335
Biochimica et Biophysics Acta, 575 (1979) 0 Elsevier/North-Holland Biomedical Press
335-349
BBA 51480
METABOLISM OF PROSTAGLANDINS, PROSTAGLANDIN ANALOGS AND THROMBOXANE Bz BY LUNG AND LIVER MICROSOMES FROM PREGNANT RABBITS
WILLIAM
S. POWELL
Department of Medicine, McGill University, and the Endocrine Laboratory Hospital, 687 Pine Avenue West, Montreal, Quebec, H3A 1Al (Canada) (Received May lst, 1979) (Revised manuscript received
August
*, Royal Victoria
6th, 1979)
Key words: Prostaglandin metabolism; Thromboxane
B2; (Pregnant rabbit microsome)
Liver microsomes from pregnant rabbits converted prostaglandins FZLY,El, and E, to their ZO-hydroxy metabolites along with smaller amounts of the corresponding 19-hydroxy compounds. Prostaglandins E, and E, were also reduced to prostaglandins F,, and F2&, respectively, and prostaglandin El was isomerized to S-isoprostaglandin E 1. The above products were also identified after incubation of prostaglandins with liver microsomes from non-pregnant rabbits. In this case, the yield of 20-hydroxy metabolites was much lower. Thromboxane B, and a number of prostaglandin Fzo! analogs were also hydroxylated by lung and liver microsomes from pregnant rabbits. The relative rates of hydroxylation by lung microsomes were: prostaglandin Ez = prostaglandin F,, = 16,16dimethylprostaglandin F,, = 13,14-didehydroprostaglandin F,, > thromboxane Bz > 15methylprostaglandin F,, = 17-phenyl-l&19,20-trinorprostaglandin F,, = ent-13,14didehydro-15-epiprostaglandin F,,. Similar results were obtained with liver microsomes except that thromboxane Bz was a relatively poorer substrate for hydroxylation.
Introduction One of the major steps in prostaglandin metabolism is o-oxidation. Prostaglandins Ai [ 1,2] and E, [ 21 have been shown to be converted to their 19- and 20-hydroxy derivatives by guinea pig liver microsomes in the presence of * Please send correspondence
to this address.
336 NADPH or NADH. The hydroxylated products were identified after conversion to the corresponding prostaglandin B, derivatives with base [1,2]. We have recently shown that lung microsomes from pregnant rabbits are very active in converting prostaglandins and some of their metabolites to their 20”hydroxy derivatives [3f. We have measured the rates of o-hydroxylation of prostaglandin F2@ in lung and liver microsomes from rabbits using an assay in which the products were measured by mass spectromet~ [43. Pregnancy was shown to cause 127-fold and 20”fold increases in the 20-hydroxylation of prostaglandin F,, by lung and liver microsomes, respectively. Similar increases in 20”hydroxylation by rabbit lung microsomes were induced by pseudopregnancy and progesterone treatment [4]. We have now investigated the metabolism of a number of prostaglandins, prostaglandin Bz by F 2o1analogs and thromboxane microsomal fractions from lung and liver of pregnant rabbits. Materials and Methods Radioactively labeled prostaglandin El, prostaglandin Ez, prostaglandin FZcu, arachidonic acid and sodium borohydride were purchased from Amersham/ Searle. Unlabeled prostaglandins, prostaglandin analogs and thromboxane B2 were kindly provided by Drs. J.E. Pike of the Upjohn Company and J. Fried (13,14didehydropros~glandin F 2a and ear-13,14-didehydro-15-epiprostaglandin FZO(). Rabbits were purchased from Canadian Hybrid Farms, Stanstead, Quebec, and were sacrificed by inhalation of carbon dioxide. Gas chromatography-mass spectrometry was carried out using an LKB 9000 instrument. 15-Methyl [ 9@-3H]prostaglandin F,, [5]. 16,16-dimethyl[9@-3H]prostaglandin F,, [6] and 17-phenyl-18,19,20-trinor[9/3-3H]prostaglandin Fz, [7] were prepared by reduction of the corresponding prostaglandin E, analogs with sodium [“HI borohydride as described in the literature. [1-14C]Thromboxane B, was synthesized by incubation of [l-14C]arachidonic acid with human platelets as described by Hamberg and Samuelsson [8]. Preparation of microsomal fractions Rabbit lungs were minced in 4 volumes of 0.05 M Tris-HCI, pH 7.5, containing 0.25 M sucrose. The mixture was homogenized in an ice-water bath with a Vir-Tis homogenizer at full speed for six periods of 10 s with 1 min in between to allow for cooling. Rabbit livers were minced in buffer as described above and homogenized for 1 min in a Potter-Elvehjem homogenizer in an ice-water bath. The homogenates were centrifuged at 8000 X g for 10 min at 4°C using a Sorvall model SS-3 centrifuge with an SM 24 rotor. The supernatant was centrifuged at 100 000 X g for 60 min at 2°C using a Beckman model L2-75B ultracentrifuge and a Ti 50 rotor. The pellet was resuspended in 0.05 M Tris-HCl, pH 7.5, with a Potter-Elvehjem homogenizer and centrifuged as described above. The final pellet was suspended in 0.05 M Tris-HCl, pH 7.4 (1.5 volumes/g lung or 1 volume/g liver). Incubation and purification procedures Microsomal fractions (0.5 ml or 5 ml for analytical or preparative experimen@ respectively) were incubated with prostaglandins, prostaglandin analogs or thrombox~e BZ in the presence of NADPH (1 mM) for 45 min at 37” c (15
337
min for analytical experiments). The incubations were terminated by addition of ethanol (2 volumes). The mixtures were filtered and the filtrate was concentrated in vacua to a volume equivalent to that of the original incubation mixture. After dilution with 1 volume of water, the mixtures were acidified to pH 3 with 1 N HCl and extracted with ethyl acetate (4 X 1 volume). The ethyl acetate extracts were washed with water and concentrated to dryness in vacua. The residues were subjected to chromatography on columns of silicic acid (2 g of Bio-Sil HA, Bio-Rad Laboratories, Richmond, CA), which were eluted with diethyl ether/benzene (7 : 3, v/v, 60 ml), ethyl acetate/methanol (9 : 1, v/v, 60 ml) and methanol (30 ml). The radioactive products in the ethyl acetate/ methanol fraction were further purified by thin-layer chromatography on Silica gel G (E. Merck, Darmstadt) with diethyl ether/methanol/acetic acid (100 : 6 : 1, v/v) as solvent. The thin-layer chromatography plates were scanned with a Packard model 7200 radiochromatogram scanner and the radioactive bands were scraped and eluted with ethyl acetate/methanol (9 : 1, v/v). The eluted material was methylated with diazomethane and further purified by thin-layer chromatography with various concentrations of methanol in diethyl ether as solvents. For analytical experiments, the radioactive products in the ethyl acetate extract were analyzed directly by thin-layer chromatography using plates precoated with silica gel (E. Merck, Darmstadt) with diethyl ether/ methanol/acetic acid (100 : 6 : 1, v/v) as solvent. The plates were scanned and the radioactive bands were eluted with methanol/diethyl ether (1 : 1, v/v). The solvent was evaporated and the radioactivity in each of the bands was measured by liquid scintillation counting. Gas chromatography-mass
spectrometry
The purified products from the incubations with prostaglandins E, and E, were converted to their O-methyl oxime derivatives by treatment with 1 mg of methoxylamine hydrochloride (Pierce Chemical Co., Rockford, IL) in pyridine (0.1 ml) at room temperature for 16 h. The solvent was evaporated and the residues were dissolved in benzene and filtered. The O-methyl oxime derivatives, as well as the purified products from the other incubations were then converted to their trimethylsilyl ether derivatives by treatment with Tri-Sil Z (Pierce Chemical Co.) for 5 min at 60°C. The trimethylsilyl ether and Omethyloximetrimethylsilyl ether derivatives were analyzed by gas chromatography-mass spectrometry using a 6 foot column of 1.5% OV-101, and temperatures ranging from 240 to 27O”C, depending on the C value of the product. Retention times were converted to C values by comparison with the retention times of a series of saturated fatty acid methyl esters [9]. The mass spectra of the trimethylsilyl ether derivatives of the methyl esters of some prostaglandin Fzcr analogs and their w-hydroxylated derivatives are given in Table 1. Fragment ions which are observed in the mass spectra of these derivatives are shown in Fig. 3. Results Metabolism of prostaglandins by liver microsomes Prostaglandin F,,. Fig. 1A shows a thin-layer
from pregnant
rabbits
radiochromatogram
obtained
338
after incubation of prostaglandin F,, (20 Erg/ml) with a microsomal fraction (0.5 ml; 13 mg protein/ml) from the liver of a pregnant rabbit (25 days of gestation) in the presence of NADPH (1 mM). After a similar experiment conducted on a larger scale with 250 c(g of substrate. the two bands of radio-
.
