- Email: [email protected]
Steroids in primates: 5-androstene-3β, 16α, 17β-triol excretion by a pregnant irus monkey (macaca pascicularis)
51
STEROIDS IN PRIMATES: 5-ANDROSTENE-3~,
16~
17~-TRIOL EXCRETION BY A PREGNANT IRUS
MONKEY (MACACA FASCICULARIS) M.E. Manson, C.H.L. Shackleton~ F.L. Mitchell o T! J.-A. Gustafsson and J. Sjovall Medical Research Council 2 Clinical Research Centre, Warlord Road, Harrow, Middlesex, England and Department of Chemistry~ Karolinska Instituter, Stockholm, Sweden.
Received 5/4/71 Abstract The major compound present in the steroid monosulfate fraction of urine from a macaque monkey (Macaca fascicularis) during pregnancy was identified by gas chromatography-mass spectrometry as 5-androstene-3~, 16~, 17~-triol. The excretion of this compound increased throughout the period of gestation (from i0 to 130 ~g/24 hr). Other compounds identified were 3~-hydroxy5-androsten-17-one, 5-androstene-3~, 17~-diol and three androstane-3, 16, 17-triols.
The excretion of estrogens by pregnant macaque monkeys has been studied previously (1-3) 9 but relatively little is known of the excretion of neutral steroids.
Pregnanediol has been shown not to
be an important metabolite of progesterone in macaque monkeys (4-6) and there is little evidence for the presence in urine of other C21 metabolites of progesterone.
In contrast to man and higher sub-
human primates (7) the major metabolite of progesterone in these species is androsterone
(5,6).
O,Malley and Lipsett (8) have
measured individual 17-oxosteroids excreted as glucuronide and sulfate esters by non-pregnant adult rhesus monkeys (Macaca mulatta) but
52
ST ER O I D S to date little is known of the excretion of neutral C19 steroids by pregnant animals. In human pregnancy 3~-hydroxy-A 5 steroids and steroid sulfates are important intermediates in the formation of estrogens (9,10). The purpose of the present study was to identify the major steroid sulfates which are present in urine from irus monkeys (Macaca fascicularis) during pregnancy. MATERIALS AND METHODS
[7~-3H]- 3~-hydroxy-5-androsten-17-one sulfate was obtained from the Radiochemical Centre, Amersham, England. Amberlite XAD-2 and Amberlyst A-26 were obtained from British Drug Houses~ Ltd., Poole, Dorset, England. Sephadex LH-20 was obtained from AB Pharmacia, Uppsala, Sweden. Urine samples (24 hr) were collected without preservatives and stored at -30°Cas soon as possible. ~ - 3 H ] - 3~-hydroxy-5-androsten-17-one sulfate was added to the urine samples prior to extraction. Free and conjugated steroids were extracted using columns of Amberlite XAD-2 resin by a method described previously (11,12). After the urine (volume v = 100-300 ml) had been poured through columns of XAD-2 resin (i00 x 2.5 cm) the columns were washed with water (2 v) and the steroid conjugates recovered by elution with methanol (4 v). The methanol extracts were dried and chromatographed on Sephadex LH-20 columns (35 g) using methanol: chloroform (1:17 0.01 M with respect to sodium chloride) as the eluting solvent. Fractions of i0 ml were collected~ and a small portion of each was removed for the measurement of radioactivity. Labelled 3~-hydroxy-5-androsten-17-one sulfate appeared in fractions eluted between 260 and 380 ml~ and the fractions between 200 and 460 ml were assumed to contain all the monosulfates. The pooled extract was dried and dissolved in 5 ml acetate buffer for hydrolysis of the steroid conjugates with digestive juice from Helix pomatia. The liberated steroids were extracted in a column of Amberlite XAD-2 resin (i0 g) and acidic impurities removed by passing the methanol eluate through the anion exchanger Amberlyst A-26 (bicarbonate form). The methanol extracts were dried and the samples further purified on small silicic acid columns (13). Trimethylsilyl ethers were prepared and the samples analysed by gas chromatography and combined gas chromatography-mass spectrometry (GC-MS instrument, LKB 9000, LKB Stockholm) using 02-1 and SE-30 stationary phases. Retention times (tR) were determined relative to 5~-cholestane. Mass spectra were recorded on magnetic tape (14) and an IBM computer was used to process the data (15).
