Department of Chemistry, Karolinska Institutet, Stockholm1) and Department of Obstetrics and Gynaecology, Royal Veterinary College, Stockholm2)
IDENTIFICATION AND QUANTITATIVE DETERMINATION OF STEROIDS IN BOVINE CORPUS LUTEUM DURING OESTROUS CYCLE AND PREGNANCY
By M. Axelson, G. Schumacher, J. Sj\l=o"\vall, B. Gustafsson and J. O. Lindell ABSTRACT Neutral steroids in bovine corpus luteum were isolated by liquid-gel chromatography on hydrophobic Sephadex, and were analyzed by computerized gas chromatography-mass spectrometry. The presence of pro-
gesterone and 20\g=b\-hydroxy-4pregne-3one
was
confirmed. In addition,
-diol, 3\g=b\-hydroxy-5\g=a\\x=req-\ hydroxy- 5- pregnen -20- one, 5- pregnenewere fully identified, and pregnan-20-one and 3-hydroxy-4-pregnen-20-onne, 4 pregnene-3,20-diol, 22-hydroxycholesterol and 20,22-dihydroxycholesterol were partially characterized. Steroid sulphates were not detected. Quantification of the six fully identified steroids was based on peak areas in specific fragment ion current chromatograms constructed by the computer. During the 4th\p=n-\19thday of the oestrous cycle the steroid concentrations varied as follows: progesterone 6.0\p=n-\36.7\g=m\g/g wet luteal weight, 20\g=b\-hydroxy-4-pregnen-3-one 0.8\p=n-\5.5 \g=m\g/g,3\g=b\-hydroxy-5pregn -20one 1.0\p=n-\7.1 \g=m\g/g,5-pregnene-3\g=b\,20\g=b\-diol< 0.2\p=n-\0.9 \g=m\g/g, 3\g=b\-hydroxy-5\g=a\-pregnan-20-one 1.7\p=n-\8.6 \g=m\g/g, and 5\g=a\-pregnane-3\g=b\,20\g=b\-diol< 0.2-1.2 \g=m\g/g,
3\g=b\-
3\g=b\,20\g=b\
5\g-a\pregna e-3\g=b\,20\g=b\-diol,32
The concentrations of progesterone and 3\g=b-hydrox-5pregn -20one seemed to vary in parallel and were low during days 11\p=n-\17. During this and 5\g=a\-pregnane\x=req-\ period the concentrations of 3\g=b\,20\g=b\-diolwere highest as was the relative contribution of all three 20\g=b\\x=req-\ hydroxysteroids to the total amount of steroids. The relative amount of 3\g=b-hydroxy-5\g=a-pregna-20one seemed to be highest during days 4\p=n-\6. The total steroid concentration in corpora lutea taken in early pregnancy 5-pregnene-3\g=b\,20\g=b\-diol
(75\p=n-\105 days) was 18\p=n-\47 \g=m\g/g. In the period 75\p=n-\90 days, progesterone only 35\p=n-\42 % of the total steroids, 3\g=b\-hydroxy-5\g=a\-pregnan\x=req-\
constituted
20-one as much as 23\p=n-\40 % and the total 20\g=b\-hydroxysteroids 18\p=n-\30%. The total steroid concentration in corpora lutea taken in midterm and late pregnancy was 21\p=n-\77 \g=m\g/g. In this period progesterone was by far the predominant steroid and constituted about 80\p=n-\90 % of the total steroids in corpora lutea taken between days 150 and 240. Possible correlations between luteal growth, steroid oxidoreductases and steroid concentrations are discussed.
and 20/3-hydroxy-4-pregnen-3-one have been identified in bovine luteum corpus (see Gomes Sc Erb 1965). The concentration of these steroids in the oestrous cycle and during pregnancy have been measured in many studies (see Gomes 8c Erb 1965; Erb et al. 1968e, 1971) and variations under different conditions have been investigated (Garverick et al. 1971; Wagner et al. 1972). The presence of 3/3-hydroxy-zl5-, 3/3-hydroxy-, 17/?-hydroxy-, and 20/?-hydroxysteroid oxidoreductases in corpus luteum has been demonstrated in several studies (see Savard 1973), and variations in the activities of these enzymes during the cycle have been reported (Brandan et al. 1972). However, the im¬ portance of these variations for luteal steroid production and metabolism is difficult to assess since substrates and products other than the two steroids mentioned above have not been found. For this reason it seemed important to make a more detailed study of steroids in luteal tissue. This paper describes the application of liquid-gel chromatography and computerized gas chromatography-mass spectrometry to the analysis of steroids in bovine corpus luteum. Six steroids have been identified and results of quantitative determinations of these steroids in the cycle and in pregnancy are reported.
