- Email: [email protected]
The excretion of urinary steroids by the owl monkey (Aotus trivirgatus) studied using open tubular capillary column gas chromatography and mass spectrometry
THE EXCRETION OF URINARY STEROIDS BY THE OWL MONKEY (AOTUS ~R~~~RG~~U~~ STUDIED USING OPEN TUBULAR CAPILLARY COLUMN GAS CHROMATOGRAPHY AND MASS SPECTROMETRY K. D. R. SEWHELL and R. C. BONNEY Division of Clinical Chemistry, Clinical Research Centre. Watford Road, Harrow, Middlesex, England and Wellcome Laboratories of Comparative Physiology, Zoological Society of London, Regent’s Park. London, England (Received 23 July 1979) SUMMARY A qualitative analysis of steroids excreted in urine by the owl monkey (Aorus ~ri~irg~r~s) ispresented. The study was carried out using the high resolving power of wall coated open-tubular glass capillary column gas chromatography and mass spectrometry. Oestrone was the major oestrogen excreted by the female animal and was also present in greater amounts than testosterone in the male monkey. The principal progesterone metabolites were identified lo be 68-hydroxypregnanolone and 16_hydroxypregnanolone isomers. Corticosteroid metabolism was also characterized by the presence of 16-hydroxylated in addition to C-20 reduced metabolites.
(lotus triuirgatus) fulfils an important role as a laboratory model for research into human malaria [28-3 I]. Studies of the reproductive endocrinology of this speties are necessary for the establishment of a captive breeding programme, and essential to such studies is an accurate qualitative assessment of steroid metab-
INTRODUCTION There have been a number of investigations
of steroid metabolism and excretion in several species of Old World monkeys and apes [l-26]. Similar studies in New Wdrld monkeys have to date been restricted to the marmoset (Callithrix jacchus) [IS, 191 and the squirrel monkey (Saimari sciureus) [27]. The work of these authors indicates that metaboiic routes of steroid excretion by New World monkeys differ from those of other primates. New World monkeys are being used increasingly in medical research, and among them, the owl monkey
Olhll. The present communication
describes the identification, using wall coated open tubular glass capillary column gas chromato~aphy and mass spectrometry (GC-MS), of the major steroid metabolites excreted in urine from adult monkeys of this species. MATERIALS AND METHODS
The following trivial names and abbreviations are used in this presentation: Oestrone. 3-hydroxy-~,3,S(lO~estratrien-l7-one; Oestradiol-I 78, ~~3,S(lO~es~ra~riene-3,~7~diol; Testosterone, 17/J-hydroxy-4-androsten-3-one; Androsterone. 3z-hydroxy-S/7-androstan-17-one; Dehydre epiandrosterone, 3/I-hydroxy-S-androsten-17-one; I l-oxoandrosterone. 32.hydroxy-5&androstane-3.1 I-dione; 1l&Hydroxyandrosterone, 31.1 Ifi-dihydroxy-5&androstan- 1‘I-one; Pregnanediol, SB-pregnane-3z,20ct-diol; alloPregnanediol, Sz-pre~ane-3~2~-diol; Progesterone, 4-pregnene-3,20-dione; Pregnanolone, 3a-hydroxy-5/?-pregnan-2%one; 68-Hydroxypregnanolone. 3r,6B-DihydroxySz-pregnan-20-one; 17-Hydroxypregnanolone. 32.172dihydroxy-S/f-pregnan-20-one; l&Hidroxypregnanolone, 32.16z-dihydroxy-5n(B)-pregnan-2O-one; Tetrahydrocortisol. 3x.1 I/?.17z,21-tetrahydroxy-5&pregnan-20-one; at/oTeetrahydrocortisol. 32.1 l~,ij~,2l-tet~~ydroxy-5=-pre~ nan-20-one; Cortolone, 32,17cr20a(BLZI-tetrahydroxv-5& pregnan-1 I-one; Cortisol, I I/?,1‘Isti&ihydrox$4_pr&&ne-3,20-dione; 6&Hydroxycortisol. @,I 1&17a,21-tetrahydroxy-Qpregnene- 3,20-dione; ZO-Dihydrocortisol, 11/?,172, 202&%,21-tetrahydroxy-Q_pregnene- 32O-dione; MO-TMS ether, methyloxime-trimethyisilyl ether derivative; m/z mass/charge ratio; rR = retention time. 37
Animals
Animals were housed in individual cages in conditions of controlled temperature (2s25°C) and lighting (12 h artificial light, 12 h dim red light). The cages were fitted with removable sloping trays with funnels in the base to assist in the collection of urine. Daily urine collections were made and the samples stored at -20°C until required for analysis. Extraction The
of steroids
analytical procedures have been described in detail elsewhere [24,32] and are therefore outlined briefly. Steroids were extracted from urine samples using small columns of the neutral resin Amberlite XAD-2. Following hydrolysis of steroid conjugates with a mixed /?-glucuronidase and sulphatase enzyme prep aration (Helix pomatia, L’lndustrie Biologique Francaise) the “freed” steroids were re-extracted on
K. D. R. SETCHELLand R. C. BONNEY
38
Amberlite XAD-2 and this extract was then passed through a column of Sephadex LH-20 (6g, cyclohexane: ethanol, 4: 1, v/v) to effect a purification [33]. Chromate oxidation
The dried steroid extract was dissolved in acetone (0.5 ml) and 3 drops of Jones reagent (prepared by dissolving 26.7 g of chromium trioxide in 23 ml of concentrated sulphuric acid and diluting to 100 ml with distilled water) added. The sample was shaken for 2 h at room temperature and then diluted with lOOm1 distilled water. The reaction products were then extracted on small columns of Amberlite XAD-2 resin. A methyloxime derivative was prepared of the dried extract. Derivatisation
0-Methyloximetrimethylsilyl (MO-TMS) ether derivatives were prepared of proportions of the tottal steroid extract {34] and removal of the non-volatile silylating reagent and further purification of the extract was carried out using small columns of Lipidex 5000 [353; this step being essential to enable GC analysis on open tubular capillary columns where the samples are introduced by solid injection. Gas chromatography (CC)
Gas chromatographic analysis was performed using a Pye 104 gas chromatograph equipped with flame
ionisation detectors and modified to accept a 25 m wall coated open-tubular glass (WCOT) capillary column coated with Silicone OV-1. Samples were applied to the column via a solid injection’system similar to that described by Van de Berg & Cox[36]. Helium was used as a carrier gas and the flow rate through the column was approx. 2ml/min. GC was carried out using temperature programming conditions; 185-275°C with increments of Z”C/min. Gas chromatography-mass spectrometry (CC-MS)
GC-MS was performed using a Varian MAT-731 double focusing mass spectrometer. The conditions for mass spectrometry were as follows: temperature of separator and transfer line, 250°C; temperature of ion source, 250°C; ionization current, 800 PA; ionization voltage, 70 eV; accelerating voltage, 8 kV. Repetitive magnetic scanning over the mass range &loo0 atomic mass units (amu) was the mode of analysis. Steroid identification The identification of a steroid was based upon the gas chromatographic retention time (tR) of the steroid derivative relative to homologous series of n-alkanes, C24-C34 (methylene units, MU) and the complete mass spectrum when compared with the authentic steroid. In many instances, due to the non-availability of reference steroids and particularly with compounds present at extremely low levels, it was only possible to
20 Time (min) Fig. 1. The gas chromatographic separation using a wall coated open tubular capillary column of steroids excreted in the urine of the adult female owl monkey (Aotus triciryatus) during the peri-ovulatory period. The analysis was performed by a Pye 104 gas chromatograph housing a 25m capillary column coated with silicone OV-1. Samples were introduced Ga an automatic solid injection system. Helium was the carrier gas and the flow rate through the column was ca. Zml/min. Temperature programmed operation from 175-275°C in increments Gf 2,2’C/min. was used. The principal steroids are indicated as follows: 1. androsterone, 2 aetiocholanolone, 3. oestrone, 4 oestradiol-17J, 5. 17-hydroxypregnanolone. 6. Sa-pregnane-3a,2Oa-diol, 7. 5/Lpregnane-3a.2Oa-dial, 8. pregnanetriol, 9. 6/?-hydroxypregnanolone, 10. 5-pregnene-3/?,2Oa-dial, 11. 3a,16a-dihydroxy-Sa-pregan-20_one, 12 3a,l6a-dihydroxy-5fi-pregnan-20-one.