PGF2a
10
i
Distance from origin
(cm)
Fig. 1. A. Thin-layer radiochromatogram of the products obtained after incubation of [9@-3Hlprostaglandin Fzol (10 pg) with a microsomal fraction (0.5 ml: 13 mg protein/ml) from the liver of a pregnant rabbit (25 days of gestation) in the presence of NADPH (1 mM). After incubation at 37’C for 15 min the reaction was terminated by the addition of ethanol and the products were extracted. Solvent: diethyl ether/ methanol/acetic acid (100 : 6 : 1, v/v). B. Thin-layer radiochromatogram of the products obtained after incubation of Il-14Clprostaglandin El (250 fig) with a microsomal fraction (5 ml; 28 mg protein/ml) from the liver of a male rabbit in the presence of NADPH (1 mM). In this experiment, the microsomal fraction was resuspended in the boiled (5 min) and filtered supernatant from the 100 000 X g centrifugation. Subsequent experiments in which the microsomal fraction was resuspended in 0.05 M Tris, PH 7.5, gave identical results. After incubation for 60 min at 37°C the products were extracted and purified by column chromatography. Solvent: diethyl ether/methanol/acetic acid (100 : 6 : 1. v/v). C. Thin-layer radiochromatogmm of the products obtained after incubation of a lung microsomal fraction (0.5 ml; 2 mg protein/ml) from a pregnant rabbit (25 days of gestation) with [1-‘4Clthromboxane B2 (10 tig) in the presence of NADPH (1 mM) for 15 min at 37’C. Solvent: diethyl ether/methanol/acetic acid (100 : 6 : 1, v/v).
339
active material were eluted, methylated, and rechromatographed by thin-layer chromatography with methanol/ether as solvent. The purified products were then trimethylsilylated and analyzed by gas chromatography-mass spectrometry. The less polar material (RF 0.21) was identified as unreacted prostaglandin Fzo, on the basis of the C value (24.0) and mass spectrum of the trimethylsilyl ether derivative of its methyl ester. The more polar material (RF 0.05) gave two peaks after gas chromatography, with C values of 26.2 and 27.0. The major product (C value 27.0) had a C value and mass spectrum identical to that which we previously reported for 20-hydroxyprostaglandin Fza, synthesized by lung microsomes from pregnant rabbits [3]. The minor product (C value 26.2) had a C value and mass spectrum identical to that of authentic 19 (R)-19hydroxyprostaglandin F,, . Liver microsomes from male and non-pregnant female rabbits also converted prostaglandin F,, to its 19- and 20-hydroxy metabolites which were identified by gas chromatography-mass spectrometry. In both cases, the overall yield was much lower than with liver microsomes from pregnant rabbits. Comparable amounts of 19-hydroxyprostaglandin F 20 and 20-hydroxyprostaglandin F,, were formed. Prostuglandin E,. The metabolism of prostaglandin El by liver microsomes from pregnant rabbits was somewhat more complicated than that of prostaglandin F,,. [ 1-14C]Prostaglandin E, (500 pg) was incubated with a liver microsomal fraction (10 ml; 10 mg protein/ml) from a pregnant rabbit (21 days of gestation) in the presence of NADPH (1 mM) for 60 min. The radioactive products were extracted and purified by column and thin-layer chromatography. Three bands (RF 0.10, 0.23 and 0.37) of radioactive material were obtained after thin-layer chromatography with ether/methanol/acetic acid (100 : 6 : 1, v/v) as solvent. The material in each band was eluted, methylated and rechromatographed by thin-layer chromatography with methanol/ether as solvent. The purified products were converted to their O-methyloximetrimethylsilyl ether derivatives and analyzed by gas chromatography-mass spectrometry . The most polar product (22% of recovered radioactivity) gave two pairs of 0-methyloxime isomers. The more abundant pair had C values (26.9 and 27.4) and mass spectra which were identical to those which we reported for the Omethyloxime-trimethylsilyl ether derivative of the methyl ester of 20-hydroxyprostaglandin E, [3]. The second pair of isomers had C values of 26.1 and 26.6 and mass spectra nearly identical to those reported for the O-methyloximetrimethylsilyl ether derivatives of the methyl ester of 19-hydroxyprostaglandin E,, which was isolated from semen [lo]. The product (4% of recovered radioactivity) with an RF value of 0.23 with ether/methanolic/acetic acid (100 : 6 : 1, v/v) as solvent did not form an Omethyloxime derivative. It had a C value (24.1) and mass spectrum identical to that of the trimethylsilyl ether derivative of the methyl ester of authentic prostaglandin F,,. The material in the least polar band (RF 0.37; 74% of recovered radioactivity) was methylated and further purified by thin-layer chromatography using ether/methanol (49 : 1, v/v) iis solvent. Two bands of radioactivity (RF 0.23 and 0.33) were detected in a ratio of about 10 : 1. These products were
340
eluted and converted to their 0-methyloxime-trimethylsilyl ether derivatives. They were identified as prostaglandin E, (major product) and B-isoprostaglandin E, (minor product) on the basis of the identity of their C values and mass spectra with those of authentic standards. All of the above products were identified by gas chromatography-mass spectrometry after incubation of prostaglandin E, with liver microsomes from non-pregnant rabbits. Fig. 1B shows a radiochromatogram obtained after incubation of [ l-‘4C]prostaglandin E, (50 pg/ml) with a corresponding fraction from a mature male rabbit. Three bands of radioactive material were obtained which were identified as unreacted prostaglandin E, along with a small amount of 8isoprostaglandin E, (RF 0.37), prostaglandin F,, (RF 0.23) and a 1 : 1 mixture of 19- and 20-hydroxyprostaglandin El. In this case the major products were prostaglandin F,, and B-isoprostaglandin E,. Liver microsomes from male and non-pregnant female rabbits formed much less 20-hydroxyprostaglandin El than those from pregnant rabbits. Prostuglandin E,. Prostaglandin E, was converted by liver microsomal fractions from pregnant rabbits in the presence of NADPH to prostaglandin F20r, 19-hydroxyprostaglandin E, and 20-hydroxyprostaglandin E,, which were identified by comparison of their chromatographic properties and mass spectra with those of authentic standards. NADPH was required for the formation of the major product (20-hydroxyprostaglandin E2). The formation of prostaglandin F,, occurred in the absence of added cofactors, but was stimulated by NADH and NADPH. Metabqlism rabbits
of thromboxane
B2 by lung and liver microsomes
from pregnant
Thromboxane B, was metabolized by lung and liver microsomes from pregnant rabbits in a manner similar to prostaglandins. Fig. 1C shows a radiochromatogram obtained after incubation of [l-14C]thromboxane B, (20 .ug/ml) with a lung microsomal fraction obtained from a pregnant rabbit (25 days of gestation). Three bands of radioactive material were obtained. The products in each band were further purified by thin-layer chromatography as described above and analyzed by gas chromatography-mass spectrometry after conversion to the trimethylsilyl ether derivatives of their methyl esters. The least polar product (RF 0.43) was identified by chromatography and mass spectrometry as thromboxane BZ. The trimethylsilyl ether derivative of the methyl ester of the most polar product (RF 0.12) had a C value of 27.5, 2.8 units higher than that of the corresponding derivative of thromboxane B,. By comparison, the substitution of a trimethylsilyloxy group in either the 19- or the 20-position of trimethylsilyl ether derivatives of prostaglandin methyl esters augments their C values by about 2.0 [l] or 2.8-3.0 [1,3] units, respectively. Because of the similarity of the structure of thromboxane B2 with those of prostaglandins, this suggests that the product with an RF value of 0.12 is 20-hydroxythromboxane B2. The mass spectrum (Fig. 2) of the trimethylsilyl ether derivative of the methyl ester of this product confirms the presence of a hydroxyl group in the terminal pentyl group of the alkyl side chain. Intense ions are observed at m/e 598 (M90), 508 (M-2X 90), 454 (M-234), 439 (M-159-90), 389 (Me,SiO’=
341
CH-CH=CH-CH(OSiMe3)-(CH,)4-CH~OSiMe3), 349 (M-159-2 X 90), 323, 313 (M-234-141), 295 (M-234-159), 256 (base peak; Me,SiO+JH=CHCH,-CH= CH-( CH2)3-COzMe+), 217 (Me,SiO’= CH-CH= CH-OSiMe3), 191 (Me,SiO’=CH--OSiMe,), 129, and 103. This mass spectrum is very similar to that of the trimethylsilyl ether derivative of the methyl ester of thromboxane Bz, except that ions (m/e 598, 508, 454, 389 and 313) in which the alkyl side chain is intact have m/e values 88 mass units higher than the corresponding ions of the thromboxane B2 derivative. The trimethylsilyl ether derivative of the second product (RF 0.17) had a C value of 27.4, which, by analogy with prostaglandins [ll] would be consistent with the presence of an o-carboxylic acid group. The mass spectrum of the material in this peak suggested the presence of two components with identical retention times. The amounts of these compounds which were formed varied from one experiment to the other, and in many cases they were not detectable. Sufficient quantities could not be isolated to provide conclusive evidence for their structures. The mass spectral evidence suggests, however, that the 2 components may be the trimethylsilyl ether derivatives of the methyl esters of the ocarboxylic acid derivative of thromboxane Bz (i.e., 9,11,15-trihydroxythromba8,13diene-1,20dioic acid), and either 20,21-epoxy-w-homothromboxane Bz or 20-methyl-20-oxothromboxane Bz. The latter compounds could have arisen from the reaction of diazomethane with 20-oxothromboxane Bz. Diazomethane is known to react with aldehydes to give epoxides and methyl ketones [12]. The major ions in the mass spectrum of the products with a C value of 27.4 can be explained in terms of these compounds. Intense ions of the following m/e values can be attributed to the trimethylsilyl ether derivative of the methyl ester of 9,11,15-trihydroxythromba-5,13-diene-1,20-dioic acid:
Fig. 2. Mass spectrum of the trimethylsilyl ether derivative of the methyl ester of the major metabolite isolated after incubation of thromboxane B2 with lung and liver microsomes from Pregnant rabbits.
