Metabolism in vitro of androstenedione and dehydroepiandrosterone by normal ovarian tissue

4.50

SHORT

COMMUNICATIONS

BnA 5323.5

Metabolism in vitro of androstenedione and dehydroepiandrosterone normal ovarian tissue

by

It has been amply documented that the biosynthesis of androgens and estrogens in adrenals and gonads proceeds from both As and 44 C,, precursors and that pregnenolone (representing the “As pathway”) is the preferred substrate under the various conditions employed in vitro. It has not been equally clearly established whether A5 or A4 substrates of the C,, type hold the same relationship as far as metabolism and biosynthesis in vitro of C,, compounds is concerned. Previous studies with dehydroepiandrosterone and androstenedione as substrates have either used single precursors or quantities of two precursors that were not equimolar and therefore not comparable. To investigate this question, we have carried out an incubation study on ovarian tissue from a healthy 36-year-old nullipara with regular menstrual cycles and dysmenorrhea (indicating consistent ovulation). The tissue was removed on cycle Day 20; endometrium obtained at the same time showed a progestational effect corresponding to Day ZI of a z&day cycle. The section tissue weighed 1.02 g and contained no follicular cysts or luteal tissue.

of ovarian

The incubation flask was prepared by evaporating the substrates (freshly chromatographed [4-14C]androstenedione (I &, 6.4 pg) and [7&H]dehydroepiandrosterone (IO & 6.4 pg)) as a thin film on its bottom. The chilled ovarian tissue was minced with sharp scissors, suspended in 2 vol. of Krebs-Ringer phosphate buffer (pH 7.4), and preweighed cofactors (NAD+, NADH, NADPf, NADPH, ATP; approx. IO ,uM each) were added. The mixture was incubated with shaking at 37.5” for 30 min in an atmosphere of O,-CO, (95 : 5, v/v). The reaction was stopped with acetone, and the mixture was stored temporarily in the deep freeze. Presently, the solvent was filtered off and the solids extracted 3 times with additional acetone; the combined

solutions were evaporated under N, in vacuum. The watery remainder with ethyl acetate, which was evaporated in similar fashion. The residue in 90% methanol and extracted 3 times with light petroleum (b.p. backwashing. The light petroleum was discarded. The methanol solution

was extracted was dissolved 30-60”) with was partially

evaporated and re-extracted with ethyl acetate, which was dried with Na,SO, and evaporated under N, in vacuum. This residue was chromatographed as outlined in Fig. I, employing systems and techniques described previouslyl. The chromatographic papers were scanned in a 4-n Atomic accessory scanner, and the radioactive zones were cut out and eluted. Identification of the various radioactive substances was carried out by recrystallization to constant specific activity with authentic carrier steroids according to the methods and criteria of AXELROD et aL2. At least 3 crystallizations, but in many cases as many as 5, were carried out. Table I lists the compounds isolated and the proof of their radiochemical purity. The total amount of purified 3H radioactivity recovered was 63%, that of 14C radioactivity was 77%. No major unidentified areas of radioactivity were encountered; therefore this probably represents methodological losses. In addition to the reported metabolites, specific search was made for r6-hydroxyandrostenedione, rg-aldoandrostenedione, As-androstene-3/3,17P-diol, and estriol. No radioactivity corresponding to these substances was found. This indicates either that they were Biochim.

Biophys.

A&z,

187 (1969)450-453

SHORT COMMUNICATIONS

4.51

Extract

-r

I 19-Hydroxyandrostenedione

Benzene-formamide (1:l) 18 h, estradiol marker I I Overflow

I Methylcyclohexanepropylene glycol (1:l) 4 days, testosterone marker

Toluene-

propylene glycol ( 1: 1) 6 h, e&radio1 marker

I

6/3-Hydroxy&drostenedione

Estradiol

I

I

Dehydroepiandrosterone

19-Nortestosterone

L

OW,rflow

Testosterone

Estrone

Methylcyclohexanepropylene glycol (1:l) 24 h, androstenedione marker

1 3/j-Hydroxyandrosta-5,16-diene

Androstenkdione Fig.

I

not biosynthesized under the conditions of this incubation or that they were synthesized but were rapidly transformed into other metabolites. The percent conversion of the 2 substrates into various metabolites is shown in Table II. The percentages are derived by calculating the total disint./min in each TABLE

I

RADIOCHEMICAL

PURITY

Compound

OF ISOLATED

COMPOUNDS

Total disint. lmin

Recrystallizations

3H Dehydroepiandrosterone 3b-Hydroxyandrosta-5, I 6-diene Androstenedione ~g-Hydroxyandrostenedione $9-Hydroxyandrostenedione Testosterone ~g-Nortestosterone Estrone Estradiol

1%

II43000 12600

Iro400000 (49 500)

*

-

-

12I0000

I

1%

-

040 000

122000 2

5920 65 200

I 230 48 700

255 000

50000

12800

341000

* SH counts in successive recrystallizations the data establishing radiochemical purity.

8940

77 000

1%

115000

2260

7960

go10

aH

117000

47 600 486000 44 300 I540000

n

n-r

SH

-

per mg)

(disint.lmin

n-2

030

156 6590 2 450 1750 16goo

-

2 150

1040 000

122 000

_*

1970 I 160 47400 12 800

8770 75 500

I45 6330 2 490 1790 16700

=H

1%

110000

-

ggo000

116000

_*

2 070

1930 I 180

46 800 12 300 8 880

77 600

143 6520 2 500 I 820 17000

did not meet the * 5% criterion, and are therefore omitted from

Biochim.

Biophys.