18:1
53
STEROIDS
July 1971
A semi-quantitative estimation of 5-androstene-3~, 16~,17~ L triol was carried out by the addition of an internal standard~ 5~-cholane-3~, 24-dioi to the samples prior to formation of trimethyl silyl ethers.
RESULTS The major compound excreted in the steroid monosulfate fraction gave a mass spectrum (as trimethylsilyl ether) which indicated a 5-androstene-3,16~17-triol
structure (Fig. I).
Retention times
on QF-I and SE-30 stationary phases were~ within experimental error, identical to those of 5-androstene-3~,16~17~-triol ether (tR SE-30, 1.25; t R QF-I, 0.99).
trimethylsilyl
Other epimers of this
compound are well separated on these stationary phases (16).
The
parent ion of the mass spectrum is at m/e 522 and the loss of one two and three trimethylsilanol groups gives rise to the peaks at m/e 432, 342 and 252.
A loss of 103 mass units is characteristic
for steroids with vicinal trimethylsiloxy groups and results in peaks at m/__~e329 and 239. ~- 2001
so
M-(2,90+103)
•
,29
:I 0
lO0
"
/
I ,252
M/F
IM-90
M-2 90
,l/
!i;J
200
M-(90403)
522
" 300
400
500
Fig. 1 Mass spectrum of the trimethylsilyl ether of 5-androstene3~,16~,17~-trioi isolated from urine of a pregnant irus monkey. Three androstane-3~16,17-triols were also detected by GC-MS (tR SE-30~ 0.90, 0.97 and 1.05) but the stereochemistry of the functional groups was not established.
The mass spectrometric
fragmentation patterns are very similar to that of 5-androstene-
S T E R O I D S
54 3~,16~,17~-triol, the
18:1
corresponding fragments having a mass of
two units greater than those seen in the unsaturated compound (17). 3~-Hyd~oxy-5-androsten-17-one 3~17~-dioi
(tR SE-30~ 0.45) and 5-androstene-
(tR SE-30, 0..59) were also identified in the monosulfate
fraction using GC-MS.
The mass spectra of the trimethylsilyl
ethers of these compounds have been previously reported
(18).
Results of a semi-quantitative measurement of urinary 5-androstene3~,16~,17~-trioi at different stages of pregnancy and in a non-pregnant irus monkey are given in Table i.
The total excretion of the three
androstane-3,16,17-triols was much lower than that of the unsaturated compound and ranged only from 3 to 13 ~g per 24 hr at the same periods of pregnancy. Table 1 Urinary excretion of 5-androstene-3~,16~,17~-triol by an irus monkey at various stages of pregnancy~ and in one non-pregnant animal. Period of gestation (week)
steroid excretion (~24 hr)
8
i0
9
13
13
16
18
36
20
130
Non-pregnant monkey
3
DISCUSSION Estrogen synthesis in man during pregnancy is principally carried out by the feto-placental unit.
Neither fetus nor placenta
has the full complement of enzymes necessary for the transformation
July 1971
ST ER O I D S
of 3~-hydroxy-5-pregnen-20-one to estrogen.
55 The fetus or mother
must supply steroid precursors ~ (e. g. 3 ~-hydroxy-5-androsten-17one or 3 ~, 16~-dihydroxy-5-androsten-17-one)
for conversion into
estrogen in the placenta (i0). Sub-human primates also require a complete feto-placental unit for estrogen synthesis. homogenates of adrenal
Davies and co-workers
(19) found that
(fetal and maternal) or placental tissue
from irus monkeys could not form estrone or estradiol from labelled 3~-hydroxy-5-pregnen-20-one but mixed placental and adrenal tissue could effect this transformation.
It can therefore
be assumed that the irus monkey fetus supplies steroid precursors for estrogen synthesis. In other respects, however, the synthesis of estrogen by pregnant macaque monkeys is very different from that in man.