Progesterone
EXPERIMENTAL
Corpora
lutea
Sixteen corpora lutea from non-pregnant cycling cows in different stages of the cycle and 17 corpora lutea from pregnant cows in different pregnancy stages were used. The cows were of the Swedish Red and White Breed (SRB). Four of the cycling animals were heifers, the others and the pregnant cows had calved once or twice. The cyclic corpora lutea were secured either at laparotomy under local anaesthesia or immediately after slaughter of the animals. They were directly cooled to -20°C and kept at that temperature until analyzed. The stage of the oestrous cycle was calculated from data of previous cycles in 11 cases (see Tables) with day 1 denoting the day of oestrus. The corpora lutea marked 25 and 27 in the Tables are regressing corpora lutea from the preceding cycle. In 5 cows the cyclic stage of the corpus luteum was evaluated by macroscopic examination of the genital organs, and by taking into particular consideration the size, vascularization and colour of corpus luteum and the status of the ovarian follicles. oestrous
The corpora lutea of pregnancy were in two cases secured immediately after slaughter. The pregnancy stage was determined by measuring the length of the foetus. In the remaining cases the conception date was exactly known and corpora lutea were obtained through laparotomy under local anaesthesia. The ovary was removed using an ecraseur and the corpus luteum was then immediately enucleated and frozen.
Chemicals
grade and were redistilled before use. Hexamethyltrimethylchlorosilane (Applied Science Laboratories, State College, were redistilled. Trimethylsilylimidazol was from Pierce Chemical Co. Pennsylvania) (Rockford, Illinois) and methoxyamine hydrochloride from Eastman Organic Chemicals (Rochester, New York). Amberlyst A-26, a strong anion exchanger from Rohm and Haas (Philadelphia, Pennsylvania) was washed and stored in the bicarbonate form in water (Sandberg et al. 1965). Hydroxyalkoxypropyl Sephadex (containing 50-55 °/o hydroxyalkyl (Cjj-C^) chains) was synthesized according to Ellingboe et al. (1970), or was obtained as Lipidex®-5000 from Packard-Becker Chemical Division (Groningen, Holland). Unlabelled steroids were from Ikapharm (Ramat-Gan, Israel) and Steraloids (Paw¬ ling, New York). They were purified by chromatography on alumina columns and were then crystallized and dried at 70°C in vacuum. All solvents disilazane and
were
of reagent
Radioactive steroids
[7a-3H] Progesterone (9.6 Ci/mmole), 3/?-hydroxy-5-[7a-3H]pregnen-20-one (18 Ci/ mmole) and 5/J-[l,2-3H]pregnane-3a,20a-diol (50 Ci/mmole) were from the Radio¬ chemical Centre (Amersham, England). 5-[7a-3H]Pregnene-3a,20/?-diol was obtained by NaBHj reduction of the labelled pregnenolone. Purity was assessed by chromato¬ graphy on Lipidex®-5000. Radioactivity was determined in a Packard Tri-Carb Liquid Scintillation Spectrometer using Instagel® as the scintillation liquid. Quenching cor¬ rection
was
made with
Isolation and
internal standard.
purification of steroids
The corpus luteum walled flask.
an
was
weighed
and
was
Chloroform/methanol, 1:1 (v/v),
sliced with a razor-blade into a thick100 ml, and 103 dpm of tritium-labelled
progesterone, 3/?-hydroxy-5-pregnen-20-one, 5-pregnene-3/5,20/?-diol
or
5/i-pregnane-
added. The mixture was homogenized with an Ultra-Turrax homogenizer (Janke-Kunkel, Staufen i. Br., Germany) and the slurry was kept in an ultra¬ sonic bath for 15 min. The extract was filtered into a round-bottomed flask, the residue was re-extracted in the same way, and finally 50 ml of chloroform/methanol was used to rinse the homogenizer and the filter. The solvents were removed in vacuum and the residue was weighed. Unconjugated neutral steroids in the extract were purified by chromatography on hydroxyalkoxypropyl Sephadex (Lipidex®-5000) (Axelson et al. 1974). Briefly, neutral lipids and cholesterol were first removed on a column in a reversed phase solvent system (methanol/water/chloroform, 9:1:2 (by vol.)) where steroids were eluted in the front fractions. Further purification was achieved on a column of Lipidex®-5000 in hexane/chloroform, 9:1 (v/v). All steroids with polarities between those of progesterone and corticosterone were collected from this column by elution with hexane/chloroform, 6:4 (v/v).