39
The excretion of urinary steroids by the owl monkey Table 1. List of steroids identified by gas chromatography-mass spectrometry trivirgatus) Retention time (MU)
in the urine of the owl monkey (Aotus
Male?
Steroid
3a-Hydroxy-S-androsten-17-one 3x-Hydroxy-SB-androstan-17-one (Androsterone) 3/3-Hydroxy-5-androsten-17-one (DHA) 3@Hydroxy-Sa-androstan-17-one 3%.Hydroxy-5&androstanc- : I,l7-dione (1 I-oxoandrosteronc) 3-Hydroxy-l,3,S(lO~estratrien-l7-one (Oestrone) 1,3,4(lO~Estratiene-3,17/$diol (Oestradiol) I ‘I/?-Hydroxy-4-androsten-3-one (Testosterone) Androstane-dial-I l-one 26.70 3a-Hydroxy-S/3-pregnan-20-one (Pregnanolone) 3c(,l7a-Dihydroxy-S/l-pregnan-20-one (17-Hydroxypregnanolone) 27.00 3x.1 l/3-Dihydroxy-5/l-androstan-17-one 27.08 3a,l l,%Dihydroxy-Sa-androstan-1Fone 27.18 27.20 Pregnene-dial-one 27.25 S-Pregnene-3,I lJO-trio1 27.25 Pregnane-diol-one 27.30 5-Androstene-38,16,17-trio1 Androstane-3,16,17-trio1 27.40 27.45 5c(-Pregnane-3a,20a-diol (a/lo-pregnanediol) Unknown Compound 180/442$ 27.50 27.61 SB-Pregnane-3a,20a-dial (Pre~ane~ol) 27.90 S~-Pre~ane-3a,l7~,2Oa-triol (Pre~anet~ol) 28.18 346B-Dihydroxy-5a-pregnan-20-one(6~-Hydroxypregnanoione) 28.33 5-Pregnene-3p,20a-diol 3~.16a-Dihydroxy-5a-pregnan-20-one 28.40 3a,l6a-Dihydroxy-5fl-pregnan-20-one 28.50 35,162-Dihdyroxy-5-pregnan-20-one 28.60 29.00 Pregnane-triol-one 29.10 Pregnane-trio1 Pregnane-tetrol (16-hydroxysteroid) 29.25 3a,l7~,2l-Trihydroxy-S~-pre~ane-l1,20-dione (tetrahydroco~isone) 29.60 3r,l1/3,17z,21-Tetrahydroxy-5/?-pregnan-1O-one (tetrahydrocortisol) 30.10 30.51 3x,1 7a,20a,21-Tetrahydroxy-S/3-pregnan-1 l-one (a-cortolone) 30.73 3a,l7a,20/l,21-Tetrahydroxy-S~-pregnan-ll-one (/3-cortolone) 30.73 5~-Pregnane-3a,11/?,17a,20/?-21-pent01 (/l-cortol) 31.20 5~-Ptegnane-3a,l1~,17a,2Oa,21-pento1 (cc-cortol) 31.02 5a.6a,ll~,l7a,2l-Pentahydroxy-S~-pre~~-~one (~-hydroxy-tetrahydrocortis 32.88 17a,2O~,Zl-Trihydroxy-4-pre~ene-3,ll-dione (~~-dihydrocortisone) 32.95 17a,20a,2l-Trihydroxy-4-pregnene-3,1l-dione (20x-dihydrocortisone) 32.54 + 32.60 1lfi,17a,21-Trihydroxy-4-pregnene-3,20-dione (cortisol& 32.70 + 32.88 6&l lfl,l7a,2t-Tetrahydroxy-4-pregnene-3,2Odione (6/i-hydroxycortisol) 32.91 + 33.03 11~,17c1,20~,21-Tetrahydroxy-4-pregnen-3-one (208-dihydrocortisol)§ 33.34 + 33.47 1lj,l7a,20a,Zl-Tetrahydroxy-4-pregene-3-one (20a-dihydrocortisol)$
Female
+ + c + c + +
24.70 25.3 I 25.73 25.82 26.12 26.16 26.30 26.36
+ +
4
+ f
+ + +
c f +
+
-Iif f + f f
+ +
+ + -+ f f f -k f +
+ + + + + + + + + + + + +
f + + + + -t + + + + + f f
* In some cases it is only possible to give a general structure to the steroid identified. For example, pre~ane-dial-one implies a pregnane steroid having two hydroxyt and one 0x0 group. t A steroid found to be present by GC-MS in urine is indicated by +. 3 Details of this compound have been presented elsewhere [39] # These steroids give rise to two peaks which are resolved in the chromatogram due to the separation of the syn- and anti- forms of the methyloxime function on the 3-0~0 group.
assign a general structure based upon the mass spectrometric data; thus for example, androstanediolone implies the steroid has a C,9 nucleus with two hydroxyl groups and one oxo group.
Table I indicates those steroids which have been identified by GC-MS in the urine from both male and female animals, and the principal urinary steroids excreted are discussed in more detail below. Oestrogens
RESULTS AND DISCUSSION
Figure 1 illustrates a GC chromatogram MO-TMS ether derivatives of a urinary extract from an adult female animal typical peri-ovulatory period and analyzed using coated open tubular glass capillary column.
of the steroid of the a wall
Oestrone and oestradiol-178 were the principal oestrogens excreted by the female animal. Quantitative data on the excretion of oestrone and oestradiol-178 in the urine of non-pregnant female animals are reported elsewhere [373 and have demonstrated that the amounts of oestrone excreted in the peri-ovula-
40
K. D. R. SETCHELL and R. C. BONNEY
tory period are approximately 3-fold greater than oestradiol-I 78 and this is evident from the gas chromatogram of the female urine obtained during this period (Fig. 1). Studies of oestrogen excretion by other New World monkeys are limited to the marmoset in which oestradiol-178 is excreted in greater amounts than both oestrone and oestriol[18]. Compared with other subhuman primates both the qualitative and quantitative patterns seem to resemble that of the orang utan [ 163. A positive identification of oestrone in the urine from male animals was obtained from a comparison of the complete mass spectrum and retention data of its MO-TMS ether derivative (tR = 26.16 MU) with that of the authentic steroid (tR = 26.16 MU). It is therefore evident from this observation that a relatively large amount of oestrone must be excreted in urine of male animals (although quantitative data were not obtained) because when GC-MS is employed in the repetitive magnetic scanning mode, due to its limited sensitivity (10 ng) it is generally not possible to obtain complete mass spectra of oestrogens in small volumes of urine from human male subjects or for that matter non-pregnant women. When computer reconstructed fragment ion current chromatograms are plotted of ions (m/z) characteristic of MO-TMS ether derivatives of oestrone and testosterone after the repetitive magnetic scanning GC-MS analysis of an extract from a male ahimal (Fig. 2), it is evident from the peak areas of the characteristic ions for each steroid, that there are “apparently” greater amounts of oestrone than testosterone in the urine. The apparent large amounts of oestrone present in the urine of male animals is an interesting finding and could be supported by data obtained using radioimmunoassay which have shown high levels of oestrone (1 ng/ml) in the plasma of male animals (Bonney, unpublished).
C,&eroids
‘Androsterone and dehydroepiandrosterone were identified in urine of both male and female animals together with the 1I-oxygenated-17-oxosteroids, 1l-oxoandrosterone and 1l/%hydroxyandrosterone. Testosterone was identified as the MO-TMS ether in extracts of urine from male animals (Fig. 2). However, no evidence was obtained for the presence of testosterone in the urine of female animals in spite of finding that plasma testosterone levels in female animals were as high as long/ml when radioimmunoassay was used (Bonney, Dixson & Fleming unpublished data). Progesterone
metabolites
Qualitatively the major proportion of steroids excreted by the female animal comprised matabolites of progesterone. pregnanediol (SD-pregnane-3a,20a-dial; Both tR = 27.61 MU, as TMS ether derivative) and allopregnanediol (Sa-pregnane-3aJOa,-diol; tR = 27.45 MU, as the TMS ether derivative) were identified, the latter isomer being present in greater amounts. However, these metabolites quantitatively accounted for less than 30% of the total progesterone metabolites excreted [37]. The major steroid excreted in the urine gave a retention time of 28.18 MU as the MO-TMS ether derivative and the mass spectrum of this steroid is seen in Fig. 3. The molecular ion M+ was at m/z 507 indicating the general structure to be that of a hydroxypregnanolone. The existence of the ion at m/z 476 formed by the loss of 31 mass units is due to the presence of a derivatized 0x0 group. The presence of the intense ions of m/z 100, 87 and 70 confirms that the 0x0 group is at the C-20 position and that there are no other functional groups in the side chain [43].