Estriol is the
dominant urinary estrogen excreted during pregnancy by women and it is largely formed in the placenta from fetal 3~,16~-dihydroxy5-androsten-17-one
(i0) but in rhesus monkeys (3) estrone is more
important~ suggesting that little 16~-hydroxylated precursor of estriol is synthesised in the monkey fetus.
This concept is
supported by the work of Heinrichs and Col~s (20) who have shown that liver tissue taken from fetal rhesus monkeys has little 16~-hydroxylase.
Very small amounts of 3 ~ 1 6 ~ - d i h y d r o x y - 5 -
androsten-17-one were formed following the incubation of 3~hydroxy-5-androsten-17-one with hepatic microsomes.
ST ER O ID S
56
18:1
The excretion of 16~-hydroxylated steroids does however increase during pregnancy in macaques.
Estriol excretion
increases about 4.5 fold in rhesus monkeys (3) and it has been shown in the present study that 5-androstene-3~16~17~-triol excretion appears to increase during pregnancy in irus monkeys. In the light of the evidence demonstrating the low activity of fetal hepatic 16~-hydroxylase it seems probable that the 16hydroxylation is taking place in the mother~ with the overall increasing excretion of 16~-hydroxylated compounds being due to an increased production of precursors by the fetus. The situation in higher primates is more similar to that in man.
In the gorilla and chimpanzee estriol is the major urinary
estrogen (21 ~22) and newborn chimpanzees excrete relatively large amounts of 3~16~-dihydroxy-5-androsten-17-one~ the major fetal precursor of estriol (12). It has been shown that 5-androstene-3~16~17~-triol
is the
major constituent of the steroid monosulfate fraction of urine excreted during pregnancy by an irus monkey.
This steroid was
also detected in urine collected from a non-pregnant animal where its concentration was much lower.
It must be stressed that these
results were obtained from only one pregnant and one non-pregnant animal and the individual variation is not known.
July 1971
s T ER O I D S
ACKNOWLEDGEMENTS We wish to thank Dr. C. Coid and Mr. P. Dawson for supplying the urine samples and Mr. J. Hancock for technical assistance. Part of the study was financed by the Swedish Medical Research Council (grant No. 13X-219). J.-~. G. is indebted to "Syskonen Wessen Stiftelse" for a fellowship. REFERENCES i. 2. 3. 4. 5. 6. 7. 8.
9. i0.
Ii. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22.
Short, R.V., and Eckstein, P., J. ENDOCRINOL. 2 2 7 15 (1961). Laumas, K.R., J. ENDOCR/_NOL. 31, 297 (1965). Hopper, B.R., and Tullner, W.W., STEROIDS 97 517 (1967). Chamberlain, J.~ Knights, B.A., and Thomas, G.H., J. ENDOCRINOL. 28, 235 (1964). Jeffrey, J.D.A., J. ENDOCRINOL. 34, 387 (1966). Plant, T.M., James, V.H.T., and Michael, R.P., J. ENDOCRINOL. 43, 493 (1969). Goldzieher, J.W.~ and Axelrod, L.R., GEN. COMP. ENDOCRINOL. 13, 201 (1969). O'Malley, B.W.~ and Lipsett, M.B., STEROIDS 8, 711 (1966). Mitchell, F.L., VITAMINS HORMONES 25, 191 (1967). Diczfalusy, E. in THE FOETO-PLACENTAL UNIT (ed. A. Pecile and C. Finzi) Excerpta Medica Foundation, Amsterdam, 1968~ p. 65. Bradlow, H.L., STEROIDS ii, 265 (1968). o Shackler.n, C.H.L., Mitchell, F.L., Gustafsson, J.-A., and Sj~vall, J., FEBS LETTERS, ii, 129 (1970b). ,, Shackler.n, C.H.L., Gustafsson, J.-~.~ and Sjovall, J., STEROIDS 17, 265 (1971). He---dfjall, B., Jansson, P.-~., ~ r d e , Y., Ryhage, R., and • t! Wikstrom, S., J. SCI. INSTR. 2, 1031 (1969). Reimendal, R., and Sj~vall, J., Proc. III Intern. Congr. Hormonal Steroids Hamburg 1970, Excerpta Medica, Amsterdam (in the press) (1971). Shackleton, C.H.L., Gustafsson, J.-~., and Sjovall, J., STERO
57
STEROIDS IN PRIMATES: 5-ANDROSTENE-3~,
16~
17~-TRIOL EXCRETION BY A PREGNANT IRUS
MONKEY (MACACA FASCICULARIS) M.E. Manson, C.H.L. Shackleton~ F.L. Mitchell o T! J.-A. Gustafsson and J. Sjovall Medical Research Council 2 Clinical Research Centre, Warlord Road, Harrow, Middlesex, England and Department of Chemistry~ Karolinska Instituter, Stockholm, Sweden.