3«,20a-diol
was
Gas
chromatography-mass speclrometry
The steroids were analyzed as O-methyloxime trimethylsilyl (MO-TMS) derivatives, prepared as described by Gardiner Se Homing (1966). The methoximes were purified by filtration through a small column of Amberlyst A-26 in OH-form in methanol (Axelson et al. 1974). Aliquots of the eluate were taken for determination of radio¬ activity and the recovery of the added 3H-labelled steroid was calculated. TMS ethers were then prepared in pyridine/hexamethyldisilazane/trimethylchlorosilane, 3:2:1 (by
vol.).
8- Sjövall 1972) was used with a Chromosorb W H. P., 80-100 mesh. Column temperature was 210°C, temperatures of molecule separator and ion source were 250°C and 290°C, respectively. The energy of the bombarding electrons was 22.5 eV. Mass spectra were taken every 5.4 second and were recorded on magnetic tape. Background subtraction, normalization, construction of fragment ion current (FIC) chromatograms, and integration of peak areas in these chromatograms were made on an IBM 1800( computer (Reimendal & Sjövall 1972; Axelson et al. 1974). Quantification of steroids was based on peak areas in specific FIC chromatograms. The following fragment ions were used for MO-TMS derivatives of the identified steroids: m/e 341, 273, 286 (progesterone), mie 417,301 (20/?-hydroxy-4-pregnen-3-one), mie 402,386 (3/?-hydroxy5-pregnen-20-one), mie 462 (5-pregnene-3/?,20/i-diol), mie 419, 388 (3/î-hydroxy-5apregnan-20-one). and mie 449, 269 (5a-pregnanc-3/J,20/?-diol). The peak areas given by the steroids in the sample were compared with those given by known amounts of the authentic steroids carried through the same derivatization procedure. The measured amounts of steroids were then corrected for losses as calculated from the recovery of 3H-labelled steroid added at the beginning of the analysis. Recoveries in 27 analyses were 80.3 ± 7.9 "lo (mean + sd). 3
A modified LKB 9000 instrument 4 mm column of 1.5% SE-30
m
(Reimcndal
on
column gas chromatography Glass capillary columns, 20 m 0.3 mm, coated with OV-1 and giving about 40 000 theoretical plates for cholestane, were prepared according to Rutten Se Luyten (1972). They were used with a glass solid injection system (van den Berg Se Cox 1972) in a Pye 104 gas Chromatograph. Column temperature was 250°C and carrier gas (N-« *j
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IDENTIFICATION AND QUANTITATIVE DETERMINATION OF STEROIDS IN BOVINE CORPUS LUTEUM DURING OESTROUS CYCLE AND PREGNANCY
By M. Axelson, G. Schumacher, J. Sj\l=o"\vall, B. Gustafsson and J. O. Lindell ABSTRACT Neutral steroids in bovine corpus luteum were isolated by liquid-gel chromatography on hydrophobic Sephadex, and were analyzed by computerized gas chromatography-mass spectrometry. The presence of pro-
gesterone and 20\g=b\-hydroxy-4pregne-3one
was
confirmed. In addition,
-diol, 3\g=b\-hydroxy-5\g=a\\x=req-\ hydroxy- 5- pregnen -20- one, 5- pregnenewere fully identified, and pregnan-20-one and 3-hydroxy-4-pregnen-20-onne, 4 pregnene-3,20-diol, 22-hydroxycholesterol and 20,22-dihydroxycholesterol were partially characterized. Steroid sulphates were not detected. Quantification of the six fully identified steroids was based on peak areas in specific fragment ion current chromatograms constructed by the computer. During the 4th\p=n-\19thday of the oestrous cycle the steroid concentrations varied as follows: progesterone 6.0\p=n-\36.7\g=m\g/g wet luteal weight, 20\g=b\-hydroxy-4-pregnen-3-one 0.8\p=n-\5.5 \g=m\g/g,3\g=b\-hydroxy-5pregn -20one 1.0\p=n-\7.1 \g=m\g/g,5-pregnene-3\g=b\,20\g=b\-diol< 0.2\p=n-\0.9 \g=m\g/g, 3\g=b\-hydroxy-5\g=a\-pregnan-20-one 1.7\p=n-\8.6 \g=m\g/g, and 5\g=a\-pregnane-3\g=b\,20\g=b\-diol< 0.2-1.2 \g=m\g/g,
3\g=b\-
3\g=b\,20\g=b\
5\g-a\pregna e-3\g=b\,20\g=b\-diol,32
The concentrations of progesterone and 3\g=b-hydrox-5pregn -20one seemed to vary in parallel and were low during days 11\p=n-\17. During this and 5\g=a\-pregnane\x=req-\ period the concentrations of 3\g=b\,20\g=b\-diolwere highest as was the relative contribution of all three 20\g=b\\x=req-\ hydroxysteroids to the total amount of steroids. The relative amount of 3\g=b-hydroxy-5\g=a-pregna-20one seemed to be highest during days 4\p=n-\6. The total steroid concentration in corpora lutea taken in early pregnancy 5-pregnene-3\g=b\,20\g=b\-diol
(75\p=n-\105 days) was 18\p=n-\47 \g=m\g/g. In the period 75\p=n-\90 days, progesterone only 35\p=n-\42 % of the total steroids, 3\g=b\-hydroxy-5\g=a\-pregnan\x=req-\
constituted
20-one as much as 23\p=n-\40 % and the total 20\g=b\-hydroxysteroids 18\p=n-\30%. The total steroid concentration in corpora lutea taken in midterm and late pregnancy was 21\p=n-\77 \g=m\g/g. In this period progesterone was by far the predominant steroid and constituted about 80\p=n-\90 % of the total steroids in corpora lutea taken between days 150 and 240. Possible correlations between luteal growth, steroid oxidoreductases and steroid concentrations are discussed.