AOTUS tlONKEY MILE Fig 2. Selected ion current chromatograms of ions characteristic of MO-TMS ether derivatives of oestrone (m/z 371, 340) and testosterone (m/z 389, 358, 268) recorded during the repetitive magnetic scanning GC-MS analysis of a urine extract from a male owl monkey.
The excretion of urinary steroids by the owl monkey
3aci
41
476
1
I
296
m
Fig. 3. The complete mass spectrum of the MO-TMS ether derivative of major steroid excreted in the urine of the female owl monkey, and subsequently identified as 3a,6@Iihydroxy-Sa-pregnan-20-one. This triplet of ions arises from the loss of the side chain together with carbon atom C-16 and its subsequent fragmentation, and is typical of MO derivatives of all 21-deoxy-20-oxosteroids. The presence of two derivatized hydroxyl groups is evident from the ions m/z 386 [M-(90 + 31)] and m/z 296 [M-(2 x 90 + 31)]. The relative ease of formation of the ion at m/z 386 is evident, from the mass spectrum, in the large pro portion of the total ionization carried by this ion, suggesting its loss from a labile position in the molecule and since the mass spectra of 21-hydroxy-, 17-hydroxy-, 16_hydroxy- and 1S-hydroxy- pregnant lones showed significant differences in their fragmentation patterns it was considered that this steroid was possibly a 6-hydroxy-pregnanolone structure. Furthermore, 6hydroxylation is commonplace in many primate species, the baboon 123-261 and the marmoset [ 183 both excrete a large amount of 6-hydroxylated steroids in urine. Confirmation that this urinary steroid was a 6-hydroxy-pregnanolone was obtained following chromate oxidation to the pregnane-3,6,20-trione structure, a well documented procedure [23,40] which in addition enables the stereochemistry at the C-S position to be determined from the gas chromatographic behaviour and fragmentation pattern of the methyloxime derivative of the product [40]. The oxidation reaction was checked using the reference steroids 3a&-dihydroxyS/?-androstan-17-one and 3x,6x,1 l&trihydroxy-5& androstan-17-one to ensure that epimerization of these S/Y-steroids to the corresponding Sa-compounds did not occur under the acid conditions employed The gas chromatographic behaviour of the methyloxime derivative of the product of chromate oxidation gave rise to two peaks of equal intensity with retention indices te = 30.00 MU and r,r = 30.23 MU indicating the stereochemistry of the hydrogen at C-5 to be a configuration [4O]. The gas chromatographic behaviour and retention indices where shown to be identical to those described by Allen et al. [&I] for methyloxime derivative of the authentic steroid 5a-pregnane-3,6,2@trione. Furthermore the mass +1.14/l--o
spectrum was similar to the authentic steroid reported elsewhere[40] exhibiting the characteristic ion m/z 138 which as suggested by Allen et al. [40] arises out of an initial cleavage of the C-5:6 bond followed by the transfer of a hydrogen atom from a site in ring A to a position in ring B and finally homolytic fission of the C-9:10 bond. The relatively low intensity of the m/z 138 ion in the spectrum further conhrtned the stereochemistry of the hydrogen at C-5 to be of the a-configuration. The retention time of the MG-TMS ether derivative of the urinary steroid (ts = 28.18 MU) was found to be less than any reference 6a-hydroxy-pregnant lone-MGTMS ether and identical with that reported for the same derivative of 6/?-hydroxy-pregnanolone identified in the urine of marmosets [18 191. Since the order of GC elution of MG-TMS ether derivatives of pregnanolone isomers on the non-selective phase, silicone OV-1, is 3a,5a, > 345/f > 3/?,5a (3/%5/3steroids, although rarely occurring in animals have the shortest tR values) and having proven a Sa- steroid it was deduced that the major urinary steroid excreted by this animal had the structure 3a,6fidihydroxy-Sapregnan-2@one. 6/?-Hydroxypregnanolone has also been identified as one of the major steroids excreted by non-pregnant marmosets Cl93 and in pregnancy is one of the major progesterone metabolites of that species [18]. While the pregnant owl monkey has not been studied, quantitatively this steroid accounts for > so”/, of the pro gesterone metabolites in non-pregnant animals 1373 and the GC urinary steroid profile (Fig. 1) resembles that of the pregnant marmoset monkey [ 183. Other hydroxylated pregnanolone metabolites were also found in relatively large amounts in the urine of the female animals, notably 17-hydroxypregnanolone (3aJ7a-dihydroxy-5fl-pregnan-2O-one) and two 16hydroxypregnanolone isomers. The mass spectra of the MO-TMS ether derivatives of all of these hydroxylated pregnanolones are very similar; the parent ion being at mJz 507 and base peaks at mjz 476, however, from our experience [32] they can be differentiated by the presence of prominent structurally sig-
42
K. D. R.
SETCHEU
nificant ions of m/r 156. I58 and 188 for 17-hydroxypregnanolones arising out of cleavage between carbon bonds C-17/C- IS and C-l S/C-l 6 and fragmentation of the side chain and methyloxime group [41]. It should be noted that MO-TMS ethers of 2l-hydroxypregnanolones in addition to all 21-hydroxy- 1‘I-deoxysteroids are readily distinguished because they give rise to prominent ions, which are often the base peaks at m/z 175 and 188, while 15-hydroxy-pregnanolones exhibit intense ions at m/z 170 and 201 in the mass spew tra [32,42]. Although pregnanediol isomers are formed by the owl monkey, it is interesting that 6- and 16-hydroxylation are the most important metabolic transformations undergone by progesterone in the non-pregnant animal When compared with other species of sub-human primates, progesterone metabolism by the owl monkey most closely resembfes that of the marmoset Cl93 and differs from “Old World“ macaque monkeys in which side chain cleavage occurs giving rise to androsterone [IO, 38’J. Polar steroids
A wide spectrum of polar steroid metabolites was identified. A number of unusual tetrahydrocorticosteroids were present. The molecular ion (M+) in their spectra was at m/z 683 indicting a MO-TMS ether of a pregnane-tetrolone, however due to the absence of any ions characteristic of a dihydroxyacetone side chain, glycol side chain or a 21-hydroxy-20.oxo-l7-deoxy side chain and due to the presence of ions of m/z 156, 158 and 188, it is tentatively suaested that these steroids are possibly 3,6,16,17-tetrahydroxypregnan-20-one or 3,11,16,17-tetrahydroxyprcgnan-20.one isomers. Due to the lack of authentic steroids however, their identification is tentative and remains to he confirmed. Corticosteroid metabolism was characterized by the presence of tetrahydrocortisone, tetrahydrocortisol and a number of cortolone isomers Cortisol, 20.dihydrocortisol, and not surprisingly, in view of the efiicient 6~-hydroxyIation system, 6~hydroxycortisol, were the most prominent corticosteroid metabelites excreted and in general the qualitative excretion of corticosteroid metabolites was similar to that for other New World primates [ 19,2A.
Acknowledgements-The authors are grateful for the technical assistance given by Mr M. J. Mad&an in the work,
and to Mr D. Fleming for colkction of urine sampkgThis investigation received support from the Malaria Component of the UNDP~~~ Bank/WHO Special Programme for Reserach and Training in Tropical Diseases.