Acta,

187 (1969) 450-453

SHORT COMMUNICATIONS

4.52 TABLE II CONVERSION

OF

SUBSTRATES

AS

PERCENT

Conzpound Dehydroepiandrosterone 3B-Hydroxyandrosta-5,16-diene Androstenedione 19-Hydroxyandrostenedione 6b-Hydroxyandrostenedione Testosterone 19-Nortestosterone Estrone Estradiol

RECOVERED

RADIOACTIVITY

y0 of $H dehydroepiandrosterone recovered 8.22

0.09 74.57 0.34 3.50 1.83 0.31 11.08

O/’of 14Candrostenedione recovered

71.72 0.47 0.34 3.54 2.95 0.53 20.12

compound from the average specific activity of the final 3 recrystallizations and the weight of added carrier. The total purified activity in each label was considered as IOO%, and the percentages in the various metabolites calculated accordingly. In the relatively short period of incubation, many reactions proceeded to substantial extent. It must be kept in mind that the amount of substrate added was 6.4 ,ug in each instance; the endogenous nonradioactive substrate available is unknown but is likely to be negligible, based on known measurements of endogenous steroid levels in steroidogenic tissue3+. Two compounds not previously described in ovarian incubations were isolated: a minor amount of the Al6 dehydration product of dehydroepiandrosterone (o.og”h) and a substantial amount of an important metabolite, Ig-nortestosterone. In looking at the major end-product, estradiol, it can be seen that androstenedione was a substantially better substrate than dehydroepiandrosterone. However, the isotope ratio of the intermediate, testosterone, at the end of the incubation is unity. This suggests that there was an initially rapid conversion of androstenedione to testosterone to estradiol at a time when relatively little 3H-labeled material was entering the system. At that moment, the 14C: 3H ratio of testosterone should also have been greater than I. However, this material was apparently further converted to Ig-nortestosterone and estrogens. Androstenedione substrate available later in time had an isotope ratio approaching unity. This hypothesis is strengthened by the relatively similar isotope ratios of Iq-nortestosterone, estrone and estradiol. Similarly, it may be inferred that 6#?-hydroxyandrostenedione was formed late in the incubation, as its isotope ratio is unity. The formation of Iq-nortestosterone is of particular interest in view of the suggestion, made previously5, that alternate pathways of aromatization may exist in human ovarian tissue. The small amount of dehydroepiandrosterone remaining at the end of the incubation clearly indicates an effective transformation of dehydroepiandrosterone into 44 compounds by means of the 3&ol-dehydrogenase system. Thus, the 6.4 peg androstenedione added could not have exerted any marked inhibitory effect. Aromatization of androstenedione proceeded initially at a very rapid rate, even though the substrate was originally extracellular. Aromatization of androstenedione biosynthesized from dehydroepiandrosterone must have occurred somewhat later, since a prior enzymatic step is required. It is difficult to tell whether the lower yield of estradiol from dehydroepiandrosterone is due to this delay or whether the aromatizing Biochim. Biophys. Acta, 187 (1969) 450-453

SHORTCOMMUNICATIONS

453

system is unstable under these conditions and proceeds with rapidity only at the very beginning. Finally, it must be recognized that these results are derived from only a single selected sample of tissue, and must be interpreted with appropriate caution. Southwest Foundation for Research and Education, San Antonio, Texas 78228

LEONARDR. AXELROD JOSEPH W. GOLDZIEHER (U.S.A.)

1 L. R. AXELROD AND J. W. GOLDZIEHER, J. CL&. Endocrinol. Metab., 22 (1962) 431. 2 L. R. AXELROD, C. MATTHIJSSEN, J. W. GOLDZIEHER AND J. E. PULLIAM, Acta Endocvi~zol. SuPpl.> gg (1965). 3 A. R. SOHVAL AND J. L. GABRILOVE, J. Ural., 93 (1965) 711. 4 G.J. RACE, H.M.WU,C.F.HAMILTON.J.E.THOMPSONAND D.J. AUSTIN,]. Am.Med.Assoc., 181 (1962) 104. 5 L. R. AXELROD AND J. W. GOLDZIEHER, J. C&z. Endocrinol. Metab.. 25 (1965) 1275.

Received June znd, 1969 Biochim.

Biophys.

Acta,

187 (1969)450-453

BBA 53234

The isolation and identification of 5a-cholestan-3/?-ol from the human atheromatous aorta 5cr-Cholestan-3/&ol (cholestanol) has been identified as a companion of cholesterol in several human tissuesl-s, including human arteries4. There is, however, considerable doubt as to the quantities of this compound present in the total sterol fractions. The older literature on atheroma5*B contains reports of proportions of cholestanol as high as 13% but recent reports l,‘, based on improved analytical methods, suggest that it amounts only to about 0.5-1~/~of the total sterol in tissues. In certain previous studies19s9eper-acids have been used to oxidise unsaturated sterols and facilitate the isolation of stanols. Here we have preferred to avoid irreversible alteration of the sterols and to rely on chromatographic separations (cf. ref. 7). Plaques from grossly diseased aortas were processed, and sterol and sterol ester fractions were isolated, as previously described lo. ‘Polar’ sterols were removed from the total sterol fraction by repeated crystallisation from methanol. Sterol esters were subjected to mild alkaline hydrolysis, in a reagent made up from 6 ml of aq. KOH (33%, w/v) in 50 ml of ethanol, at 50” for 15 h. Both sterol fractions were then treated with redistilled trifluoroacetic anhydride in chloroform. Sterol trifluoroacetates are quite as satisfactory as propionate+l for thin-layer chromatography on AgNO,-silica gel; we have found them advantageous because of their more convenient retention times in gas-liquid chromatography and because their mass spectra normally show clear molecular ions. All thin-layer chromatography was carried out on layers (0.5 mm for preparative, 0.25 mm for analytical separations) Biochim.

Biophys.

Acta,

187 (1969) 453-456