and 20/3-hydroxy-4-pregnen-3-one have been identified in bovine luteum corpus (see Gomes Sc Erb 1965). The concentration of these steroids in the oestrous cycle and during pregnancy have been measured in many studies (see Gomes 8c Erb 1965; Erb et al. 1968e, 1971) and variations under different conditions have been investigated (Garverick et al. 1971; Wagner et al. 1972). The presence of 3/3-hydroxy-zl5-, 3/3-hydroxy-, 17/?-hydroxy-, and 20/?-hydroxysteroid oxidoreductases in corpus luteum has been demonstrated in several studies (see Savard 1973), and variations in the activities of these enzymes during the cycle have been reported (Brandan et al. 1972). However, the im¬ portance of these variations for luteal steroid production and metabolism is difficult to assess since substrates and products other than the two steroids mentioned above have not been found. For this reason it seemed important to make a more detailed study of steroids in luteal tissue. This paper describes the application of liquid-gel chromatography and computerized gas chromatography-mass spectrometry to the analysis of steroids in bovine corpus luteum. Six steroids have been identified and results of quantitative determinations of these steroids in the cycle and in pregnancy are reported.
Progesterone
EXPERIMENTAL
Corpora
lutea
Sixteen corpora lutea from non-pregnant cycling cows in different stages of the cycle and 17 corpora lutea from pregnant cows in different pregnancy stages were used. The cows were of the Swedish Red and White Breed (SRB). Four of the cycling animals were heifers, the others and the pregnant cows had calved once or twice. The cyclic corpora lutea were secured either at laparotomy under local anaesthesia or immediately after slaughter of the animals. They were directly cooled to -20°C and kept at that temperature until analyzed. The stage of the oestrous cycle was calculated from data of previous cycles in 11 cases (see Tables) with day 1 denoting the day of oestrus. The corpora lutea marked 25 and 27 in the Tables are regressing corpora lutea from the preceding cycle. In 5 cows the cyclic stage of the corpus luteum was evaluated by macroscopic examination of the genital organs, and by taking into particular consideration the size, vascularization and colour of corpus luteum and the status of the ovarian follicles. oestrous
The corpora lutea of pregnancy were in two cases secured immediately after slaughter. The pregnancy stage was determined by measuring the length of the foetus. In the remaining cases the conception date was exactly known and corpora lutea were obtained through laparotomy under local anaesthesia. The ovary was removed using an ecraseur and the corpus luteum was then immediately enucleated and frozen.
Chemicals
grade and were redistilled before use. Hexamethyltrimethylchlorosilane (Applied Science Laboratories, State College, were redistilled. Trimethylsilylimidazol was from Pierce Chemical Co. Pennsylvania) (Rockford, Illinois) and methoxyamine hydrochloride from Eastman Organic Chemicals (Rochester, New York). Amberlyst A-26, a strong anion exchanger from Rohm and Haas (Philadelphia, Pennsylvania) was washed and stored in the bicarbonate form in water (Sandberg et al. 1965). Hydroxyalkoxypropyl Sephadex (containing 50-55 °/o hydroxyalkyl (Cjj-C^) chains) was synthesized according to Ellingboe et al. (1970), or was obtained as Lipidex®-5000 from Packard-Becker Chemical Division (Groningen, Holland). Unlabelled steroids were from Ikapharm (Ramat-Gan, Israel) and Steraloids (Paw¬ ling, New York). They were purified by chromatography on alumina columns and were then crystallized and dried at 70°C in vacuum. All solvents disilazane and
were
of reagent
Radioactive steroids
[7a-3H] Progesterone (9.6 Ci/mmole), 3/?-hydroxy-5-[7a-3H]pregnen-20-one (18 Ci/ mmole) and 5/J-[l,2-3H]pregnane-3a,20a-diol (50 Ci/mmole) were from the Radio¬ chemical Centre (Amersham, England). 5-[7a-3H]Pregnene-3a,20/?-diol was obtained by NaBHj reduction of the labelled pregnenolone. Purity was assessed by chromato¬ graphy on Lipidex®-5000. Radioactivity was determined in a Packard Tri-Carb Liquid Scintillation Spectrometer using Instagel® as the scintillation liquid. Quenching cor¬ rection
was
made with
Isolation and
internal standard.