REFERENCES
l. Kirdani R. V., Gleason T. L and Layne D. S.: The metabolism of aldosterone in the chimpanzee. Gen. camp. Endocr. 3 (1963) 616-620.
and R. C. BONNEY 2. Layne D. S., Kirdani R. V., Gleason T. L. and Pincus G.: The secretion of cortisol in immature chimpanzees. Gen. camp. Endoer. 4 119631155-188. 3. Shackle& C. H. L.,‘M&hell F. L., Gus&son J.-A. and Sjavall J.: Steroids in primates: Excretion of 31-hydroxy-A’ steroids by a newborn chimpazee (Pan rriglcdytes). FEBS L41t. 2 (1970) 129-131. 4. Pal S. 8.: The pattern of urinary excretion of individual l7-ketosteroids in rhesus monkeys. Acta en&r. Cope&. 67 (1971) 71 I-720. 5. Shackleton C. H. L and Gustafsson J-A: Steroids in newborns and infants. Identification by combined gas chromatography-mass spectrometry of the major steroids excreted by a newborn chimpanzee (Pun triglodyres) Steroids 18 (1971) 175-186. 6. Manson M. E., Shackkton C. H. L., Mitchell F. L, Gustafsson J.-A. and Sj6vall J.: Steroids in primates: 5-Androstene-3/?,16al7@iol excretion by a pregnant irus monkey (Monaco jascicularis). Steroids 18 (1971) 51-57. 7. Gokizeiher J. W. and Axelrod L. R.: Urinary metabotites of4-‘*C-progesterone in the baboon (Papio spp.). Gen. camp. Endocr. 13 (1969) 201-205. 8. Townsley J. D.: Steroid hormones in pregnancy: The baboon as a model for studies on oestrogen synthesis. Acta endocr,, Cope& Suppl. 166 (1972) 191-197. 9. Solomon S. and Leung K.: Steroid hormones in nonhuman primates during pregnancy. Acta endocr., Cope& Suppf. 166 (1972) 178-190. 10. Reddy V. V., Balin H. and Nes W. R.: Tbe metabolism of progesterone ‘by the rhesis monkey (Mocacn mulutta). Steroids 17 (1971) 493-502. Il. Ishihara M., Gsawa Y., Kirdani R. Y. and Sandberg A. A.: Progesterone metabolism in the baboon. J. steroid B&hem. 6 (1975) 1213-1218. 12. Yamamoto Y., Manyon A., Kirdani R. Y. and Sandberg A. A.: Androgei metabolism in the rhesus monkev. Sleroids 31 1197817 I l-729. 13. Tiwnsley J. D.:‘Oes&ogen excretion of the pregnant baboon (Papio pap&). E~d~ri~~l~y 95 (1974) l759- 1762. 14. Pepe G. J. and Townsley J. D.: Cortisol metabolism in female baboons (Popio papio). Endocrinology 95 (1974) 1658-1663. 15, Pepe G. J. and Townsley J. D.: The metabolism of cortisoi by term baboon neonates (fapio pupjo).E~d~* rinology 99 (1976)-9. 16. Collins D. C., Graham C. E. and Reedy J. R. K.: Identification and measurement of urinary oestrone, oestradiol- I ‘I/?,oestriol, pregnanediol and androsterone during the menstrual cyck of the orang utan. EndocrinoIogy96 (1975) 93-101: 17 Shackkton C. H. L: Steroid excretion in the neonatal period: A comparative study of the excretion of steroids by human ape and rhesus monkey infants. J. steroid Biochem. 5 (I 974) I I 3- 1IS. 18. Shackkton C. H. L: progesterone and oestrogen metabolism in the pregnant marmoset (C~l~f~r~x jac&us). .I. steroid B&hem. 5 (1974) 597-600. 19. Shackkton C. H. L: The excretion of steroids by the adult marmoset monkey (Collithhrix jacchur). J. steroid B&hem. 6 (1975) 1429-1432. 20. Shackkton C. H. L and Mitchell F. L: The comparison of perinatal steroid endocrinology in simians with a view to finding a suitabk animai model to study human probkms Lab. Anim. Jfrmdbks (1975) 159-181. 21. Setchell K. D. R.. Gontscharow N. P., Ax&on M. and Sjiivall J. : The characterisation of polar corticosteroids in the urine of the macaque monkey (Macaca&seieutaris) and the baboon (Pupio homadryos). Acto endocr., copenic. 79 (1975) 53~550. 22. Setchell K. D. R. and Shackkton C. H. L.: Cortisol and corticosterone metabolism in the macaque mon-
The excretion of urinary steroids by the owl monkey
23.
24.
25.
26.
27
28
29
30
31
32
key (Macaca muk~~ta). Actu endocr.. Copenh. 78 (1975) 91-109. Setchell K. D. R., Axelson M., Sjiivall J. and Gontsharrow N. P.: The identification of 3a.61,l I/I.l7a,2I-pentahydroxy-S/?-pregnan-2&one (6/?-hydroxy-THFb The major urinary steroid of the baboon (Papio papio). J. sreroid Biochem. 7 (1976) 801-808. Setchell K. D. R.. Axelson M., Simarina A. I. and Gontscharow N. P.: Urinary steroid excretion and conjugation by the babooc (Papio hamodryo+A comprehensive study. J. sreroid Biochem. 7 (1976) 809-8 16. Setchell K. D. R.: 6_Hydroxylation, an important route in the metabolism of corticosteroids by the baboon: The fate of administered ietrahydrocortisol. FEES Lerr. 70 (1976) 197-200. Setchell K. D. R., Axelson M., SjGvall J., Kirk D. N. and Morgan R. E.: The identification of 6a-hydroxytetrahydrocortisol (3a,6a,l I/?,l7a,21-pentahydroxy-S/3pregnan-20-one) in urine. FEES Lett. 88 (1978) 215-218. Setchell K. D. R., Chua K.-S. and Himsworth R. L.: Urinary steroid excretion by the squirrel monkey (Saimuri sciureus). J. Endocr. 73 (1977) 365-375. Geiman Q. M. and Meagher M. J.: Susceptibility of New World monkey to PIasmodium falciparum from man. Nature, Land. 215 (1967) 437. Voller A., Green G. J. and Richards W. H. G.: Cross immunity studies with East and West African strains of Plasmodium falciparum in owl monkeys (Aotus rriuirgarus). Am. J. Trop. Med. Hyg. 76 (1973) 135-139. Hayes D. E. and Ward R. A.: Sporozoite transmission of falciparum malaria (Burma-Thai strain) from man to Aotus monkey. Am. J. Trop. Med. Hyg. 26 (1977) 184-185. Schmidt L. H.: PIasmadiumfalciparum and PIasmodium uiua.x infections in the owl monkey (AOWS triuirgatus). 1. The course of untreated infections, II. Responses to chloroquinine, quinine 11. and pyrimethamine. Methods employed in the search for new blood schizonticidal drugs. Am. J. Trop. Med. Hyg. 27 (1978) 671-737. Setchell K. D. R., Almt B., Axelson M. and SjGvall J.: The multicomponent analysis of conjugates ol neutral steroids in urine by lipophilic ion exchange chroma-
33.
34.
35.
36.
37.
38.
39.
tography and computerized gas chromatography-mass spectrometry. J. steroid Biochem. 7 (1976) 615-629. Setchell K. D. R. and Shackleton C. H. L.: The group separation of plasma and urinary steroids by column chromatography on Sephadex LH-20. Clin. Chim. Acm 47 (1973) 381-388. Thenot J.-P. and Horning E. C.: MC%TMS derivatives of human urinary steroids for GC and GC-MS studies. Analyt. ht. 5 (1972) 21-33. Axelson M. and Sjiivall J.: Separation and computerized gas chomatography-mass spectrometry of unconjugated neutral steroids in plasma. J. steroid &o&em. 5 (1974) 733-738. Van den Berg P. M. J. and Cox T. P. H.: An all glass solid sampling device for open-tubular columns in gas chromatography. Chromorogrophia S (I 972) 301-304. Bonney R. C. and S&hell K. D. R.: The excretion of gonadal steroids during the reproductive cycle of the owl monkey (Aotus rribirgotus). J. steroid Biochem 12 (1980) 417421. Plant T. M., James V. H. T. and Michael R. P.: Conversion of [4-‘*Cl-progesterone to androsterone by female rhesus monkeys (Macaca mulatta). J. Endocr. 51 (1971) 751-761. Setchell K. D. R., Bull. R. and Adlercreutz, H.: Steroid excretion during the reproductive cycle and in pregnancy of the v&vet m&key (Cercopithecus aeth;op;s ovaervthrus). J. steroid Biochem. 12 (1980) 375-384. Allen, J. G:, ‘Thomas, G. H.. Brooks. c. J. W. and Knights, B. A.: The determination of stereochemistry at C-5 of 3,6-dioxygenated steroids. Steroids 13 (1969) 133-142. Vouros P. and Harvey, D. J.: Method for selective introduction of trimethylsilyl and perdeuterotrimethylsilyl groups in hydroxy steroids and its utility in mass spcctrometric interpretations. Analyt. Chem. 45 (1973) 7-12. Gustafsson, J.-A. and Sjiivall, J.: Steroids in germfree and conventional rats-6. Identification of l52- and 2l-hydroxylated C1, steroids in faeczs from germfree rats. Eur. J. Biochem. 6 (1968) 236247. Brooks, C. J. W. and Harvey, D. J.: Gas chromatographic and mass spectrometric studies of oximes derived from 20-oxosteroids. Steroids 15 (1970) 283-301. ..”