Received 5/4/71 Abstract The major compound present in the steroid monosulfate fraction of urine from a macaque monkey (Macaca fascicularis) during pregnancy was identified by gas chromatography-mass spectrometry as 5-androstene-3~, 16~, 17~-triol. The excretion of this compound increased throughout the period of gestation (from i0 to 130 ~g/24 hr). Other compounds identified were 3~-hydroxy5-androsten-17-one, 5-androstene-3~, 17~-diol and three androstane-3, 16, 17-triols.
The excretion of estrogens by pregnant macaque monkeys has been studied previously (1-3) 9 but relatively little is known of the excretion of neutral steroids.
Pregnanediol has been shown not to
be an important metabolite of progesterone in macaque monkeys (4-6) and there is little evidence for the presence in urine of other C21 metabolites of progesterone.
In contrast to man and higher sub-
human primates (7) the major metabolite of progesterone in these species is androsterone
(5,6).
O,Malley and Lipsett (8) have
measured individual 17-oxosteroids excreted as glucuronide and sulfate esters by non-pregnant adult rhesus monkeys (Macaca mulatta) but
52
ST ER O I D S to date little is known of the excretion of neutral C19 steroids by pregnant animals. In human pregnancy 3~-hydroxy-A 5 steroids and steroid sulfates are important intermediates in the formation of estrogens (9,10). The purpose of the present study was to identify the major steroid sulfates which are present in urine from irus monkeys (Macaca fascicularis) during pregnancy. MATERIALS AND METHODS
[7~-3H]- 3~-hydroxy-5-androsten-17-one sulfate was obtained from the Radiochemical Centre, Amersham, England. Amberlite XAD-2 and Amberlyst A-26 were obtained from British Drug Houses~ Ltd., Poole, Dorset, England. Sephadex LH-20 was obtained from AB Pharmacia, Uppsala, Sweden. Urine samples (24 hr) were collected without preservatives and stored at -30°Cas soon as possible. ~ - 3 H ] - 3~-hydroxy-5-androsten-17-one sulfate was added to the urine samples prior to extraction. Free and conjugated steroids were extracted using columns of Amberlite XAD-2 resin by a method described previously (11,12). After the urine (volume v = 100-300 ml) had been poured through columns of XAD-2 resin (i00 x 2.5 cm) the columns were washed with water (2 v) and the steroid conjugates recovered by elution with methanol (4 v). The methanol extracts were dried and chromatographed on Sephadex LH-20 columns (35 g) using methanol: chloroform (1:17 0.01 M with respect to sodium chloride) as the eluting solvent. Fractions of i0 ml were collected~ and a small portion of each was removed for the measurement of radioactivity. Labelled 3~-hydroxy-5-androsten-17-one sulfate appeared in fractions eluted between 260 and 380 ml~ and the fractions between 200 and 460 ml were assumed to contain all the monosulfates. The pooled extract was dried and dissolved in 5 ml acetate buffer for hydrolysis of the steroid conjugates with digestive juice from Helix pomatia. The liberated steroids were extracted in a column of Amberlite XAD-2 resin (i0 g) and acidic impurities removed by passing the methanol eluate through the anion exchanger Amberlyst A-26 (bicarbonate form). The methanol extracts were dried and the samples further purified on small silicic acid columns (13). Trimethylsilyl ethers were prepared and the samples analysed by gas chromatography and combined gas chromatography-mass spectrometry (GC-MS instrument, LKB 9000, LKB Stockholm) using 02-1 and SE-30 stationary phases. Retention times (tR) were determined relative to 5~-cholestane. Mass spectra were recorded on magnetic tape (14) and an IBM computer was used to process the data (15).