342
554 (M-90), 529 (M-115; loss of Cle--CZO side chain), 464 (M-2 X 90), 439 (M-115-90), 410 (M-234; cf. mass spectrum of 20-hydroxythromboxane BZ, Fig. 2), 349 (M-115-2 X 90), 345 (Me3SiO’=CH-CH=CH-CH(OSiMe,)(CH,),-CO,Me), 295 (M-234-115), 269 (M-234-141), 256 (base peak; cf. Fig. 2), 217, 191. Ions at the following m/e values can be attributed to the methyl esters of the trimethylsilyl ether derivatives of either the epoxymethano or 20-methyl derivative of 20-oxothromboxane B,: 628 (M), 570 (M-58), 538 (M-90), 529 (M-99, C1s-CZ, side chain), 523 (M-90-15), 448 (M-2 X 90), 394 (M-234), 349 (M-99-2 X 90), 329 (C,,-C,, side chain; cf. ion at m/e 345 in the spectrum of the o-carboxylic acid derivative of thromboxane B,), 295 (M-234-99), 253 (M-234-141), 217, 191. Ions were also observed at m/e 337 and 323, which also occur in the mass spectrum of the trimethylsilyl ether derivative of the methyl ester of thromboxane B,. It is difficult to distinguish between the two derivatives of 20-oxothromboxane B2 on the basis of these results. In support of the 20-methyl derivative, there is an intense ion at m/e 43 (CH,C=O+) but not at m/e 71 (as would be expected from the y-fission of an epoxide). Further experiments are necessary to investigate the oxidation of 20-hydroxyprostaglandins and thromboxanes to the corresponding wcarboxylic acids. Liver microsomes from pregnant rabbits converted thromboxane Bz to a more polar material with an RF value (0.12) identical to that of 20-hydroxythromboxane Bz. Analysis by gas chromatography-mass spectrometry of this material, after methylation and trimethylsilylation, revealed two substances. The major product had a C value and mass spectrum identical to that of the corresponding derivative of 20-hydroxythromboxane BZ as described above. The C value (26.8) of the minor product was 2.1 units higher than that of the trimethylsilyl ether derivative of the methyl ester of thromboxane B,. The mass spectrum of this product was almost identical to that of the trimethylsilyl ether derivative of the methyl ester of 20-hydroxythromboxane B2, with the exception that intense ions were also observed at m/e 364 (M-234-90) and 117 (base peak, Me3SiO’=CH-CHJ). Ions with an m/e value of 117 are also observed in the mass spectra of the trimethylsilyl ether derivatives of 19-hydroxyprostaglandins. Thus it would appear that the minor product is 19-hydroxythromboxane Bz. Metabolism of prostaglandin pregnant rabbits
F,,
analogs
by lung and liver microsomes
from
The induction of enzymes which metabolize prostaglandins during pregnancy could affect the potencies of prostaglandin analogs used as abortifacients, especially those, such as 15-methylprostaglandin F,, and 16,16-dimethylprostaglandin Fzcv [13] which are not substrates for 15-hydroxyprostaglandin dehydrogenase. We therefore investigated the metabolism of some prostaglandin F,, analogs with anti-fertility properties. 15-Methyl [9P-3H]prostaglandin F 20, 16,16dimethyl [9fl-3H]prostaglandin F 2a and 17-phenyl [ 9/3-3H] prostaglandin Fza were incubated in the presence of NADPH (1 mM) with lung and liver microsomes from pregnant rabbits (25 days gestation). The products of these reactions were purified by column and thin-layer chromatography and analyzed by gas chromatography-mass spectrometry as described above. In each
343
MesSi
:
,d
Fig. 3. Fragment ions observed Fzol derivatives (cf. Table I).
in the trimethylsilyl
ether derivatives
of the methyl
esters of prostaglandin
case two bands of radioactive materials were obtained after thin-layer chromatography, the less polar of which was identified by gas chromatography-mass spectrometry as unreacted substrate. The polar products obtained after incubation of 15methylprostaglandin Fzo, with liver microsomes from pregnant rabbits were converted to the trimethylsilyl derivatives of their methyl esters and analyzed by gas chromatographymass spectrometry. Two peaks were obtained with C values which were 2.2 and 2.9 (major product) units higher than that of the corresponding derivative of 15methylprostaglandin Fza! (C v al ue, 24.2). The mass spectra of these two products are quite similar to that of the corresponding derivative of 15-methylprostaglandin F,, (Table I). The only major difference is that ions in which the terminal pentyl group is present (m/e 686 (A!), 671 (M-15), 581 (M-15-90) and 506 (M-2 X 90)) are observed at m/e values 88 units higher than the corresponding ions in the mass spectrum of the trimethylsilyl derivative of the methyl ester of 15-methylprostaglandin F,,. The mass spectral evidence indicates the presence of a hydroxyl group in the terminal pentyl group of each of these derivatives. The C values suggest that the major product (C value, 27.1), is 20-hydroxy-15-methylprostaglandin Fzol, whereas the minor product (C value 26.4) is 19-hydroxy-15-methylprostaglandin F,,. Incubation of 15-methylprostaglandin F,, with lung microsomes from pregnant rabbits gave rise to one major product which had chromatographic properties and a mass spectrum identical to the product with a C value of 27.1 (20-hydroxy-15-methylprostaglandin F,,) described above. Similar results were obtained with 16J6dimethylprostaglandin Fza. Liver microsomes converted this analog to two products with C values which were 2.3 and 3.2 (major product) units greater than that of 16,16dimethylprostaglandin F,, (C value 24.8; trimethylsilyl ether, methyl ester derivatives). The mass spectra of the two products were very similar to that of the corresponding derivative of 16,16-dimethylprostaglandin F,, except that ions (685 (M-15), 610 (M-90) and 595 (M-15-90)) in which the alkyl side chain is retained are observed at m/e values 88 units higher than the corresponding ions of the derivative of 16,16dimethylprostaglandin Fza. In addition, there is an intense ion at m/e 117 (Me&O’= CH--CHJ) in the mass spectrum of the minor product (C value 27.1). These results suggest that the products with C values of 27.1 and 28.0 are the trimethylsilyl ether, methyl ester derivatives of 19-hydroxy16,lGdimethylprostaglandin F 2o1and 20-hydroxy-16,16-dimethylprostaglandin F Za, respectively. Lung microsomes from pregnant rabbits converted 16,16dimethylprostaglandin F,, to a single major product which had chromatog-
IN
LI to
[141.
prostaglandin
20-Hydroxy-16.16
Fga
Fza
lost
Fza
**
dimethyl-
16,16-Dimethylprostaglandin
methylprostaglandin
20-Hydmry-l5-
MASS
THEIR
SPECTRA
Fzor
*
arising
’
3
(430)
’
(520)
(610)
’
(506)
(432)
’
’
(596)
(595)
’
(90
units)
457
256
307
397
457
117
143
243
270
143
256
3
191
191
191
147
243
270
Other
The
ions
in the
(M-b-c)
(M-b-c)
ion. reported
parent
(243)
217
217
217
191
CH(OSiMe3)2
C3H3
217
the those
(OSiMeg)2
to
PROSTAGLANDIN
307
b
conform
OF
2
M-d
patterns
below
ESTERS
in brackets
METHYL
are shown
Fragmentation
mass
THE
397
M--c
underlined.