40.
41.
42.
43.
43
_
(lotus triuirgatus) fulfils an important role as a laboratory model for research into human malaria [28-3 I]. Studies of the reproductive endocrinology of this speties are necessary for the establishment of a captive breeding programme, and essential to such studies is an accurate qualitative assessment of steroid metab-
INTRODUCTION There have been a number of investigations
of steroid metabolism and excretion in several species of Old World monkeys and apes [l-26]. Similar studies in New Wdrld monkeys have to date been restricted to the marmoset (Callithrix jacchus) [IS, 191 and the squirrel monkey (Saimari sciureus) [27]. The work of these authors indicates that metaboiic routes of steroid excretion by New World monkeys differ from those of other primates. New World monkeys are being used increasingly in medical research, and among them, the owl monkey
Olhll. The present communication
describes the identification, using wall coated open tubular glass capillary column gas chromato~aphy and mass spectrometry (GC-MS), of the major steroid metabolites excreted in urine from adult monkeys of this species. MATERIALS AND METHODS
The following trivial names and abbreviations are used in this presentation: Oestrone. 3-hydroxy-~,3,S(lO~estratrien-l7-one; Oestradiol-I 78, ~~3,S(lO~es~ra~riene-3,~7~diol; Testosterone, 17/J-hydroxy-4-androsten-3-one; Androsterone. 3z-hydroxy-S/7-androstan-17-one; Dehydre epiandrosterone, 3/I-hydroxy-S-androsten-17-one; I l-oxoandrosterone. 32.hydroxy-5&androstane-3.1 I-dione; 1l&Hydroxyandrosterone, 31.1 Ifi-dihydroxy-5&androstan- 1‘I-one; Pregnanediol, SB-pregnane-3z,20ct-diol; alloPregnanediol, Sz-pre~ane-3~2~-diol; Progesterone, 4-pregnene-3,20-dione; Pregnanolone, 3a-hydroxy-5/?-pregnan-2%one; 68-Hydroxypregnanolone. 3r,6B-DihydroxySz-pregnan-20-one; 17-Hydroxypregnanolone. 32.172dihydroxy-S/f-pregnan-20-one; l&Hidroxypregnanolone, 32.16z-dihydroxy-5n(B)-pregnan-2O-one; Tetrahydrocortisol. 3x.1 I/?.17z,21-tetrahydroxy-5&pregnan-20-one; at/oTeetrahydrocortisol. 32.1 l~,ij~,2l-tet~~ydroxy-5=-pre~ nan-20-one; Cortolone, 32,17cr20a(BLZI-tetrahydroxv-5& pregnan-1 I-one; Cortisol, I I/?,1‘Isti&ihydrox$4_pr&&ne-3,20-dione; 6&Hydroxycortisol. @,I 1&17a,21-tetrahydroxy-Qpregnene- 3,20-dione; ZO-Dihydrocortisol, 11/?,172, 202&%,21-tetrahydroxy-Q_pregnene- 32O-dione; MO-TMS ether, methyloxime-trimethyisilyl ether derivative; m/z mass/charge ratio; rR = retention time. 37
Animals
Animals were housed in individual cages in conditions of controlled temperature (2s25°C) and lighting (12 h artificial light, 12 h dim red light). The cages were fitted with removable sloping trays with funnels in the base to assist in the collection of urine. Daily urine collections were made and the samples stored at -20°C until required for analysis. Extraction The
of steroids
analytical procedures have been described in detail elsewhere [24,32] and are therefore outlined briefly. Steroids were extracted from urine samples using small columns of the neutral resin Amberlite XAD-2. Following hydrolysis of steroid conjugates with a mixed /?-glucuronidase and sulphatase enzyme prep aration (Helix pomatia, L’lndustrie Biologique Francaise) the “freed” steroids were re-extracted on
K. D. R. SETCHELLand R. C. BONNEY
38
Amberlite XAD-2 and this extract was then passed through a column of Sephadex LH-20 (6g, cyclohexane: ethanol, 4: 1, v/v) to effect a purification [33]. Chromate oxidation
The dried steroid extract was dissolved in acetone (0.5 ml) and 3 drops of Jones reagent (prepared by dissolving 26.7 g of chromium trioxide in 23 ml of concentrated sulphuric acid and diluting to 100 ml with distilled water) added. The sample was shaken for 2 h at room temperature and then diluted with lOOm1 distilled water. The reaction products were then extracted on small columns of Amberlite XAD-2 resin. A methyloxime derivative was prepared of the dried extract. Derivatisation
0-Methyloximetrimethylsilyl (MO-TMS) ether derivatives were prepared of proportions of the tottal steroid extract {34] and removal of the non-volatile silylating reagent and further purification of the extract was carried out using small columns of Lipidex 5000 [353; this step being essential to enable GC analysis on open tubular capillary columns where the samples are introduced by solid injection. Gas chromatography (CC)
Gas chromatographic analysis was performed using a Pye 104 gas chromatograph equipped with flame
ionisation detectors and modified to accept a 25 m wall coated open-tubular glass (WCOT) capillary column coated with Silicone OV-1. Samples were applied to the column via a solid injection’system similar to that described by Van de Berg & Cox[36]. Helium was used as a carrier gas and the flow rate through the column was approx. 2ml/min. GC was carried out using temperature programming conditions; 185-275°C with increments of Z”C/min. Gas chromatography-mass spectrometry (CC-MS)
GC-MS was performed using a Varian MAT-731 double focusing mass spectrometer. The conditions for mass spectrometry were as follows: temperature of separator and transfer line, 250°C; temperature of ion source, 250°C; ionization current, 800 PA; ionization voltage, 70 eV; accelerating voltage, 8 kV. Repetitive magnetic scanning over the mass range &loo0 atomic mass units (amu) was the mode of analysis. Steroid identification The identification of a steroid was based upon the gas chromatographic retention time (tR) of the steroid derivative relative to homologous series of n-alkanes, C24-C34 (methylene units, MU) and the complete mass spectrum when compared with the authentic steroid. In many instances, due to the non-availability of reference steroids and particularly with compounds present at extremely low levels, it was only possible to
20 Time (min) Fig. 1. The gas chromatographic separation using a wall coated open tubular capillary column of steroids excreted in the urine of the adult female owl monkey (Aotus triciryatus) during the peri-ovulatory period. The analysis was performed by a Pye 104 gas chromatograph housing a 25m capillary column coated with silicone OV-1. Samples were introduced Ga an automatic solid injection system. Helium was the carrier gas and the flow rate through the column was ca. Zml/min. Temperature programmed operation from 175-275°C in increments Gf 2,2’C/min. was used. The principal steroids are indicated as follows: 1. androsterone, 2 aetiocholanolone, 3. oestrone, 4 oestradiol-17J, 5. 17-hydroxypregnanolone. 6. Sa-pregnane-3a,2Oa-diol, 7. 5/Lpregnane-3a.2Oa-dial, 8. pregnanetriol, 9. 6/?-hydroxypregnanolone, 10. 5-pregnene-3/?,2Oa-dial, 11. 3a,16a-dihydroxy-Sa-pregan-20_one, 12 3a,l6a-dihydroxy-5fi-pregnan-20-one.
39
The excretion of urinary steroids by the owl monkey Table 1. List of steroids identified by gas chromatography-mass spectrometry trivirgatus) Retention time (MU)
in the urine of the owl monkey (Aotus
Male?