18:1
53
STEROIDS
July 1971
A semi-quantitative estimation of 5-androstene-3~, 16~,17~ L triol was carried out by the addition of an internal standard~ 5~-cholane-3~, 24-dioi to the samples prior to formation of trimethyl silyl ethers.
RESULTS The major compound excreted in the steroid monosulfate fraction gave a mass spectrum (as trimethylsilyl ether) which indicated a 5-androstene-3,16~17-triol
structure (Fig. I).
Retention times
on QF-I and SE-30 stationary phases were~ within experimental error, identical to those of 5-androstene-3~,16~17~-triol ether (tR SE-30, 1.25; t R QF-I, 0.99).
trimethylsilyl
Other epimers of this
compound are well separated on these stationary phases (16).
The
parent ion of the mass spectrum is at m/e 522 and the loss of one two and three trimethylsilanol groups gives rise to the peaks at m/e 432, 342 and 252.
A loss of 103 mass units is characteristic
for steroids with vicinal trimethylsiloxy groups and results in peaks at m/__~e329 and 239. ~- 2001
so
M-(2,90+103)
•
,29
:I 0
lO0
"
/
I ,252
M/F
IM-90
M-2 90
,l/
!i;J
200
M-(90403)
522
" 300
400
500
Fig. 1 Mass spectrum of the trimethylsilyl ether of 5-androstene3~,16~,17~-trioi isolated from urine of a pregnant irus monkey. Three androstane-3~16,17-triols were also detected by GC-MS (tR SE-30~ 0.90, 0.97 and 1.05) but the stereochemistry of the functional groups was not established.
The mass spectrometric
fragmentation patterns are very similar to that of 5-androstene-
S T E R O I D S
54 3~,16~,17~-triol, the
18:1
corresponding fragments having a mass of
two units greater than those seen in the unsaturated compound (17). 3~-Hyd~oxy-5-androsten-17-one 3~17~-dioi
(tR SE-30~ 0.45) and 5-androstene-
(tR SE-30, 0..59) were also identified in the monosulfate
fraction using GC-MS.
The mass spectra of the trimethylsilyl
ethers of these compounds have been previously reported
(18).
Results of a semi-quantitative measurement of urinary 5-androstene3~,16~,17~-trioi at different stages of pregnancy and in a non-pregnant irus monkey are given in Table i.
The total excretion of the three
androstane-3,16,17-triols was much lower than that of the unsaturated compound and ranged only from 3 to 13 ~g per 24 hr at the same periods of pregnancy. Table 1 Urinary excretion of 5-androstene-3~,16~,17~-triol by an irus monkey at various stages of pregnancy~ and in one non-pregnant animal. Period of gestation (week)
steroid excretion (~24 hr)
8
i0
9
13
13
16
18
36
20
130
Non-pregnant monkey
3
DISCUSSION Estrogen synthesis in man during pregnancy is principally carried out by the feto-placental unit.
Neither fetus nor placenta
has the full complement of enzymes necessary for the transformation
July 1971
ST ER O I D S
of 3~-hydroxy-5-pregnen-20-one to estrogen.
55 The fetus or mother
must supply steroid precursors ~ (e. g. 3 ~-hydroxy-5-androsten-17one or 3 ~, 16~-dihydroxy-5-androsten-17-one)
for conversion into
estrogen in the placenta (i0). Sub-human primates also require a complete feto-placental unit for estrogen synthesis. homogenates of adrenal
Davies and co-workers
(19) found that
(fetal and maternal) or placental tissue
from irus monkeys could not form estrone or estradiol from labelled 3~-hydroxy-5-pregnen-20-one but mixed placental and adrenal tissue could effect this transformation.
It can therefore
be assumed that the irus monkey fetus supplies steroid precursors for estrogen synthesis. In other respects, however, the synthesis of estrogen by pregnant macaque monkeys is very different from that in man.
Estriol is the
dominant urinary estrogen excreted during pregnancy by women and it is largely formed in the placenta from fetal 3~,16~-dihydroxy5-androsten-17-one
(i0) but in rhesus monkeys (3) estrone is more
important~ suggesting that little 16~-hydroxylated precursor of estriol is synthesised in the monkey fetus.