OF
’
(231)
(321)
:
’
(231)
411
’
are
(321)
411
M-b
peaks
DERIVATIVES
(423) (333)
513
(243)
:
(333)
685
I
(423)
3
:
(507)
(257)
(347)
(437)
513 ’
’
597
(581)
521
(257)
611
2 ’
(347)
686
l
527 (437)
’
583
M-a
ETHER
of trimethylsilanol
base
loss
The
from
(493)
’
M-15
a superscript.
598
by
3.
TRIMETHYLSILYL
(508)
M
in Fig.
is indicated
THE
Ions
OF
METABOLITES
d are defined
OF
THE
molecules
compounds
ions
SOME
15-Methylprostaglandi
similar
methylsilanol
Fragment
AND
IONS
LOGS
I
TABLE
MAJOR
103
129
147
129
147
103
117
for
of tri-
ANA-
literature
number
Fz,
670 (580) (490)
582 (492) (402)
’
’ 2
1 ’
’ ’
’ ’ 3
655 (565)
561
(543)
691
603
’
’
’
511 (421) (331)
513 (423) (333) (243) 513 (423) (333) (243) 513 (423) (333) (243) 511 (421) (331)
’ 2
’ 2
’ 2 3
’ 2 3
1 2 3
(321) (231)
411 (321) (231)
(321)
’ ’
’ ’
’
(295)
385
(295)
’
’
1 ’
’ 2
507 (417) 1 (327)2
565 (475) (385)
477 (387) (297)
173
295
207
217
217
217
217
217
191
191
191
191
-191
* The spectrum for the 19-hydroxy derivative is similar except that the base peak is at m/e 143 and the ion at m/e 103 is less intense. ** The spectrum of the 19-hydroxy derivative is similar except for the presence of an intense ion at m/e 117.
13,14Didehydro-20-hydroxyprostaglandin Fzol ent-13.14~Didehydro-15-epi-20hydroxyprostac&ndin FzOl
13J4Dfdehydroprostaglandin ent-13,14Didehydro-15-epiprostaglandin Fzor
Fzol
706 (616) (526) (436) 648 (558) (468)
17-(Hydroxyphenyl)-18,19,20trinorprostaglandin Fzcl
17-(Methoxyphenyl)-18.19.20trinorprostaglandin Fzol
618 (528)
17-Phenyl-18,19,20_trinorprostaglandin Fza 438 397 347 307 397 359 308 307 397 308 301 301 376 325 147 129 103 413 147 129 103 269 205 180 179 211 147 121
271 255 233 (c)
346
raphic properties and a mass spectrum identical to those described above for 20-hydroxy-16,16-dimethylprostaglandin F,,. Both lung and liver microsomes converted 17-phenyl-18,19,20-trinorprostaglandin F,, to a more polar product. Methylation with diazomethane converted this substance to two products with RF values of 0.20 and 0.29 after thinlayer chromatography on silica gel with ether/methanol (24 : 1, v/v) as solvent. The trimethylsilyl ether derivative of the more polar substance (RF 0.20) had a C value of 29.9, which was 2.3 units higher than that of the corresponding derivative of 17-phenyl-18,19,20-trinorprostaglandin F,,. The mass spectrum (Table I) of this product was similar to that of the trimethylsilyl ether, methyl ester derivative of 17-phenyl-18,19,20-trinorprostaglandin FZa, except that ions (706 (M), 616 (M-90), 526 (M-2 X 90), 436 (M-3 X 90), 565 (M-141; carboxylic acid side chain) and 475 (M-141-90)) which contain the intact alkyl side chain are shifted upwards by 88 mass units, indicating the presence of a trimethylsilyloxy group. The mass spectrum (Table I) of the trimethylsilyl ether derivative (C value 29.5) of the less polar product (RF 0.29) was also similar to that of the corresponding derivative of 17-phenyl-18,19,20-trinorprostaglandin FZa, except that the masses of ions (648 (M), 558 (M-90), 468 (M-2 X 90), 507 (M--141), 417 (M-141-90) and 327 (M-141-2 X 90)) in which the alkyl side chain was retained are 30 units higher, suggesting the presence of a methoxyl group. Since diazomethane would be expected to methylate only phenolic hydroxyl groups, it is presumably the phenyl group of the substrate which was hydroxylated. Thus the product of the reaction catalyzed by lung and liver microsomes is 17-(hydroxyphenyl)-18,19,20trinorprostaglandin F,,. The position of the hydroxyl group in the phenyl ring cannot be determined from the data presented here. We also investigated the metabolism of some 13,14-didehydroprostaglandin F,, analogs which are not substrates for 15-hydroxyprostaglandin dehydrogenase [13]. 13,14-Didehydroprostaglandin F,, was incubated with lung and liver microsomes from pregnant rabbits and the products were purified by column chromatography, converted to the trimethylsilyl ether derivatives of their methyl esters, and analyzed by gas chromatography-mass spectrometry. This analog was converted to one major product by both lung and liver microsomes. This product had a C value 2.8 units higher than the corresponding derivative of the substrate (C value 23.6) and a mass spectrum (Table I) which was consistent with an o-hydroxy metabolite. These results suggest that the product is 13,14-didehydro-20-hydroxyprostaglandin FZa. Similar results were obtained with ent-13,14-didehydro-15-epiprostaglandin Flcu, although this compound was metabolized much more slowly (Table I). Relative rates of hydroxylation thromboxane B2
of prostaglandins,
prostaglandin
analogs and
The relative rates of hydroxylation of radioactively labeled prostaglandins thromboxane B, and the prostaglandin Fza analogs discussed above EZ and F,,, by’ lung and liver microsomes from pregnant rabbits (25 days gestation) were also investigated. Each of these substances (60 PM) was incubated with microsomal fractions in the presence of NADPH (1 mM) for 15 min at 3’7°C. The products of the reactions were extracted and analyzed by thin-layer chromatog-
341
raphy on silica gel with ether/methanol/acetic acid (100 : 6 : 1, v/v) as solvent. The radioactivity in each of the bands was determined by liquid scintillation counting. Alternatively, a fraction con~ning both prost~l~dins and hydroxyprostaglandins was obtained by column chromatography. A column of silicic acid (0.5 g) was eluted with benzene/ethyl acetate (7 : 3, v/v) (5 ml), and acetone (5 ml), each containing 0.5% acetic acid. The material in the acetone fraction was methylated and trimethylsilylated and examined by gas chromatography. The percent conversion to hydroxypros~gl~dins was determined by measuring peak areas. Both the thin-layer chromato~aphic and gas chromatographic methods gave identical results. Table II shows the percentage of each substrate which was hydroxylated. With the exception of 17-phenyl-l&19,20as discussed trinorprostaglandin F,,, this reflects mainly 20-hydroxylation, above. At substrate concentrations of 60 J&I, the percentages of prostaglandin E,, prostaglandin Flol, 13J4didehydroprostaglandin Fzoc and 16,16_dimethylprostaglandin F,, converted to hydroxylated products are approximately the same (Table II) both in lung (about 90%) and liver (about 45%) microsomes. Thromboxane B2 is hydroxylated to an appreciable extent by lung microsomes, whereas ebb-13,14didehydro-15~piprostaglandin Fza, lo-methylprostaglandin F za! and 17-phenyl-18,19,20-t~norprostaglandin F,, are hydroxylated to a much lesser extent. All four of these substances are relatively poor substrates for hydroxylation (
Biochimica et Biophysics Acta, 575 (1979) 0 Elsevier/North-Holland Biomedical Press
335-349
BBA 51480
METABOLISM OF PROSTAGLANDINS, PROSTAGLANDIN ANALOGS AND THROMBOXANE Bz BY LUNG AND LIVER MICROSOMES FROM PREGNANT RABBITS
WILLIAM
S. POWELL
Department of Medicine, McGill University, and the Endocrine Laboratory Hospital, 687 Pine Avenue West, Montreal, Quebec, H3A 1Al (Canada) (Received May lst, 1979) (Revised manuscript received
August
*, Royal Victoria
6th, 1979)
Key words: Prostaglandin metabolism; Thromboxane
B2; (Pregnant rabbit microsome)
Liver microsomes from pregnant rabbits converted prostaglandins FZLY,El, and E, to their ZO-hydroxy metabolites along with smaller amounts of the corresponding 19-hydroxy compounds. Prostaglandins E, and E, were also reduced to prostaglandins F,, and F2&, respectively, and prostaglandin El was isomerized to S-isoprostaglandin E 1. The above products were also identified after incubation of prostaglandins with liver microsomes from non-pregnant rabbits. In this case, the yield of 20-hydroxy metabolites was much lower. Thromboxane B, and a number of prostaglandin Fzo! analogs were also hydroxylated by lung and liver microsomes from pregnant rabbits. The relative rates of hydroxylation by lung microsomes were: prostaglandin Ez = prostaglandin F,, = 16,16dimethylprostaglandin F,, = 13,14-didehydroprostaglandin F,, > thromboxane Bz > 15methylprostaglandin F,, = 17-phenyl-l&19,20-trinorprostaglandin F,, = ent-13,14didehydro-15-epiprostaglandin F,,. Similar results were obtained with liver microsomes except that thromboxane Bz was a relatively poorer substrate for hydroxylation.