Steroid
3a-Hydroxy-S-androsten-17-one 3x-Hydroxy-SB-androstan-17-one (Androsterone) 3/3-Hydroxy-5-androsten-17-one (DHA) 3@Hydroxy-Sa-androstan-17-one 3%.Hydroxy-5&androstanc- : I,l7-dione (1 I-oxoandrosteronc) 3-Hydroxy-l,3,S(lO~estratrien-l7-one (Oestrone) 1,3,4(lO~Estratiene-3,17/$diol (Oestradiol) I ‘I/?-Hydroxy-4-androsten-3-one (Testosterone) Androstane-dial-I l-one 26.70 3a-Hydroxy-S/3-pregnan-20-one (Pregnanolone) 3c(,l7a-Dihydroxy-S/l-pregnan-20-one (17-Hydroxypregnanolone) 27.00 3x.1 l/3-Dihydroxy-5/l-androstan-17-one 27.08 3a,l l,%Dihydroxy-Sa-androstan-1Fone 27.18 27.20 Pregnene-dial-one 27.25 S-Pregnene-3,I lJO-trio1 27.25 Pregnane-diol-one 27.30 5-Androstene-38,16,17-trio1 Androstane-3,16,17-trio1 27.40 27.45 5c(-Pregnane-3a,20a-diol (a/lo-pregnanediol) Unknown Compound 180/442$ 27.50 27.61 SB-Pregnane-3a,20a-dial (Pre~ane~ol) 27.90 S~-Pre~ane-3a,l7~,2Oa-triol (Pre~anet~ol) 28.18 346B-Dihydroxy-5a-pregnan-20-one(6~-Hydroxypregnanoione) 28.33 5-Pregnene-3p,20a-diol 3~.16a-Dihydroxy-5a-pregnan-20-one 28.40 3a,l6a-Dihydroxy-5fl-pregnan-20-one 28.50 35,162-Dihdyroxy-5-pregnan-20-one 28.60 29.00 Pregnane-triol-one 29.10 Pregnane-trio1 Pregnane-tetrol (16-hydroxysteroid) 29.25 3a,l7~,2l-Trihydroxy-S~-pre~ane-l1,20-dione (tetrahydroco~isone) 29.60 3r,l1/3,17z,21-Tetrahydroxy-5/?-pregnan-1O-one (tetrahydrocortisol) 30.10 30.51 3x,1 7a,20a,21-Tetrahydroxy-S/3-pregnan-1 l-one (a-cortolone) 30.73 3a,l7a,20/l,21-Tetrahydroxy-S~-pregnan-ll-one (/3-cortolone) 30.73 5~-Pregnane-3a,11/?,17a,20/?-21-pent01 (/l-cortol) 31.20 5~-Ptegnane-3a,l1~,17a,2Oa,21-pento1 (cc-cortol) 31.02 5a.6a,ll~,l7a,2l-Pentahydroxy-S~-pre~~-~one (~-hydroxy-tetrahydrocortis 32.88 17a,2O~,Zl-Trihydroxy-4-pre~ene-3,ll-dione (~~-dihydrocortisone) 32.95 17a,20a,2l-Trihydroxy-4-pregnene-3,1l-dione (20x-dihydrocortisone) 32.54 + 32.60 1lfi,17a,21-Trihydroxy-4-pregnene-3,20-dione (cortisol& 32.70 + 32.88 6&l lfl,l7a,2t-Tetrahydroxy-4-pregnene-3,2Odione (6/i-hydroxycortisol) 32.91 + 33.03 11~,17c1,20~,21-Tetrahydroxy-4-pregnen-3-one (208-dihydrocortisol)§ 33.34 + 33.47 1lj,l7a,20a,Zl-Tetrahydroxy-4-pregene-3-one (20a-dihydrocortisol)$
Female
+ + c + c + +
24.70 25.3 I 25.73 25.82 26.12 26.16 26.30 26.36
+ +
4
+ f
+ + +
c f +
+
-Iif f + f f
+ +
+ + -+ f f f -k f +
+ + + + + + + + + + + + +
f + + + + -t + + + + + f f
* In some cases it is only possible to give a general structure to the steroid identified. For example, pre~ane-dial-one implies a pregnane steroid having two hydroxyt and one 0x0 group. t A steroid found to be present by GC-MS in urine is indicated by +. 3 Details of this compound have been presented elsewhere [39] # These steroids give rise to two peaks which are resolved in the chromatogram due to the separation of the syn- and anti- forms of the methyloxime function on the 3-0~0 group.
assign a general structure based upon the mass spectrometric data; thus for example, androstanediolone implies the steroid has a C,9 nucleus with two hydroxyl groups and one oxo group.
Table I indicates those steroids which have been identified by GC-MS in the urine from both male and female animals, and the principal urinary steroids excreted are discussed in more detail below. Oestrogens
RESULTS AND DISCUSSION
Figure 1 illustrates a GC chromatogram MO-TMS ether derivatives of a urinary extract from an adult female animal typical peri-ovulatory period and analyzed using coated open tubular glass capillary column.
of the steroid of the a wall
Oestrone and oestradiol-178 were the principal oestrogens excreted by the female animal. Quantitative data on the excretion of oestrone and oestradiol-178 in the urine of non-pregnant female animals are reported elsewhere [373 and have demonstrated that the amounts of oestrone excreted in the peri-ovula-
40
K. D. R. SETCHELL and R. C. BONNEY
tory period are approximately 3-fold greater than oestradiol-I 78 and this is evident from the gas chromatogram of the female urine obtained during this period (Fig. 1). Studies of oestrogen excretion by other New World monkeys are limited to the marmoset in which oestradiol-178 is excreted in greater amounts than both oestrone and oestriol[18]. Compared with other subhuman primates both the qualitative and quantitative patterns seem to resemble that of the orang utan [ 163. A positive identification of oestrone in the urine from male animals was obtained from a comparison of the complete mass spectrum and retention data of its MO-TMS ether derivative (tR = 26.16 MU) with that of the authentic steroid (tR = 26.16 MU). It is therefore evident from this observation that a relatively large amount of oestrone must be excreted in urine of male animals (although quantitative data were not obtained) because when GC-MS is employed in the repetitive magnetic scanning mode, due to its limited sensitivity (10 ng) it is generally not possible to obtain complete mass spectra of oestrogens in small volumes of urine from human male subjects or for that matter non-pregnant women. When computer reconstructed fragment ion current chromatograms are plotted of ions (m/z) characteristic of MO-TMS ether derivatives of oestrone and testosterone after the repetitive magnetic scanning GC-MS analysis of an extract from a male ahimal (Fig. 2), it is evident from the peak areas of the characteristic ions for each steroid, that there are “apparently” greater amounts of oestrone than testosterone in the urine. The apparent large amounts of oestrone present in the urine of male animals is an interesting finding and could be supported by data obtained using radioimmunoassay which have shown high levels of oestrone (1 ng/ml) in the plasma of male animals (Bonney, unpublished).
C,&eroids
‘Androsterone and dehydroepiandrosterone were identified in urine of both male and female animals together with the 1I-oxygenated-17-oxosteroids, 1l-oxoandrosterone and 1l/%hydroxyandrosterone. Testosterone was identified as the MO-TMS ether in extracts of urine from male animals (Fig. 2). However, no evidence was obtained for the presence of testosterone in the urine of female animals in spite of finding that plasma testosterone levels in female animals were as high as long/ml when radioimmunoassay was used (Bonney, Dixson & Fleming unpublished data). Progesterone
metabolites
Qualitatively the major proportion of steroids excreted by the female animal comprised matabolites of progesterone. pregnanediol (SD-pregnane-3a,20a-dial; Both tR = 27.61 MU, as TMS ether derivative) and allopregnanediol (Sa-pregnane-3aJOa,-diol; tR = 27.45 MU, as the TMS ether derivative) were identified, the latter isomer being present in greater amounts. However, these metabolites quantitatively accounted for less than 30% of the total progesterone metabolites excreted [37]. The major steroid excreted in the urine gave a retention time of 28.18 MU as the MO-TMS ether derivative and the mass spectrum of this steroid is seen in Fig. 3. The molecular ion M+ was at m/z 507 indicating the general structure to be that of a hydroxypregnanolone. The existence of the ion at m/z 476 formed by the loss of 31 mass units is due to the presence of a derivatized 0x0 group. The presence of the intense ions of m/z 100, 87 and 70 confirms that the 0x0 group is at the C-20 position and that there are no other functional groups in the side chain [43].
AOTUS tlONKEY MILE Fig 2. Selected ion current chromatograms of ions characteristic of MO-TMS ether derivatives of oestrone (m/z 371, 340) and testosterone (m/z 389, 358, 268) recorded during the repetitive magnetic scanning GC-MS analysis of a urine extract from a male owl monkey.
