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The metabolism of pregnenolone and 17~-hydroxyprogesterone by homogenates of human term placentas

The hmnan placenta secretes large amounts of progesterone and estrogens at tile end of pregnancy. To determine the activities of certain enzymes involved in the synthesis of these steroids, homogenates of term placenta were incubated with [4-14C]pregne - nolone, a precursor of steroid hormones, and I7a-hydroxy [4-1'~C] progesterone, a postu- lated intermediate in the biosynthesis of androgens and estrogens.

Term placentas, obtained within 15 rain of delivery, were homogenized at 4 ° for I min in a Waring blendor with the sucrose-nicot inamide-phosphate buffer described by RYAN ]. The homogenate was centrifuged at 650 ;.: g for 20 rain to remove nuclei and cellular debris and the resulting supernatant fraction was used for the incubation. Each incubation vessel contained the equivalent of z2 g (wet wt.) of tissue, IO t~moles NAD, 2o/,moles ATP, 5 t *moles NADP, 5o t~moles glucose 6-phosphate, and IO Kornberg units of glucose-6-t)hosphate dehydrogenase (EC 1.1.1.49 ). To four vessels was added I ~C of I4-14C~pregnenolone (Nuclear Chicago, 19.77 mC/mmole), found to be free of contaminants by thin-layer and gradient-elution partition chro- matography. To another four vessels were added I . lO G disintegrations/min of I7=- hydroxy[4-14C!progesterone (New England Nuclear Co., 14 mC/mmole), found tree of contaminants by thin-layer chromatography. Of each set of four, two vessels were incubated for I h in a Dubnoff shaking incubator at 37 ° in air, and two were im- mediately frozen to serve as controls. The incubation mixtures were extracted three times with 5 vol. of chloroform. The extract was taken to dryness under reduced pressure, the residue was dissolved in peroxide-free diethyl ether, and extracted three times with o.5 vol. of I N NaOH. The alkaline extracts were pooled and backwashed once with ether and the ether fractions were pooled. The ether fraction was washed with water to neutrality and the solvent evaporated to yield the neutral fraction. The base phase was adjusted to pH 8.5-9.o, extracted three times with equal volumes of chloroform and the solvent evaporated to yield the phenolic fraction.

Steroids were characterized by thin-layer chromatography, as described by LISBOA .aND DICZFALUSY 2. The plates were scanned for radioactivity with a modified Nuclear Chicago Actigraph II . Further purification was achieved by gradient-elution partition chromatography on a celite column, with 9 ° % methanol as stationary phase and 2,2,4-trimethyl pentane as mobile phase with a gradient of 1,2-dichloroethane a.

The phenolic fractions were chromatographed in the benzene-methanol (85:I5, v/v) thin-layer system at full saturation, with authentic standards. No radioactivity was found associated with the estrogens. Preliminary experiments with this same incubation system, however, had shown almost quantitative conversion of testo- sterone to estradiol.

The neutral fraction of the pregnenolone incubation was chromatographed in the benzene-methanol (85:15, v/v) system at full saturation and four radioactive peaks became evident upon scanning. The first had an RE of o.62, identical with authentic progesterone. I t was rechromatographed on System A of LISBOA and again moved as a single peak, with an RE of o.59, identical with progesterone. This was eluted and recrystallized to constant specific activity with authentic progesterone. The yield, based on the specific acti~dty of the crystals, was 5.8 % of the initial radio- activity of the pregnenolone.

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666 SHORT C O M M U N I C A T I O N S

The second peak had a n R F of 0.43. Pregnenolone, 2oa-hydroxy-pregn-4-ene- 3-one, I7c~-hydroxyprogesterone and dehydroepiandrosterone have similar Rv's in this system. This peak was eluted and subjected to gradient-elution partition chromatog- raphy. A single radioactive peak that corresponded to carrier 2oa-hydroxy-pregn- 4-ene-3-one was found. The specific activities of the fractions of this peak were constant. The fractions were pooled and recrystatlized with authentic 2oa-hydroxy- pregn-4-ene-3-one to constant specific activity. The peak of carrier dehydroepiandro- sterone appears four fractions before the radioactive peak and calculation of its specific activity differed for each one of the tubes. When an aliquot of this peak was recrystallized with authentic dehydroepiandrosterone, the radioactivity remained in the mother liquor. The yield of 2oa-hydroxy-pregn-4-ene-3-one was 1.7 % of the initial pregnenolone for each incubation flask.

The third peak of the thin-layer chromatogram had an RF of o.27. On elution and rechromatography on System A it had an RF of o-37 with some tailing. On the chloroform-ethyl ether (6o:4o, v/v) system the RE was o.26, identical with i7c~,2o fl- dihydroxy-pregn-4-ene-3-one.

The fourth peak, with an RE of o.2o, was subsequently run in several thin-layer chromatographic systems, but could not be identified. I t is not ITa,2oc~-dihydroxy- pregn-4-ene-3-one, nor I7a,I6a-dihydroxy-pregn-4-ene-3-one.