Introduction One of the major steps in prostaglandin metabolism is o-oxidation. Prostaglandins Ai [ 1,2] and E, [ 21 have been shown to be converted to their 19- and 20-hydroxy derivatives by guinea pig liver microsomes in the presence of * Please send correspondence
to this address.
336 NADPH or NADH. The hydroxylated products were identified after conversion to the corresponding prostaglandin B, derivatives with base [1,2]. We have recently shown that lung microsomes from pregnant rabbits are very active in converting prostaglandins and some of their metabolites to their 20”hydroxy derivatives [3f. We have measured the rates of o-hydroxylation of prostaglandin F2@ in lung and liver microsomes from rabbits using an assay in which the products were measured by mass spectromet~ [43. Pregnancy was shown to cause 127-fold and 20”fold increases in the 20-hydroxylation of prostaglandin F,, by lung and liver microsomes, respectively. Similar increases in 20”hydroxylation by rabbit lung microsomes were induced by pseudopregnancy and progesterone treatment [4]. We have now investigated the metabolism of a number of prostaglandins, prostaglandin Bz by F 2o1analogs and thromboxane microsomal fractions from lung and liver of pregnant rabbits. Materials and Methods Radioactively labeled prostaglandin El, prostaglandin Ez, prostaglandin FZcu, arachidonic acid and sodium borohydride were purchased from Amersham/ Searle. Unlabeled prostaglandins, prostaglandin analogs and thromboxane B2 were kindly provided by Drs. J.E. Pike of the Upjohn Company and J. Fried (13,14didehydropros~glandin F 2a and ear-13,14-didehydro-15-epiprostaglandin FZO(). Rabbits were purchased from Canadian Hybrid Farms, Stanstead, Quebec, and were sacrificed by inhalation of carbon dioxide. Gas chromatography-mass spectrometry was carried out using an LKB 9000 instrument. 15-Methyl [ 9@-3H]prostaglandin F,, [5]. 16,16-dimethyl[9@-3H]prostaglandin F,, [6] and 17-phenyl-18,19,20-trinor[9/3-3H]prostaglandin Fz, [7] were prepared by reduction of the corresponding prostaglandin E, analogs with sodium [“HI borohydride as described in the literature. [1-14C]Thromboxane B, was synthesized by incubation of [l-14C]arachidonic acid with human platelets as described by Hamberg and Samuelsson [8]. Preparation of microsomal fractions Rabbit lungs were minced in 4 volumes of 0.05 M Tris-HCI, pH 7.5, containing 0.25 M sucrose. The mixture was homogenized in an ice-water bath with a Vir-Tis homogenizer at full speed for six periods of 10 s with 1 min in between to allow for cooling. Rabbit livers were minced in buffer as described above and homogenized for 1 min in a Potter-Elvehjem homogenizer in an ice-water bath. The homogenates were centrifuged at 8000 X g for 10 min at 4°C using a Sorvall model SS-3 centrifuge with an SM 24 rotor. The supernatant was centrifuged at 100 000 X g for 60 min at 2°C using a Beckman model L2-75B ultracentrifuge and a Ti 50 rotor. The pellet was resuspended in 0.05 M Tris-HCl, pH 7.5, with a Potter-Elvehjem homogenizer and centrifuged as described above. The final pellet was suspended in 0.05 M Tris-HCl, pH 7.4 (1.5 volumes/g lung or 1 volume/g liver). Incubation and purification procedures Microsomal fractions (0.5 ml or 5 ml for analytical or preparative experimen@ respectively) were incubated with prostaglandins, prostaglandin analogs or thrombox~e BZ in the presence of NADPH (1 mM) for 45 min at 37” c (15
337
min for analytical experiments). The incubations were terminated by addition of ethanol (2 volumes). The mixtures were filtered and the filtrate was concentrated in vacua to a volume equivalent to that of the original incubation mixture. After dilution with 1 volume of water, the mixtures were acidified to pH 3 with 1 N HCl and extracted with ethyl acetate (4 X 1 volume). The ethyl acetate extracts were washed with water and concentrated to dryness in vacua. The residues were subjected to chromatography on columns of silicic acid (2 g of Bio-Sil HA, Bio-Rad Laboratories, Richmond, CA), which were eluted with diethyl ether/benzene (7 : 3, v/v, 60 ml), ethyl acetate/methanol (9 : 1, v/v, 60 ml) and methanol (30 ml). The radioactive products in the ethyl acetate/ methanol fraction were further purified by thin-layer chromatography on Silica gel G (E. Merck, Darmstadt) with diethyl ether/methanol/acetic acid (100 : 6 : 1, v/v) as solvent. The thin-layer chromatography plates were scanned with a Packard model 7200 radiochromatogram scanner and the radioactive bands were scraped and eluted with ethyl acetate/methanol (9 : 1, v/v). The eluted material was methylated with diazomethane and further purified by thin-layer chromatography with various concentrations of methanol in diethyl ether as solvents. For analytical experiments, the radioactive products in the ethyl acetate extract were analyzed directly by thin-layer chromatography using plates precoated with silica gel (E. Merck, Darmstadt) with diethyl ether/ methanol/acetic acid (100 : 6 : 1, v/v) as solvent. The plates were scanned and the radioactive bands were eluted with methanol/diethyl ether (1 : 1, v/v). The solvent was evaporated and the radioactivity in each of the bands was measured by liquid scintillation counting. Gas chromatography-mass
spectrometry
The purified products from the incubations with prostaglandins E, and E, were converted to their O-methyl oxime derivatives by treatment with 1 mg of methoxylamine hydrochloride (Pierce Chemical Co., Rockford, IL) in pyridine (0.1 ml) at room temperature for 16 h. The solvent was evaporated and the residues were dissolved in benzene and filtered. The O-methyl oxime derivatives, as well as the purified products from the other incubations were then converted to their trimethylsilyl ether derivatives by treatment with Tri-Sil Z (Pierce Chemical Co.) for 5 min at 60°C. The trimethylsilyl ether and Omethyloximetrimethylsilyl ether derivatives were analyzed by gas chromatography-mass spectrometry using a 6 foot column of 1.5% OV-101, and temperatures ranging from 240 to 27O”C, depending on the C value of the product. Retention times were converted to C values by comparison with the retention times of a series of saturated fatty acid methyl esters [9]. The mass spectra of the trimethylsilyl ether derivatives of the methyl esters of some prostaglandin Fzcr analogs and their w-hydroxylated derivatives are given in Table 1. Fragment ions which are observed in the mass spectra of these derivatives are shown in Fig. 3. Results Metabolism of prostaglandins by liver microsomes Prostaglandin F,,. Fig. 1A shows a thin-layer
from pregnant
rabbits
radiochromatogram
obtained
338
after incubation of prostaglandin F,, (20 Erg/ml) with a microsomal fraction (0.5 ml; 13 mg protein/ml) from the liver of a pregnant rabbit (25 days of gestation) in the presence of NADPH (1 mM). After a similar experiment conducted on a larger scale with 250 c(g of substrate. the two bands of radio-
.
