(A) 1
2
0.04
1
2 xo.oi
45
;”
i=i
-0.04
t;
?I 0.02
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o.oc
0
10
20
30
(mini
40
I,~~
50
0
20
d
II-*
40
60
80
i J,.,,
fmin)
Fig. 2. HPLC profiles of reaction products of 1I-deoxycorticosterone and cytochrome P-450,,8. (A) Normal phase HPLC and (B) reverse phase HPLC. The peak numbers correspond to the positions of the authentic steroids on HPLC, (1) 1I-Deoxycorticosterone, (2) corticosterone, (3) IS-hydroxy-1 l-deoxy~rti~sterone, (4) 19-hydroxy-1 l-d~xycorti~s~rone, and (5) cortisol (the internal standard). The chromatogtaphic conditions were as in Figs l(A) and (B). 11-Deoxycorticosterone (1OOpM) was incubated with cytochrome P-450,,, (39 pmol) in the presence of adrenodoxin, adrenodoxin reductase and a NADPH-generating system (a final volume of 0.5 ml). The extract of the reaction mixture was subjected to (A) normal phase HPLC and (B) reverse phase HPLC, respectively, and the eluate was monitored as to the absorbance at 254 nm, The detailed conditions for this experiment are given under Experimental.
MIHO OHTA et
76
al.
a- (El)
-1.6
7-
-1.4
-c
-1.2
T ‘E \
-0.8
“0
0 -
(A)
E
1 0.4
L 0’
10.2
L 1
2
Incubation
3
4
5
time (min)
0
I 20
40
P-450,,~
60
80
100
2 a
0.0
(pmol)
Fig. 3. (A) Time-course of the formation of 19-hydroxy-1 I-deoxycorticosterone (Product [I]). 1I-Deoxycorticosterone (100 NM) was incubated with the reconstituted cytochrome P-450,,, (44 pmol)-system in a final volume of 0.5 ml at 37°C for the indicated periods. For the detailed conditions and the analytical method, see under Experimental. (B) Effect of the concentration of cytochrome P-450,,, on the formation of 19-hydroxy-1 I-deoxycorticosterone (Product [I]). 1I-Deoxycorticosterone (100 FM) was incubated with the indicated amounts of cytochrome P-450,,8-system in a final volume of 0.5ml. The products were analyzed as described under Experimental. Closed and open circles show the rate of production of 19-hydroxy-1 I-deoxycorticosterone (Product [I]) and that of corticosterone, respectively. The value obtained on incubation without cytochrome P-450,,, is due to a noise on HPLC.
19-hydroxy- 1 1-deoxycorticosterone formation under these conditions was determined to be 7.0 mol/ min/mol P-450. The dose dependency of the formation of 19-hydroxy- 11-deoxycorticosterone (Product [I]) as to the cytochrome P-450,,, concentrations was studied. As shown in Fig. 3(B), a linear relationship was observed between the 19-hydroxy1I-deoxycorticosterone formation and the amount of cytochrome P-450,,, in a range below 100 PM. After heat-treatment, cytochrome P-450,,, (treated for 5 min at 100°C) did not produce any corticosterone, 1%hydroxy-1 l-deoxycorticosterone or 19-hydroxyI I-deoxycorticosterone (data not shown). Metabolism of 19-hydroxy- 11 -deoxycorticosterone through the cytochrome P-450,,p-catalyzed reaction
When a large amount of cytochrome P-450,,, was used for the reaction and the reaction products were analyzed by HPLC, the peak of 19-hydroxy-lldeoxycorticosterone (Product [I]) unexpectedly decreased in the chromatogram and new unidentified peaks appeared instead (one of the new peaks is denoted as Product (II) in Fig. 4). Figure 5 shows the amounts of corticosterone, 1%hydroxy-1 l-deoxycorticosterone and 19-hydroxy- 11-deoxycorticosterone (Product [I]) produced on the incubation of 11-deoxycorticosterone with a high concentration of cytochrome P-450, iD. The addition of more than 200 pmol of cytochrome P-450,,, to the incubation medium resulted in a decrease in the production of 19-hydroxy- 11-deoxycorticosterone, while the amounts of corticosterone and 1%hydroxy- ll-