The excretion of urinary steroids by the owl monkey
3aci
41
476
1
I
296
m
Fig. 3. The complete mass spectrum of the MO-TMS ether derivative of major steroid excreted in the urine of the female owl monkey, and subsequently identified as 3a,6@Iihydroxy-Sa-pregnan-20-one. This triplet of ions arises from the loss of the side chain together with carbon atom C-16 and its subsequent fragmentation, and is typical of MO derivatives of all 21-deoxy-20-oxosteroids. The presence of two derivatized hydroxyl groups is evident from the ions m/z 386 [M-(90 + 31)] and m/z 296 [M-(2 x 90 + 31)]. The relative ease of formation of the ion at m/z 386 is evident, from the mass spectrum, in the large pro portion of the total ionization carried by this ion, suggesting its loss from a labile position in the molecule and since the mass spectra of 21-hydroxy-, 17-hydroxy-, 16_hydroxy- and 1S-hydroxy- pregnant lones showed significant differences in their fragmentation patterns it was considered that this steroid was possibly a 6-hydroxy-pregnanolone structure. Furthermore, 6hydroxylation is commonplace in many primate species, the baboon 123-261 and the marmoset [ 183 both excrete a large amount of 6-hydroxylated steroids in urine. Confirmation that this urinary steroid was a 6-hydroxy-pregnanolone was obtained following chromate oxidation to the pregnane-3,6,20-trione structure, a well documented procedure [23,40] which in addition enables the stereochemistry at the C-S position to be determined from the gas chromatographic behaviour and fragmentation pattern of the methyloxime derivative of the product [40]. The oxidation reaction was checked using the reference steroids 3a&-dihydroxyS/?-androstan-17-one and 3x,6x,1 l&trihydroxy-5& androstan-17-one to ensure that epimerization of these S/Y-steroids to the corresponding Sa-compounds did not occur under the acid conditions employed The gas chromatographic behaviour of the methyloxime derivative of the product of chromate oxidation gave rise to two peaks of equal intensity with retention indices te = 30.00 MU and r,r = 30.23 MU indicating the stereochemistry of the hydrogen at C-5 to be a configuration [4O]. The gas chromatographic behaviour and retention indices where shown to be identical to those described by Allen et al. [&I] for methyloxime derivative of the authentic steroid 5a-pregnane-3,6,2@trione. Furthermore the mass +1.14/l--o
spectrum was similar to the authentic steroid reported elsewhere[40] exhibiting the characteristic ion m/z 138 which as suggested by Allen et al. [40] arises out of an initial cleavage of the C-5:6 bond followed by the transfer of a hydrogen atom from a site in ring A to a position in ring B and finally homolytic fission of the C-9:10 bond. The relatively low intensity of the m/z 138 ion in the spectrum further conhrtned the stereochemistry of the hydrogen at C-5 to be of the a-configuration. The retention time of the MG-TMS ether derivative of the urinary steroid (ts = 28.18 MU) was found to be less than any reference 6a-hydroxy-pregnant lone-MGTMS ether and identical with that reported for the same derivative of 6/?-hydroxy-pregnanolone identified in the urine of marmosets [18 191. Since the order of GC elution of MG-TMS ether derivatives of pregnanolone isomers on the non-selective phase, silicone OV-1, is 3a,5a, > 345/f > 3/?,5a (3/%5/3steroids, although rarely occurring in animals have the shortest tR values) and having proven a Sa- steroid it was deduced that the major urinary steroid excreted by this animal had the structure 3a,6fidihydroxy-Sapregnan-2@one. 6/?-Hydroxypregnanolone has also been identified as one of the major steroids excreted by non-pregnant marmosets Cl93 and in pregnancy is one of the major progesterone metabolites of that species [18]. While the pregnant owl monkey has not been studied, quantitatively this steroid accounts for > so”/, of the pro gesterone metabolites in non-pregnant animals 1373 and the GC urinary steroid profile (Fig. 1) resembles that of the pregnant marmoset monkey [ 183. Other hydroxylated pregnanolone metabolites were also found in relatively large amounts in the urine of the female animals, notably 17-hydroxypregnanolone (3aJ7a-dihydroxy-5fl-pregnan-2O-one) and two 16hydroxypregnanolone isomers. The mass spectra of the MO-TMS ether derivatives of all of these hydroxylated pregnanolones are very similar; the parent ion being at mJz 507 and base peaks at mjz 476, however, from our experience [32] they can be differentiated by the presence of prominent structurally sig-
42
K. D. R.
SETCHEU
nificant ions of m/r 156. I58 and 188 for 17-hydroxypregnanolones arising out of cleavage between carbon bonds C-17/C- IS and C-l S/C-l 6 and fragmentation of the side chain and methyloxime group [41]. It should be noted that MO-TMS ethers of 2l-hydroxypregnanolones in addition to all 21-hydroxy- 1‘I-deoxysteroids are readily distinguished because they give rise to prominent ions, which are often the base peaks at m/z 175 and 188, while 15-hydroxy-pregnanolones exhibit intense ions at m/z 170 and 201 in the mass spew tra [32,42]. Although pregnanediol isomers are formed by the owl monkey, it is interesting that 6- and 16-hydroxylation are the most important metabolic transformations undergone by progesterone in the non-pregnant animal When compared with other species of sub-human primates, progesterone metabolism by the owl monkey most closely resembfes that of the marmoset Cl93 and differs from “Old World“ macaque monkeys in which side chain cleavage occurs giving rise to androsterone [IO, 38’J. Polar steroids
A wide spectrum of polar steroid metabolites was identified. A number of unusual tetrahydrocorticosteroids were present. The molecular ion (M+) in their spectra was at m/z 683 indicting a MO-TMS ether of a pregnane-tetrolone, however due to the absence of any ions characteristic of a dihydroxyacetone side chain, glycol side chain or a 21-hydroxy-20.oxo-l7-deoxy side chain and due to the presence of ions of m/z 156, 158 and 188, it is tentatively suaested that these steroids are possibly 3,6,16,17-tetrahydroxypregnan-20-one or 3,11,16,17-tetrahydroxyprcgnan-20.one isomers. Due to the lack of authentic steroids however, their identification is tentative and remains to he confirmed. Corticosteroid metabolism was characterized by the presence of tetrahydrocortisone, tetrahydrocortisol and a number of cortolone isomers Cortisol, 20.dihydrocortisol, and not surprisingly, in view of the efiicient 6~-hydroxyIation system, 6~hydroxycortisol, were the most prominent corticosteroid metabelites excreted and in general the qualitative excretion of corticosteroid metabolites was similar to that for other New World primates [ 19,2A.
Acknowledgements-The authors are grateful for the technical assistance given by Mr M. J. Mad&an in the work,
and to Mr D. Fleming for colkction of urine sampkgThis investigation received support from the Malaria Component of the UNDP~~~ Bank/WHO Special Programme for Reserach and Training in Tropical Diseases.
REFERENCES
l. Kirdani R. V., Gleason T. L and Layne D. S.: The metabolism of aldosterone in the chimpanzee. Gen. camp. Endocr. 3 (1963) 616-620.