PGF2a
10
i
Distance from origin
(cm)
Fig. 1. A. Thin-layer radiochromatogram of the products obtained after incubation of [9@-3Hlprostaglandin Fzol (10 pg) with a microsomal fraction (0.5 ml: 13 mg protein/ml) from the liver of a pregnant rabbit (25 days of gestation) in the presence of NADPH (1 mM). After incubation at 37’C for 15 min the reaction was terminated by the addition of ethanol and the products were extracted. Solvent: diethyl ether/ methanol/acetic acid (100 : 6 : 1, v/v). B. Thin-layer radiochromatogram of the products obtained after incubation of Il-14Clprostaglandin El (250 fig) with a microsomal fraction (5 ml; 28 mg protein/ml) from the liver of a male rabbit in the presence of NADPH (1 mM). In this experiment, the microsomal fraction was resuspended in the boiled (5 min) and filtered supernatant from the 100 000 X g centrifugation. Subsequent experiments in which the microsomal fraction was resuspended in 0.05 M Tris, PH 7.5, gave identical results. After incubation for 60 min at 37°C the products were extracted and purified by column chromatography. Solvent: diethyl ether/methanol/acetic acid (100 : 6 : 1. v/v). C. Thin-layer radiochromatogmm of the products obtained after incubation of a lung microsomal fraction (0.5 ml; 2 mg protein/ml) from a pregnant rabbit (25 days of gestation) with [1-‘4Clthromboxane B2 (10 tig) in the presence of NADPH (1 mM) for 15 min at 37’C. Solvent: diethyl ether/methanol/acetic acid (100 : 6 : 1, v/v).
339
active material were eluted, methylated, and rechromatographed by thin-layer chromatography with methanol/ether as solvent. The purified products were then trimethylsilylated and analyzed by gas chromatography-mass spectrometry. The less polar material (RF 0.21) was identified as unreacted prostaglandin Fzo, on the basis of the C value (24.0) and mass spectrum of the trimethylsilyl ether derivative of its methyl ester. The more polar material (RF 0.05) gave two peaks after gas chromatography, with C values of 26.2 and 27.0. The major product (C value 27.0) had a C value and mass spectrum identical to that which we previously reported for 20-hydroxyprostaglandin Fza, synthesized by lung microsomes from pregnant rabbits [3]. The minor product (C value 26.2) had a C value and mass spectrum identical to that of authentic 19 (R)-19hydroxyprostaglandin F,, . Liver microsomes from male and non-pregnant female rabbits also converted prostaglandin F,, to its 19- and 20-hydroxy metabolites which were identified by gas chromatography-mass spectrometry. In both cases, the overall yield was much lower than with liver microsomes from pregnant rabbits. Comparable amounts of 19-hydroxyprostaglandin F 20 and 20-hydroxyprostaglandin F,, were formed. Prostuglandin E,. The metabolism of prostaglandin El by liver microsomes from pregnant rabbits was somewhat more complicated than that of prostaglandin F,,. [ 1-14C]Prostaglandin E, (500 pg) was incubated with a liver microsomal fraction (10 ml; 10 mg protein/ml) from a pregnant rabbit (21 days of gestation) in the presence of NADPH (1 mM) for 60 min. The radioactive products were extracted and purified by column and thin-layer chromatography. Three bands (RF 0.10, 0.23 and 0.37) of radioactive material were obtained after thin-layer chromatography with ether/methanol/acetic acid (100 : 6 : 1, v/v) as solvent. The material in each band was eluted, methylated and rechromatographed by thin-layer chromatography with methanol/ether as solvent. The purified products were converted to their O-methyloximetrimethylsilyl ether derivatives and analyzed by gas chromatography-mass spectrometry . The most polar product (22% of recovered radioactivity) gave two pairs of 0-methyloxime isomers. The more abundant pair had C values (26.9 and 27.4) and mass spectra which were identical to those which we reported for the Omethyloxime-trimethylsilyl ether derivative of the methyl ester of 20-hydroxyprostaglandin E, [3]. The second pair of isomers had C values of 26.1 and 26.6 and mass spectra nearly identical to those reported for the O-methyloximetrimethylsilyl ether derivatives of the methyl ester of 19-hydroxyprostaglandin E,, which was isolated from semen [lo]. The product (4% of recovered radioactivity) with an RF value of 0.23 with ether/methanolic/acetic acid (100 : 6 : 1, v/v) as solvent did not form an Omethyloxime derivative. It had a C value (24.1) and mass spectrum identical to that of the trimethylsilyl ether derivative of the methyl ester of authentic prostaglandin F,,. The material in the least polar band (RF 0.37; 74% of recovered radioactivity) was methylated and further purified by thin-layer chromatography using ether/methanol (49 : 1, v/v) iis solvent. Two bands of radioactivity (RF 0.23 and 0.33) were detected in a ratio of about 10 : 1. These products were
340
eluted and converted to their 0-methyloxime-trimethylsilyl ether derivatives. They were identified as prostaglandin E, (major product) and B-isoprostaglandin E, (minor product) on the basis of the identity of their C values and mass spectra with those of authentic standards. All of the above products were identified by gas chromatography-mass spectrometry after incubation of prostaglandin E, with liver microsomes from non-pregnant rabbits. Fig. 1B shows a radiochromatogram obtained after incubation of [ l-‘4C]prostaglandin E, (50 pg/ml) with a corresponding fraction from a mature male rabbit. Three bands of radioactive material were obtained which were identified as unreacted prostaglandin E, along with a small amount of 8isoprostaglandin E, (RF 0.37), prostaglandin F,, (RF 0.23) and a 1 : 1 mixture of 19- and 20-hydroxyprostaglandin El. In this case the major products were prostaglandin F,, and B-isoprostaglandin E,. Liver microsomes from male and non-pregnant female rabbits formed much less 20-hydroxyprostaglandin El than those from pregnant rabbits. Prostuglandin E,. Prostaglandin E, was converted by liver microsomal fractions from pregnant rabbits in the presence of NADPH to prostaglandin F20r, 19-hydroxyprostaglandin E, and 20-hydroxyprostaglandin E,, which were identified by comparison of their chromatographic properties and mass spectra with those of authentic standards. NADPH was required for the formation of the major product (20-hydroxyprostaglandin E2). The formation of prostaglandin F,, occurred in the absence of added cofactors, but was stimulated by NADH and NADPH. Metabqlism rabbits
of thromboxane
B2 by lung and liver microsomes
from pregnant
Thromboxane B, was metabolized by lung and liver microsomes from pregnant rabbits in a manner similar to prostaglandins. Fig. 1C shows a radiochromatogram obtained after incubation of [l-14C]thromboxane B, (20 .ug/ml) with a lung microsomal fraction obtained from a pregnant rabbit (25 days of gestation). Three bands of radioactive material were obtained. The products in each band were further purified by thin-layer chromatography as described above and analyzed by gas chromatography-mass spectrometry after conversion to the trimethylsilyl ether derivatives of their methyl esters. The least polar product (RF 0.43) was identified by chromatography and mass spectrometry as thromboxane BZ. The trimethylsilyl ether derivative of the methyl ester of the most polar product (RF 0.12) had a C value of 27.5, 2.8 units higher than that of the corresponding derivative of thromboxane B,. By comparison, the substitution of a trimethylsilyloxy group in either the 19- or the 20-position of trimethylsilyl ether derivatives of prostaglandin methyl esters augments their C values by about 2.0 [l] or 2.8-3.0 [1,3] units, respectively. Because of the similarity of the structure of thromboxane B2 with those of prostaglandins, this suggests that the product with an RF value of 0.12 is 20-hydroxythromboxane B2. The mass spectrum (Fig. 2) of the trimethylsilyl ether derivative of the methyl ester of this product confirms the presence of a hydroxyl group in the terminal pentyl group of the alkyl side chain. Intense ions are observed at m/e 598 (M90), 508 (M-2X 90), 454 (M-234), 439 (M-159-90), 389 (Me,SiO’=
341
CH-CH=CH-CH(OSiMe3)-(CH,)4-CH~OSiMe3), 349 (M-159-2 X 90), 323, 313 (M-234-141), 295 (M-234-159), 256 (base peak; Me,SiO+JH=CHCH,-CH= CH-( CH2)3-COzMe+), 217 (Me,SiO’= CH-CH= CH-OSiMe3), 191 (Me,SiO’=CH--OSiMe,), 129, and 103. This mass spectrum is very similar to that of the trimethylsilyl ether derivative of the methyl ester of thromboxane Bz, except that ions (m/e 598, 508, 454, 389 and 313) in which the alkyl side chain is intact have m/e values 88 mass units higher than the corresponding ions of the thromboxane B2 derivative. The trimethylsilyl ether derivative of the second product (RF 0.17) had a C value of 27.4, which, by analogy with prostaglandins [ll] would be consistent with the presence of an o-carboxylic acid group. The mass spectrum of the material in this peak suggested the presence of two components with identical retention times. The amounts of these compounds which were formed varied from one experiment to the other, and in many cases they were not detectable. Sufficient quantities could not be isolated to provide conclusive evidence for their structures. The mass spectral evidence suggests, however, that the 2 components may be the trimethylsilyl ether derivatives of the methyl esters of the ocarboxylic acid derivative of thromboxane Bz (i.e., 9,11,15-trihydroxythromba8,13diene-1,20dioic acid), and either 20,21-epoxy-w-homothromboxane Bz or 20-methyl-20-oxothromboxane Bz. The latter compounds could have arisen from the reaction of diazomethane with 20-oxothromboxane Bz. Diazomethane is known to react with aldehydes to give epoxides and methyl ketones [12]. The major ions in the mass spectrum of the products with a C value of 27.4 can be explained in terms of these compounds. Intense ions of the following m/e values can be attributed to the trimethylsilyl ether derivative of the methyl ester of 9,11,15-trihydroxythromba-5,13-diene-1,20-dioic acid:
Fig. 2. Mass spectrum of the trimethylsilyl ether derivative of the methyl ester of the major metabolite isolated after incubation of thromboxane B2 with lung and liver microsomes from Pregnant rabbits.