deoxycorticosterone produced reached plateaus and never decreased in the cytochrome P-450,,, concentration range investigated. We considered that the substrate was exhausted within 4min in the incubation mixture with more than 200 pmol of cytochrome P-450,,,. These results suggest that the new unidentified peak (Product [II] in Fig. 4) was probably derived from 19-hydroxy-1 l-deoxycorticosterone. Figure 6 shows the time courses of the production of corti19costerone, 1%hydroxy-1 1-deoxycorticosterone, hydroxy-1 I-deoxycorticosterone (Product [I]) and the new unidentified product (Product [II]) in the presence of 318 pmol of cytochrome P-450,,,. Under these conditions, the amounts of corticosterone and 18-hydroxy-1 I-deoxycorticosterone accumulated in the reaction mixture reached plateaus after 2 min because of exhaustion of the substrate, and thereafter they did not significantly decrease up to 15 min. On the other hand, 19-hydroxy-1 I-deoxycorticosterone (Product [I]) linearly accumulated in the reaction mixture up to 2min, and then decreased. In accord with the beginning of the decrease in 1Phydroxy-1 ldeoxycorticosterone, Product (II) started to appear in the reaction mixture and increased progressively. Stoichiometry between the decrease in 19-hydroxy1 1-deoxycorticosterone and the increase in Product (II) could not be established, because Product (II) was one of several unidentified peaks seen in the chromatogram (see Fig. 4B). These results suggest that the 19-hydroxy-1 I-deoxycorticosterone produced was further metabolized to other steroids by
19-Hydroxy-DOC and IP0x0-DOC biosynthesis by P-450,,,
77 7
(AlI
(A)(2) 1
l
2
Iminl
I
_ (mini
-!
Fig. 4. Conversion of IQ-hydroxy-1I-deoxycorticosterone (Product [I] to new metabolites catalyzed by the reconstituted cytochrome P-450,,8 system. 11-Deoxycorticosterone (100 p M) was incubated with (A) a small amount (106pmol) and (B) a large amount (530pmol) of the reconstituted cytochrome P-450,,B-systemin a final volume of 0.5 ml at 37°C for 4 min. The products were analyzed as described under Ex~rimental. (1) Normal phase HPLC. (2) Reverse phase HPLC. Each peak number corresponds to the position of the corresponding authentic steroid. (1) 1I-Deoxycorticosterone, (2) corticosterone, (3) 18-hydroxy-1 I-deoxycorticosterone, (4) IQ-hydroxy-1 I-deoxycorticosterone, and (5) cortisol (the internal standard).
the cytochrome P-450,,,. Therefore we performed the following experiments. 19-Hydroxy- 1I-deoxycorticosterone was incubated as a substrate with the cytochrome F-45O,~~-reconstituted system and the products were analyzed by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram (data not shown). One of the peaks (Product [II]) showed a retention time of 4min on normal phase HPLC and one of 30min on reverse phase HPLC, respectively. These retention times coincide with those of authentic lPoxo-1 l-deoxycorticosterone. As the amount of
P- 450,,B I pm011 Fig. 5. Effect of the cytoehrome P-450,,, concentration on 19-hydroxy- i 1~eoxyco~icosterone formation. I I-Deoxycorticosterone (100 PM) was incubated with various amounts of the reconstituted cytochrome P-450,,8-system in a final volume of 0.5ml at 37°C for 4min. The amounts of 19-hydroxy-ll-deoxycorticosterone (Product [I]) (a), corticosterone (0) and It-hydroxy-I l~eoxycorticosterone [IS-(OH)DOC] (A) accumulated in the media were determined by HPLC.