This concept is
supported by the work of Heinrichs and Col~s (20) who have shown that liver tissue taken from fetal rhesus monkeys has little 16~-hydroxylase.
Very small amounts of 3 ~ 1 6 ~ - d i h y d r o x y - 5 -
androsten-17-one were formed following the incubation of 3~hydroxy-5-androsten-17-one with hepatic microsomes.
ST ER O ID S
56
18:1
The excretion of 16~-hydroxylated steroids does however increase during pregnancy in macaques.
Estriol excretion
increases about 4.5 fold in rhesus monkeys (3) and it has been shown in the present study that 5-androstene-3~16~17~-triol excretion appears to increase during pregnancy in irus monkeys. In the light of the evidence demonstrating the low activity of fetal hepatic 16~-hydroxylase it seems probable that the 16hydroxylation is taking place in the mother~ with the overall increasing excretion of 16~-hydroxylated compounds being due to an increased production of precursors by the fetus. The situation in higher primates is more similar to that in man.
In the gorilla and chimpanzee estriol is the major urinary
estrogen (21 ~22) and newborn chimpanzees excrete relatively large amounts of 3~16~-dihydroxy-5-androsten-17-one~ the major fetal precursor of estriol (12). It has been shown that 5-androstene-3~16~17~-triol
is the
major constituent of the steroid monosulfate fraction of urine excreted during pregnancy by an irus monkey.
This steroid was
also detected in urine collected from a non-pregnant animal where its concentration was much lower.
It must be stressed that these
results were obtained from only one pregnant and one non-pregnant animal and the individual variation is not known.
July 1971
s T ER O I D S
ACKNOWLEDGEMENTS We wish to thank Dr. C. Coid and Mr. P. Dawson for supplying the urine samples and Mr. J. Hancock for technical assistance. Part of the study was financed by the Swedish Medical Research Council (grant No. 13X-219). J.-~. G. is indebted to "Syskonen Wessen Stiftelse" for a fellowship. REFERENCES i. 2. 3. 4. 5. 6. 7. 8.
9. i0.
Ii. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21. 22.
Short, R.V., and Eckstein, P., J. ENDOCRINOL. 2 2 7 15 (1961). Laumas, K.R., J. ENDOCR/_NOL. 31, 297 (1965). Hopper, B.R., and Tullner, W.W., STEROIDS 97 517 (1967). Chamberlain, J.~ Knights, B.A., and Thomas, G.H., J. ENDOCRINOL. 28, 235 (1964). Jeffrey, J.D.A., J. ENDOCRINOL. 34, 387 (1966). Plant, T.M., James, V.H.T., and Michael, R.P., J. ENDOCRINOL. 43, 493 (1969). Goldzieher, J.W.~ and Axelrod, L.R., GEN. COMP. ENDOCRINOL. 13, 201 (1969). O'Malley, B.W.~ and Lipsett, M.B., STEROIDS 8, 711 (1966). Mitchell, F.L., VITAMINS HORMONES 25, 191 (1967). Diczfalusy, E. in THE FOETO-PLACENTAL UNIT (ed. A. Pecile and C. Finzi) Excerpta Medica Foundation, Amsterdam, 1968~ p. 65. Bradlow, H.L., STEROIDS ii, 265 (1968). o Shackler.n, C.H.L., Mitchell, F.L., Gustafsson, J.-A., and Sj~vall, J., FEBS LETTERS, ii, 129 (1970b). ,, Shackler.n, C.H.L., Gustafsson, J.-~.~ and Sjovall, J., STEROIDS 17, 265 (1971). He---dfjall, B., Jansson, P.-~., ~ r d e , Y., Ryhage, R., and • t! Wikstrom, S., J. SCI. INSTR. 2, 1031 (1969). Reimendal, R., and Sj~vall, J., Proc. III Intern. Congr. Hormonal Steroids Hamburg 1970, Excerpta Medica, Amsterdam (in the press) (1971). Shackleton, C.H.L., Gustafsson, J.-~., and Sjovall, J., STERO
57