342
554 (M-90), 529 (M-115; loss of Cle--CZO side chain), 464 (M-2 X 90), 439 (M-115-90), 410 (M-234; cf. mass spectrum of 20-hydroxythromboxane BZ, Fig. 2), 349 (M-115-2 X 90), 345 (Me3SiO’=CH-CH=CH-CH(OSiMe,)(CH,),-CO,Me), 295 (M-234-115), 269 (M-234-141), 256 (base peak; cf. Fig. 2), 217, 191. Ions at the following m/e values can be attributed to the methyl esters of the trimethylsilyl ether derivatives of either the epoxymethano or 20-methyl derivative of 20-oxothromboxane B,: 628 (M), 570 (M-58), 538 (M-90), 529 (M-99, C1s-CZ, side chain), 523 (M-90-15), 448 (M-2 X 90), 394 (M-234), 349 (M-99-2 X 90), 329 (C,,-C,, side chain; cf. ion at m/e 345 in the spectrum of the o-carboxylic acid derivative of thromboxane B,), 295 (M-234-99), 253 (M-234-141), 217, 191. Ions were also observed at m/e 337 and 323, which also occur in the mass spectrum of the trimethylsilyl ether derivative of the methyl ester of thromboxane B,. It is difficult to distinguish between the two derivatives of 20-oxothromboxane B2 on the basis of these results. In support of the 20-methyl derivative, there is an intense ion at m/e 43 (CH,C=O+) but not at m/e 71 (as would be expected from the y-fission of an epoxide). Further experiments are necessary to investigate the oxidation of 20-hydroxyprostaglandins and thromboxanes to the corresponding wcarboxylic acids. Liver microsomes from pregnant rabbits converted thromboxane Bz to a more polar material with an RF value (0.12) identical to that of 20-hydroxythromboxane Bz. Analysis by gas chromatography-mass spectrometry of this material, after methylation and trimethylsilylation, revealed two substances. The major product had a C value and mass spectrum identical to that of the corresponding derivative of 20-hydroxythromboxane BZ as described above. The C value (26.8) of the minor product was 2.1 units higher than that of the trimethylsilyl ether derivative of the methyl ester of thromboxane B,. The mass spectrum of this product was almost identical to that of the trimethylsilyl ether derivative of the methyl ester of 20-hydroxythromboxane B2, with the exception that intense ions were also observed at m/e 364 (M-234-90) and 117 (base peak, Me3SiO’=CH-CHJ). Ions with an m/e value of 117 are also observed in the mass spectra of the trimethylsilyl ether derivatives of 19-hydroxyprostaglandins. Thus it would appear that the minor product is 19-hydroxythromboxane Bz. Metabolism of prostaglandin pregnant rabbits
F,,
analogs
by lung and liver microsomes
from
The induction of enzymes which metabolize prostaglandins during pregnancy could affect the potencies of prostaglandin analogs used as abortifacients, especially those, such as 15-methylprostaglandin F,, and 16,16-dimethylprostaglandin Fzcv [13] which are not substrates for 15-hydroxyprostaglandin dehydrogenase. We therefore investigated the metabolism of some prostaglandin F,, analogs with anti-fertility properties. 15-Methyl [9P-3H]prostaglandin F 20, 16,16dimethyl [9fl-3H]prostaglandin F 2a and 17-phenyl [ 9/3-3H] prostaglandin Fza were incubated in the presence of NADPH (1 mM) with lung and liver microsomes from pregnant rabbits (25 days gestation). The products of these reactions were purified by column and thin-layer chromatography and analyzed by gas chromatography-mass spectrometry as described above. In each
343
MesSi
:
,d
Fig. 3. Fragment ions observed Fzol derivatives (cf. Table I).
in the trimethylsilyl
ether derivatives
of the methyl
esters of prostaglandin
case two bands of radioactive materials were obtained after thin-layer chromatography, the less polar of which was identified by gas chromatography-mass spectrometry as unreacted substrate. The polar products obtained after incubation of 15methylprostaglandin Fzo, with liver microsomes from pregnant rabbits were converted to the trimethylsilyl derivatives of their methyl esters and analyzed by gas chromatographymass spectrometry. Two peaks were obtained with C values which were 2.2 and 2.9 (major product) units higher than that of the corresponding derivative of 15methylprostaglandin Fza! (C v al ue, 24.2). The mass spectra of these two products are quite similar to that of the corresponding derivative of 15-methylprostaglandin F,, (Table I). The only major difference is that ions in which the terminal pentyl group is present (m/e 686 (A!), 671 (M-15), 581 (M-15-90) and 506 (M-2 X 90)) are observed at m/e values 88 units higher than the corresponding ions in the mass spectrum of the trimethylsilyl derivative of the methyl ester of 15-methylprostaglandin F,,. The mass spectral evidence indicates the presence of a hydroxyl group in the terminal pentyl group of each of these derivatives. The C values suggest that the major product (C value, 27.1), is 20-hydroxy-15-methylprostaglandin Fzol, whereas the minor product (C value 26.4) is 19-hydroxy-15-methylprostaglandin F,,. Incubation of 15-methylprostaglandin F,, with lung microsomes from pregnant rabbits gave rise to one major product which had chromatographic properties and a mass spectrum identical to the product with a C value of 27.1 (20-hydroxy-15-methylprostaglandin F,,) described above. Similar results were obtained with 16J6dimethylprostaglandin Fza. Liver microsomes converted this analog to two products with C values which were 2.3 and 3.2 (major product) units greater than that of 16,16dimethylprostaglandin F,, (C value 24.8; trimethylsilyl ether, methyl ester derivatives). The mass spectra of the two products were very similar to that of the corresponding derivative of 16,16-dimethylprostaglandin F,, except that ions (685 (M-15), 610 (M-90) and 595 (M-15-90)) in which the alkyl side chain is retained are observed at m/e values 88 units higher than the corresponding ions of the derivative of 16,16dimethylprostaglandin Fza. In addition, there is an intense ion at m/e 117 (Me&O’= CH--CHJ) in the mass spectrum of the minor product (C value 27.1). These results suggest that the products with C values of 27.1 and 28.0 are the trimethylsilyl ether, methyl ester derivatives of 19-hydroxy16,lGdimethylprostaglandin F 2o1and 20-hydroxy-16,16-dimethylprostaglandin F Za, respectively. Lung microsomes from pregnant rabbits converted 16,16dimethylprostaglandin F,, to a single major product which had chromatog-
IN
LI to
[141.
prostaglandin
20-Hydroxy-16.16
Fga
Fza
lost
Fza
**
dimethyl-
16,16-Dimethylprostaglandin
methylprostaglandin
20-Hydmry-l5-
MASS
THEIR
SPECTRA
Fzor
*
arising
’
3
(430)
’
(520)
(610)
’
(506)
(432)
’
’
(596)
(595)
’
(90
units)
457
256
307
397
457
117
143
243
270
143
256
3
191
191
191
147
243
270
Other
The
ions
in the
(M-b-c)
(M-b-c)
ion. reported
parent
(243)
217
217
217
191
CH(OSiMe3)2
C3H3
217
the those
(OSiMeg)2
to
PROSTAGLANDIN
307
b
conform
OF
2
M-d
patterns
below
ESTERS
in brackets
METHYL
are shown
Fragmentation
mass
THE
397
M--c
underlined.