cytochrome P-450,,, increased, the production of Product (II) increased (Fig. 7). Heat-inactivated cytochrome P-450,,, (treated for 5 min at 100°C) did not produce any new metabolites from 19-hydroxy-lldeoxycorticosterone (data not shown). Identification of Product (II) as 19-0~0 - 11 -deoxy corticosterone
To further confirm the chemical identity of Product (II), large scale of purification of it was carried out as described under Experimental. The purified sample was subjected to mass analysis involving a field desorption technique. Figure 8 shows the FD-MS spectra of authentic 19-0x0-1 I-deoxycorticosterone and Product (II). Molecular ion peaks of Product (II) as well as of authentic 19-0x0-1 l-deoxyco~icosterone were detected at mie 344. To obtain better insight into the chemical structure of Product (II), ‘H NMR spectra of authentic 19-0x0-1 I-deoxycorticosterone and Product (II) were obtained. Figure 9(A) shows the ‘H NMR spectrum of authentic 19-0x0- 11-deoxycorticosterone in CDCI,. The signal of an aldehyde proton appeared at S = 9.93 ppm. According to the results of Genard et aE.[23] and Fujii et aI.[lS], the resonance peaks around 6 = 3.23 and at zi = 4.19 and 5.99 ppm were assigned as due to 21-OH-, 21-CH,- and 4H-proton, respectively. The resonance peak that appeared at around 6 = 1.4 ppm (indicated by an asterisk) was considered to be due to a contaminant in the solvent, because further purification of this material by HPLC caused a decrease in this signal. Figure 9(B) shows the spectrum of Product (II), which is essentially identical to that of authentic 19-0x0-1 I-deoxycorticosterone. The combined characteristics of the HPLC elution profiles, the FD-MS
MIHO OHTA et al.
78
o
z
6 8 10 12 INCUBATION TIME I mini
1L
16
Fig. 6. Metabolism of 1I-deoxycorticosterone by the reconstituted cytochrome P-450,,8-system.1I-Deoxycorticosterone (100 PM) was incubated with the reconstituted cytochrome P-450,,, (318pmol)-system in a final volume of 0.5 ml at 37°C for the indicated periods. The products were analyzed as described under Experimental. The amounts of 19-hydroxy-1l-deoxycorticosterone (Product [r]) (a), corticosterone (0) 18-hydroxy-11-deoxycorticosterone (18(0H)DOC) (A) and Product (II) (0) were estimated from the absorbance at 254nm.
spectrum and the ‘H NMR spectrum provided strong evidence of the production of 19-oxo-ll-deoxycorticosterone by cytochrome P-450, i8. These results together indicate that cytochrome P-45O1,8 can catalyze the 19-hydroxylation of 1l-de19IOH)DOC~
$ Producf
(III
P_45O,,rJ
(1) 148
I
LI
0
pm01
10
20
30
40
50
60
(min) Fig. 7. HPLC profiles of reaction products of 19hydroxy-1 I-deoxycorticosterone with the reconstituted cytochrome P-450,,B-system. 19-Hydroxy-1 l-deoxycorticosterone (100 PM) was incubated with the indicated amounts of the reconstituted cytochrome P-450,,8-system at 37°C for 4min. The products were analyzed by reverse phase HPLC. The detailed conditions for this experiment are given under Experimental. The amounts of cytochrome P-450,,8 used were (1) 148 pmol, (2) 44 pmol and (3) 15 pmol in 0.5 ml of the reaction mixture. The striped peak corresponds to Product (II), and its retention time is identical with that of authentic 19-0x0-1 I-deoxycorticosterone. 19(0H)DOC, 19-hydroxyl-deoxycorticosterone. F, cortisol (the internal standard).
oxycorticosterone and the 19-hydroxy- 1l-deoxycorticosterone thus produced is further metabolized to 19-0x0- 11-deoxycorticosterone. DISCUSSION
In this study, we demonstrated that 1l-deoxycorticosterone can be hydroxylated at the C-19 position as well as the 11/I- and l&positions by cytochrome P-450,,,. By means of FD-MS and ‘H NMR spectrometric analyses, we confirmed that the structures of the products are 19-hydroxy- 1l-deoxycorticosterone and 19-0x0- 1 1-deoxycorticosterone, and found that the rate of the formation of 19hydroxy-1 I-deoxycorticosterone is comparable to that of 18-hydroxy-11-deoxycorticosterone. To our knowledge, this is the first report of the production of 1Phydroxy-1 1-deoxycorticosterone and 19-0x01I-deoxycorticosterone by a cytochrome P-450,,, system, although Sato et a1.[9] reported that this cytochrome can catalyze the 19-hydroxylation of androstenedione, and more recently Momoi et a/.[ 181 reported that this cytochrome can also catalyze the 19-hydroxylation of 18-hydroxy-1 l-deoxycorticosterone. 19-Hydroxy-1 I-deoxycorticosterone was first isolated from bovine and hog adrenal glands [25-271. Recently, 19-hydroxy-1 I-deoxycorticosterone was found in incubates of regenerating adrenal capsules 3-4 days after enucleation [28], and 19-nor-l l-deoxycorticosterone was identified in the urine of rats with regenerating adrenal glands [29]. That enucleation in the adrenal glands of rats causes adrenal regeneration hypertension has been well demonstrated [30,3 11, and the possibility that a mineralocorticoid other than aldosterone, perhaps 19-nor-l l-deoxycorti-
I9-Hydroxy-DOC
and 19-0x0-DOC biosynthesis by P-450,,,
19
CH,OH
I c=o
19-KHO)-DOC 1
H\c//o
hl+ 344
/
0
M+l
E
& ’
M+
1, x4.0
344
Product
(II
I
I---
0 300
350
400
450
500
m/e Fig. 8. The field desorption mass spectra of authentic 19-0x0-1 I-deoxycorticosterone and the Product (II). Product (II) was purified and analyzed by FD-MS as described under Experimental. 19(CHO)DOC, 19-0x0-1 I-deoxycorticosterone.