and R. C. BONNEY 2. Layne D. S., Kirdani R. V., Gleason T. L. and Pincus G.: The secretion of cortisol in immature chimpanzees. Gen. camp. Endoer. 4 119631155-188. 3. Shackle& C. H. L.,‘M&hell F. L., Gus&son J.-A. and Sjavall J.: Steroids in primates: Excretion of 31-hydroxy-A’ steroids by a newborn chimpazee (Pan rriglcdytes). FEBS L41t. 2 (1970) 129-131. 4. Pal S. 8.: The pattern of urinary excretion of individual l7-ketosteroids in rhesus monkeys. Acta en&r. Cope&. 67 (1971) 71 I-720. 5. Shackleton C. H. L and Gustafsson J-A: Steroids in newborns and infants. Identification by combined gas chromatography-mass spectrometry of the major steroids excreted by a newborn chimpanzee (Pun triglodyres) Steroids 18 (1971) 175-186. 6. Manson M. E., Shackkton C. H. L., Mitchell F. L, Gustafsson J.-A. and Sj6vall J.: Steroids in primates: 5-Androstene-3/?,16al7@iol excretion by a pregnant irus monkey (Monaco jascicularis). Steroids 18 (1971) 51-57. 7. Gokizeiher J. W. and Axelrod L. R.: Urinary metabotites of4-‘*C-progesterone in the baboon (Papio spp.). Gen. camp. Endocr. 13 (1969) 201-205. 8. Townsley J. D.: Steroid hormones in pregnancy: The baboon as a model for studies on oestrogen synthesis. Acta endocr,, Cope& Suppl. 166 (1972) 191-197. 9. Solomon S. and Leung K.: Steroid hormones in nonhuman primates during pregnancy. Acta endocr., Cope& Suppf. 166 (1972) 178-190. 10. Reddy V. V., Balin H. and Nes W. R.: Tbe metabolism of progesterone ‘by the rhesis monkey (Mocacn mulutta). Steroids 17 (1971) 493-502. Il. Ishihara M., Gsawa Y., Kirdani R. Y. and Sandberg A. A.: Progesterone metabolism in the baboon. J. steroid B&hem. 6 (1975) 1213-1218. 12. Yamamoto Y., Manyon A., Kirdani R. Y. and Sandberg A. A.: Androgei metabolism in the rhesus monkev. Sleroids 31 1197817 I l-729. 13. Tiwnsley J. D.:‘Oes&ogen excretion of the pregnant baboon (Papio pap&). E~d~ri~~l~y 95 (1974) l759- 1762. 14. Pepe G. J. and Townsley J. D.: Cortisol metabolism in female baboons (Popio papio). Endocrinology 95 (1974) 1658-1663. 15, Pepe G. J. and Townsley J. D.: The metabolism of cortisoi by term baboon neonates (fapio pupjo).E~d~* rinology 99 (1976)-9. 16. Collins D. C., Graham C. E. and Reedy J. R. K.: Identification and measurement of urinary oestrone, oestradiol- I ‘I/?,oestriol, pregnanediol and androsterone during the menstrual cyck of the orang utan. EndocrinoIogy96 (1975) 93-101: 17 Shackkton C. H. L: Steroid excretion in the neonatal period: A comparative study of the excretion of steroids by human ape and rhesus monkey infants. J. steroid Biochem. 5 (I 974) I I 3- 1IS. 18. Shackkton C. H. L: progesterone and oestrogen metabolism in the pregnant marmoset (C~l~f~r~x jac&us). .I. steroid B&hem. 5 (1974) 597-600. 19. Shackkton C. H. L: The excretion of steroids by the adult marmoset monkey (Collithhrix jacchur). J. steroid B&hem. 6 (1975) 1429-1432. 20. Shackkton C. H. L and Mitchell F. L: The comparison of perinatal steroid endocrinology in simians with a view to finding a suitabk animai model to study human probkms Lab. Anim. Jfrmdbks (1975) 159-181. 21. Setchell K. D. R.. Gontscharow N. P., Ax&on M. and Sjiivall J. : The characterisation of polar corticosteroids in the urine of the macaque monkey (Macaca&seieutaris) and the baboon (Pupio homadryos). Acto endocr., copenic. 79 (1975) 53~550. 22. Setchell K. D. R. and Shackkton C. H. L.: Cortisol and corticosterone metabolism in the macaque mon-
The excretion of urinary steroids by the owl monkey
23.
24.
25.
26.
27
28
29
30
31
32
key (Macaca muk~~ta). Actu endocr.. Copenh. 78 (1975) 91-109. Setchell K. D. R., Axelson M., Sjiivall J. and Gontsharrow N. P.: The identification of 3a.61,l I/I.l7a,2I-pentahydroxy-S/?-pregnan-2&one (6/?-hydroxy-THFb The major urinary steroid of the baboon (Papio papio). J. sreroid Biochem. 7 (1976) 801-808. Setchell K. D. R.. Axelson M., Simarina A. I. and Gontscharow N. P.: Urinary steroid excretion and conjugation by the babooc (Papio hamodryo+A comprehensive study. J. sreroid Biochem. 7 (1976) 809-8 16. Setchell K. D. R.: 6_Hydroxylation, an important route in the metabolism of corticosteroids by the baboon: The fate of administered ietrahydrocortisol. FEES Lerr. 70 (1976) 197-200. Setchell K. D. R., Axelson M., SjGvall J., Kirk D. N. and Morgan R. E.: The identification of 6a-hydroxytetrahydrocortisol (3a,6a,l I/?,l7a,21-pentahydroxy-S/3pregnan-20-one) in urine. FEES Lett. 88 (1978) 215-218. Setchell K. D. R., Chua K.-S. and Himsworth R. L.: Urinary steroid excretion by the squirrel monkey (Saimuri sciureus). J. Endocr. 73 (1977) 365-375. Geiman Q. M. and Meagher M. J.: Susceptibility of New World monkey to PIasmodium falciparum from man. Nature, Land. 215 (1967) 437. Voller A., Green G. J. and Richards W. H. G.: Cross immunity studies with East and West African strains of Plasmodium falciparum in owl monkeys (Aotus rriuirgarus). Am. J. Trop. Med. Hyg. 76 (1973) 135-139. Hayes D. E. and Ward R. A.: Sporozoite transmission of falciparum malaria (Burma-Thai strain) from man to Aotus monkey. Am. J. Trop. Med. Hyg. 26 (1977) 184-185. Schmidt L. H.: PIasmadiumfalciparum and PIasmodium uiua.x infections in the owl monkey (AOWS triuirgatus). 1. The course of untreated infections, II. Responses to chloroquinine, quinine 11. and pyrimethamine. Methods employed in the search for new blood schizonticidal drugs. Am. J. Trop. Med. Hyg. 27 (1978) 671-737. Setchell K. D. R., Almt B., Axelson M. and SjGvall J.: The multicomponent analysis of conjugates ol neutral steroids in urine by lipophilic ion exchange chroma-
33.
34.
35.
36.
37.
38.
39.
tography and computerized gas chromatography-mass spectrometry. J. steroid Biochem. 7 (1976) 615-629. Setchell K. D. R. and Shackleton C. H. L.: The group separation of plasma and urinary steroids by column chromatography on Sephadex LH-20. Clin. Chim. Acm 47 (1973) 381-388. Thenot J.-P. and Horning E. C.: MC%TMS derivatives of human urinary steroids for GC and GC-MS studies. Analyt. ht. 5 (1972) 21-33. Axelson M. and Sjiivall J.: Separation and computerized gas chomatography-mass spectrometry of unconjugated neutral steroids in plasma. J. steroid &o&em. 5 (1974) 733-738. Van den Berg P. M. J. and Cox T. P. H.: An all glass solid sampling device for open-tubular columns in gas chromatography. Chromorogrophia S (I 972) 301-304. Bonney R. C. and S&hell K. D. R.: The excretion of gonadal steroids during the reproductive cycle of the owl monkey (Aotus rribirgotus). J. steroid Biochem 12 (1980) 417421. Plant T. M., James V. H. T. and Michael R. P.: Conversion of [4-‘*Cl-progesterone to androsterone by female rhesus monkeys (Macaca mulatta). J. Endocr. 51 (1971) 751-761. Setchell K. D. R., Bull. R. and Adlercreutz, H.: Steroid excretion during the reproductive cycle and in pregnancy of the v&vet m&key (Cercopithecus aeth;op;s ovaervthrus). J. steroid Biochem. 12 (1980) 375-384. Allen, J. G:, ‘Thomas, G. H.. Brooks. c. J. W. and Knights, B. A.: The determination of stereochemistry at C-5 of 3,6-dioxygenated steroids. Steroids 13 (1969) 133-142. Vouros P. and Harvey, D. J.: Method for selective introduction of trimethylsilyl and perdeuterotrimethylsilyl groups in hydroxy steroids and its utility in mass spcctrometric interpretations. Analyt. Chem. 45 (1973) 7-12. Gustafsson, J.-A. and Sjiivall, J.: Steroids in germfree and conventional rats-6. Identification of l52- and 2l-hydroxylated C1, steroids in faeczs from germfree rats. Eur. J. Biochem. 6 (1968) 236247. Brooks, C. J. W. and Harvey, D. J.: Gas chromatographic and mass spectrometric studies of oximes derived from 20-oxosteroids. Steroids 15 (1970) 283-301. ..”
40.
41.
42.
43.
43
_