The neutral fraction of the I7a-hydroxyprogesterone incubation was ehromato- graphed on the benzene-methanol (85:15, v/v) system and two radioactive peaks were detected. The first had the RE of authentic I7a-hydroxyprogesterone (o.43) and, when rechromatographed on System E of LLqBOA ran again as a single peak with the RE of I7a-hydroxyprogesterone.

The second peak was reehromatographed in the chloroform-diethyl ether (6o : 40, v/v) thin-layer system that separates the 2o-alpha and beta hydroxy isomers. The radioactive peak had an RE identical to that of authentic I7a,2oc~-dihydroxy-pregn- 4-ene-3-one. This radioactive peak was eluted and recrystallized to constant specific activity with carrier ITcq2o~-dihydroxy-pregn-4-ene-3-one. The yield based on the specific activities of the final crystals, corrected for losses during isolation, was 33.3 % of the initial I7a-hydroxyprogesterone.

The products of the incubations with their relative yields are shown in Table I. These values for the conversions have been calculated on the basis of the specific activities on recrystallization and are corrected for losses during purification. The largest conversion was from pregnenolone to progesterone, which indicates 3fi-ol de- hydrogenase activity *. About one-third of the progesterone formed was reduced at

T A B L E I

P R O D U C T S OF M E T A B O L I S M OF F I O M O G E N A T E S OF T E R M P L A C E N T A

Substratc Products

2o~-Hydroxy- ZT~,2o~-Hvdrox3,- Progesterone progesterone progesterone

P r e g n e n o l o n e 14.3 3.7

1 7 a - H y d r o x y p r o g e s t e r o n e - - - - 33.3

* T h e p r o d u c t of 20 r e d u c t i o n s e e m s in t h i s case t h e I7~,2ofl-dihydroxyprogesterone i s o m e r .

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SHORT COMMUNICATIONS 667

position 20 to yield the a isomer 5. Hydroxylat ion at position 17 occurs to a much smaller extent 6 and in this experiment there was some evidence of the I7-hydro- xylation of progesterone. An unfruitful search was made for I7~-hydroxypregne- nolone. No products of reduction of the A4,3-keto unsaturated system were found.

Neither androgens nor estrogens were found as labeled products of these incu- bations. Since a conversion of as little as 0.35 % of the substrate to androgens or estrogens could have been detected easily in these experiments, this indicates that the I7,2o-desmolase (I7a-hydroxypregnene CI7-C2o lyase) activity in human placental homogenates is quite small or completely absent. LITTLE et al. 7 were able to isolate radioactively labeled androstenedione after incubating labeled progesterone with a "soluble" placental preparation, only when testosterone was added before the incu- bation. WARREN AND CHEATUM 8 have reported for androstenedione a yield of o . I I % from progesterone in an experiment similar to ours, but found no synthesis of estrogens despite the known high efficiency of this system in converting androgens to estrogens.

When I7a-hydroxyprogesterone is used as substrate, the product of 20 reduction is the a isomer in good yield. The fi isomer was not found despite a careful search. When pregnenolone is used as substrate, the dihydroxylated product appears to be the I7e,2o fl isomer, although the evidence is not conclusive. We were unable to find I6-hydroxylated products of either pregnenolone or progesterone.

These results indicate that the enzyme systems that cleave the side chain of pregnenolone, progesterone, or I7a-hydroxyprogesterone to form androgens and estrogens are not active in homogenates of term placenta. This supports the hypothesis that fetal or maternal androgens are the main sources of placental estrogens 9.

This work was aided by grants from the Charles A. and Marjorie King Fund, the Lalor Foundation, The Association for the Aid of Crippled Children, and Grant HD-6, National Insti tute of Child Health and Human Development, U.S. Public Health Service. L.S. is a Fellow of the Population Council, New York.

Department of Biological Chemistry, Harvard Medical School, and the Research Laboratories of the Boston Lying-in Hospital,

Boston, Mass. (U.S.A.)

LuIs SOBREVILLA D W A I N H..AGERMAN

C L A U D E V I L L E E

1 K. J. RYAN, J. Biol. Chem., 234 (I959) 268. B. P. LISBOA AND E. DICZFALUSY, Acta Endocrinol., 4 ° ( i962) 60.

3 L. ENGEL, Anal. Biochem., 2 (1961) I I 4. 4 j . ZANDER, in C. E . W T. LLOYD, Recent Progr. Endocrinol. of Reproduction, A c a d e m i c Press ,

N e w Y o r k , 1959, p. 255. 5 B. LITTLE, J. DIMARTINIS AND ]3. NYHOLM, Acta Endocrinol., 3 ° (1959) 53 o. 6 B. LITTLE AND A. SHAW, Ac/a Endocrinol., 36 ( I96 I ) 455- 7 B. LITTLE, A. SHAW AND I~. PURDY, Acta Endocrinol., 43 (I963) 51o. s j . C. WARREN AND S. G. CHEATUM, Can. f . Biochem., 42 (1964) 143. 9 E. DICZFALUSY, Federation Proc., 23 (1964) 79I .

Received August 3rd, I964

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