(A) CDCL,
(B) CDCI,
ISCHOPOC
PRODUCTUI)
Fig. 9. ‘H NMR spectra of authentic 19-0x0-1 I-deoxycorticosterone and Product (II). (A) 19-0x0-1 Ideoxycorticosterone [19(CHO)DOC] in CDCl,; (B) Product (II) in CDCl,. The peak indicated by an asterisk is due to the solvent.
80
MIHO OHTA et al.
costerone, may be important in the pathogenesis of adrenal regeneration hypertension was suggested [7,29]. 1g-Nor- 11-deoxycorticosterone has been shown to be a potent mineralocorticoid in the rat bioassay for sodium retention [l] and in sodium transport across the toad bladder [32], whereas 19-hydroxy-11-deoxycorticosterone does not exhibit significant mineralocorticoid activity. From these results it was suggested that 19-nor-l l-deoxycorticosterone may play a significant role in the pathogenesis of sodium retention following adrenal enucleation. Although the biosynthetic pathway for 19-nor- 11-deoxycorticosterone remains unknown, several intermediate products of 1l-deoxycorticosterone oxygenated at the C-19 position were found in an adrenal homogenate by Gomez-Sanchez et a1.[7]. They reported the formation of 19hydroxy-1 l-deoxycorticosterone, 19-0x0-1 l-deoxycorticosterone and 19-oic- 11-deoxycorticosterone from 1 1-deoxycorticosterone in adrenal glands of rats undergoing adrenal regeneration and of intact rats [7]. On the other hand, as 19-nor-ll-deoxycorticosterone itself was not found in the adrenal homogenate, it was proposed that 19-nor-l l-deoxycorticosterone may be generated at the target site of its mineralocorticoid action from one of these putative precursors [31]. Therefore, the existence of 19-hydroxy- 11-deoxycorticosterone and 19-0x0- 1ldeoxycorticosterone might reflect their biological significance as precursors of 1g-nor-1 1-deoxycorticosterone. P-450, 18 was reported Recently, cytochrome to catalyze the formation of aldosterone from corticosterone or 18-hydroxycorticosterone [14-161. Aldosterone is well known as the most potent physiologically-occurring mineralocorticoid. The recent description of the formation of 18,19dihydroxy- 11-deoxycorticosterone by a cytochrome P-450,,8-reconstituted system suggests the possible existence of 1g-nor- 18-hydroxy-1 l-deoxycorticosterone [17]. Okamoto et a/.[331 reported that synthetic 19-nor-18-hydroxy-l l-deoxycorticosterone showed an affinity to the aldosterone receptor of the rat renal cytosol similar to that of 18-hydroxy-1 ldeoxycorticosterone, and that its sodium retaining activity was also similar to that of 18-hydroxy-1 ldeoxycorticosterone. These reports [14-191 and our present results suggest that cytochrome P-450,,, is deeply involved in the biosynthesis of mineralocorticoids in adrenal glands. Further studies are necessary to find further metabolites of 1l-deoxycorticosterone in the cytochrome P-450,,B-catalyzed reaction and to determine if 19-hydroxy-1 l-deoxycorticosterone and 19-0x0-1 I-deoxycorticosterone are precursors of 19-nor-l l-deoxycorticosterone. Acknowledgements-A part of this study was supported by research grants from the Ministry of Education and Culture of Japan, and the Science and Technology Agency of I
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