OF
’
(231)
(321)
:
’
(231)
411
’
are
(321)
411
M-b
peaks
DERIVATIVES
(423) (333)
513
(243)
:
(333)
685
I
(423)
3
:
(507)
(257)
(347)
(437)
513 ’
’
597
(581)
521
(257)
611
2 ’
(347)
686
l
527 (437)
’
583
M-a
ETHER
of trimethylsilanol
base
loss
The
from
(493)
’
M-15
a superscript.
598
by
3.
TRIMETHYLSILYL
(508)
M
in Fig.
is indicated
THE
Ions
OF
METABOLITES
d are defined
OF
THE
molecules
compounds
ions
SOME
15-Methylprostaglandi
similar
methylsilanol
Fragment
AND
IONS
LOGS
I
TABLE
MAJOR
103
129
147
129
147
103
117
for
of tri-
ANA-
literature
number
Fz,
670 (580) (490)
582 (492) (402)
’
’ 2
1 ’
’ ’
’ ’ 3
655 (565)
561
(543)
691
603
’
’
’
511 (421) (331)
513 (423) (333) (243) 513 (423) (333) (243) 513 (423) (333) (243) 511 (421) (331)
’ 2
’ 2
’ 2 3
’ 2 3
1 2 3
(321) (231)
411 (321) (231)
(321)
’ ’
’ ’
’
(295)
385
(295)
’
’
1 ’
’ 2
507 (417) 1 (327)2
565 (475) (385)
477 (387) (297)
173
295
207
217
217
217
217
217
191
191
191
191
-191
* The spectrum for the 19-hydroxy derivative is similar except that the base peak is at m/e 143 and the ion at m/e 103 is less intense. ** The spectrum of the 19-hydroxy derivative is similar except for the presence of an intense ion at m/e 117.
13,14Didehydro-20-hydroxyprostaglandin Fzol ent-13.14~Didehydro-15-epi-20hydroxyprostac&ndin FzOl
13J4Dfdehydroprostaglandin ent-13,14Didehydro-15-epiprostaglandin Fzor
Fzol
706 (616) (526) (436) 648 (558) (468)
17-(Hydroxyphenyl)-18,19,20trinorprostaglandin Fzcl
17-(Methoxyphenyl)-18.19.20trinorprostaglandin Fzol
618 (528)
17-Phenyl-18,19,20_trinorprostaglandin Fza 438 397 347 307 397 359 308 307 397 308 301 301 376 325 147 129 103 413 147 129 103 269 205 180 179 211 147 121
271 255 233 (c)
346
raphic properties and a mass spectrum identical to those described above for 20-hydroxy-16,16-dimethylprostaglandin F,,. Both lung and liver microsomes converted 17-phenyl-18,19,20-trinorprostaglandin F,, to a more polar product. Methylation with diazomethane converted this substance to two products with RF values of 0.20 and 0.29 after thinlayer chromatography on silica gel with ether/methanol (24 : 1, v/v) as solvent. The trimethylsilyl ether derivative of the more polar substance (RF 0.20) had a C value of 29.9, which was 2.3 units higher than that of the corresponding derivative of 17-phenyl-18,19,20-trinorprostaglandin F,,. The mass spectrum (Table I) of this product was similar to that of the trimethylsilyl ether, methyl ester derivative of 17-phenyl-18,19,20-trinorprostaglandin FZa, except that ions (706 (M), 616 (M-90), 526 (M-2 X 90), 436 (M-3 X 90), 565 (M-141; carboxylic acid side chain) and 475 (M-141-90)) which contain the intact alkyl side chain are shifted upwards by 88 mass units, indicating the presence of a trimethylsilyloxy group. The mass spectrum (Table I) of the trimethylsilyl ether derivative (C value 29.5) of the less polar product (RF 0.29) was also similar to that of the corresponding derivative of 17-phenyl-18,19,20-trinorprostaglandin FZa, except that the masses of ions (648 (M), 558 (M-90), 468 (M-2 X 90), 507 (M--141), 417 (M-141-90) and 327 (M-141-2 X 90)) in which the alkyl side chain was retained are 30 units higher, suggesting the presence of a methoxyl group. Since diazomethane would be expected to methylate only phenolic hydroxyl groups, it is presumably the phenyl group of the substrate which was hydroxylated. Thus the product of the reaction catalyzed by lung and liver microsomes is 17-(hydroxyphenyl)-18,19,20trinorprostaglandin F,,. The position of the hydroxyl group in the phenyl ring cannot be determined from the data presented here. We also investigated the metabolism of some 13,14-didehydroprostaglandin F,, analogs which are not substrates for 15-hydroxyprostaglandin dehydrogenase [13]. 13,14-Didehydroprostaglandin F,, was incubated with lung and liver microsomes from pregnant rabbits and the products were purified by column chromatography, converted to the trimethylsilyl ether derivatives of their methyl esters, and analyzed by gas chromatography-mass spectrometry. This analog was converted to one major product by both lung and liver microsomes. This product had a C value 2.8 units higher than the corresponding derivative of the substrate (C value 23.6) and a mass spectrum (Table I) which was consistent with an o-hydroxy metabolite. These results suggest that the product is 13,14-didehydro-20-hydroxyprostaglandin FZa. Similar results were obtained with ent-13,14-didehydro-15-epiprostaglandin Flcu, although this compound was metabolized much more slowly (Table I). Relative rates of hydroxylation thromboxane B2
of prostaglandins,
prostaglandin
analogs and
The relative rates of hydroxylation of radioactively labeled prostaglandins thromboxane B, and the prostaglandin Fza analogs discussed above EZ and F,,, by’ lung and liver microsomes from pregnant rabbits (25 days gestation) were also investigated. Each of these substances (60 PM) was incubated with microsomal fractions in the presence of NADPH (1 mM) for 15 min at 3’7°C. The products of the reactions were extracted and analyzed by thin-layer chromatog-
341
raphy on silica gel with ether/methanol/acetic acid (100 : 6 : 1, v/v) as solvent. The radioactivity in each of the bands was determined by liquid scintillation counting. Alternatively, a fraction con~ning both prost~l~dins and hydroxyprostaglandins was obtained by column chromatography. A column of silicic acid (0.5 g) was eluted with benzene/ethyl acetate (7 : 3, v/v) (5 ml), and acetone (5 ml), each containing 0.5% acetic acid. The material in the acetone fraction was methylated and trimethylsilylated and examined by gas chromatography. The percent conversion to hydroxypros~gl~dins was determined by measuring peak areas. Both the thin-layer chromato~aphic and gas chromatographic methods gave identical results. Table II shows the percentage of each substrate which was hydroxylated. With the exception of 17-phenyl-l&19,20as discussed trinorprostaglandin F,,, this reflects mainly 20-hydroxylation, above. At substrate concentrations of 60 J&I, the percentages of prostaglandin E,, prostaglandin Flol, 13J4didehydroprostaglandin Fzoc and 16,16_dimethylprostaglandin F,, converted to hydroxylated products are approximately the same (Table II) both in lung (about 90%) and liver (about 45%) microsomes. Thromboxane B2 is hydroxylated to an appreciable extent by lung microsomes, whereas ebb-13,14didehydro-15~piprostaglandin Fza, lo-methylprostaglandin F za! and 17-phenyl-18,19,20-t~norprostaglandin F,, are hydroxylated to a much lesser extent. All four of these substances are relatively poor substrates for hydroxylation (