purification of steroids
The corpus luteum walled flask.
an
was
weighed
and
was
Chloroform/methanol, 1:1 (v/v),
sliced with a razor-blade into a thick100 ml, and 103 dpm of tritium-labelled
progesterone, 3/?-hydroxy-5-pregnen-20-one, 5-pregnene-3/5,20/?-diol
or
5/i-pregnane-
added. The mixture was homogenized with an Ultra-Turrax homogenizer (Janke-Kunkel, Staufen i. Br., Germany) and the slurry was kept in an ultra¬ sonic bath for 15 min. The extract was filtered into a round-bottomed flask, the residue was re-extracted in the same way, and finally 50 ml of chloroform/methanol was used to rinse the homogenizer and the filter. The solvents were removed in vacuum and the residue was weighed. Unconjugated neutral steroids in the extract were purified by chromatography on hydroxyalkoxypropyl Sephadex (Lipidex®-5000) (Axelson et al. 1974). Briefly, neutral lipids and cholesterol were first removed on a column in a reversed phase solvent system (methanol/water/chloroform, 9:1:2 (by vol.)) where steroids were eluted in the front fractions. Further purification was achieved on a column of Lipidex®-5000 in hexane/chloroform, 9:1 (v/v). All steroids with polarities between those of progesterone and corticosterone were collected from this column by elution with hexane/chloroform, 6:4 (v/v).
3«,20a-diol
was
Gas
chromatography-mass speclrometry
The steroids were analyzed as O-methyloxime trimethylsilyl (MO-TMS) derivatives, prepared as described by Gardiner Se Homing (1966). The methoximes were purified by filtration through a small column of Amberlyst A-26 in OH-form in methanol (Axelson et al. 1974). Aliquots of the eluate were taken for determination of radio¬ activity and the recovery of the added 3H-labelled steroid was calculated. TMS ethers were then prepared in pyridine/hexamethyldisilazane/trimethylchlorosilane, 3:2:1 (by
vol.).
8- Sjövall 1972) was used with a Chromosorb W H. P., 80-100 mesh. Column temperature was 210°C, temperatures of molecule separator and ion source were 250°C and 290°C, respectively. The energy of the bombarding electrons was 22.5 eV. Mass spectra were taken every 5.4 second and were recorded on magnetic tape. Background subtraction, normalization, construction of fragment ion current (FIC) chromatograms, and integration of peak areas in these chromatograms were made on an IBM 1800( computer (Reimendal & Sjövall 1972; Axelson et al. 1974). Quantification of steroids was based on peak areas in specific FIC chromatograms. The following fragment ions were used for MO-TMS derivatives of the identified steroids: m/e 341, 273, 286 (progesterone), mie 417,301 (20/?-hydroxy-4-pregnen-3-one), mie 402,386 (3/?-hydroxy5-pregnen-20-one), mie 462 (5-pregnene-3/?,20/i-diol), mie 419, 388 (3/î-hydroxy-5apregnan-20-one). and mie 449, 269 (5a-pregnanc-3/J,20/?-diol). The peak areas given by the steroids in the sample were compared with those given by known amounts of the authentic steroids carried through the same derivatization procedure. The measured amounts of steroids were then corrected for losses as calculated from the recovery of 3H-labelled steroid added at the beginning of the analysis. Recoveries in 27 analyses were 80.3 ± 7.9 "lo (mean + sd). 3
A modified LKB 9000 instrument 4 mm column of 1.5% SE-30
m
(Reimcndal
on
column gas chromatography Glass capillary columns, 20 m 0.3 mm, coated with OV-1 and giving about 40 000 theoretical plates for cholestane, were prepared according to Rutten Se Luyten (1972). They were used with a glass solid injection system (van den Berg Se Cox 1972) in a Pye 104 gas Chromatograph. Column temperature was 250°C and carrier gas (N-« *j
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