IDENTIFICATION AND QUANTITATION OF 5~PREGNANEDIONE IN HUMAN PREGNANCY PLASMA K. F.
STOA
and A. BESSESEN
Hormone Laboratory, University of Bergen, School of Medicine, 5000 Bergen. Norway (Rewired
30 May 1974)
SUMMARY Studies of the progesterone binding plasma protein of the pregnant guinea-pig have revealed an affinity of this protein for 5r-pregnane-3.20-dione which is practically as high as the affinity for progesterone. Using thin layer chromatography and a competitive protein binding assay based on the pregnant guinea-pig plasma protein. determinations of progesterone and 5x-pregnanedione have been carried out in the plasma of a group of normal pregnant women in the last trimester. The mean concentrations of Sa-pregnanedione in plasma were found to vary between 19.6 ng/ml at 27-29 weeks of pregnancy and 56.9 ng/ml at 40-42 weeks. The amount of 5x-pregnanedione averaged 35% of the progesterone concentration, and there was a positive correlation between the concentrations of the two steroids (r = 0.69). Identification of 5x-pregnanedionc has been made by gas chromatography and mass spectrometry.
MATERIALS AND METHODS
INTRODUCTION In the course of research on the interaction between testosterone and different serum proteins, a protein fraction showing an unusually high affinity for testosterone was detected in pregnant guinea-pig serum. Preliminary reports on this protein have been presented [I, 21 and in more recent studies [3] its identity with the progesterone-binding protein found in pregnant guinea-pigs by Diamond it a!.[41 and later described by Allouch and Milgrom [S] has been demonstrated. This protein has been shown to have a considerably higher affinity for progesterone than for testosterone and binds progesterone about 100 times more firmly than cortisol [4]. Obviously, this guinea-pig protein offers a basis for the development of a competitive protein-binding method for the assay of progesterone, and the use of this principle in analysis of human pregnancy plasma has recently been reported [6,7]. The high affinity of this guinea-pig protein for progesterone as compared with other steroids known to be present in pregnancy plasma made it reasonable to consider any chromatographic purification of the plasma extract prior to assay as unnecessary. However, as a consequence of the remarkably high values obtained [6]. it was decided to carry out a study of the specificity of the competitive protein-binding method. This work led to the detection and quantitative assessment of important amounts of 5a-pregnane-3,20-dione in pregnancy plasma.
Steroids
Progesterone, 5a-pregnane-3.20-dione and 5/&preg nane-3,20-dione-20a-hydroxy-4-pregnene-3,2O-dione were obtained from Sigma Chemical Company, St. Louis, Missouri, U.S.A. [ l,2-3H]-Progesterone (S.A. 47.X Cijmmol) was supplied by New England Nuclear, Boston, Mass., U.S.A. Its purity was checked by chromatography before use. Solvrnts and reagents
Diethyl ether, methanol, toluene and methylal (dimethoxymethane) were all Analar grade and were redistilled before use. Before distillation, toluene was washed with concentrated sulphuric acid, then with water until neutral reaction and finally dried over sodium sulphate. Ethyl acetate was washed with 8’2; sodium bicarbonate solution and then twice with saturated calcium chloride solution. After drying with calcium chloride, it was filtered and distilled. Precoated silica gel thin layer plates (Kieselgel F& were obtained from E. Merck. Darmstadt. The plates were washed 4 times with methanol and stored in a desiccator until needed. Preparation
of the binding solution
Plasma was obtained by cardiac puncture of pregnantguinea-pigsapproximately 3 weeks before delivery, using heparinized syringes. The blood was centrifuged 21
at once and the plasma diluted
I : 20 in sodium borate hu tfcr. pH 7.S. OG5 mol ~1.This ~I;~sII~;I stock solution was stored at - 20 C no longer than I month. Immcdiately prior to use. the plasma was diluted with borate huffcr to I : 10000. To this finally diluted solution was added O%“,, (w ‘v) bovine strum albumin and [-‘H Jprogcstcronc in a concentration corresponding to approximately I0’c.p.m.. ml.
The mcthanotic aliquots containing steroid to be assayed were evaporated to dryness at 45 C and 14 ml of the ditutcd guinea-pig plasma RYIS added to the tubes. The tubes were shaken mechanically for IO min and then kept for at least another IOmjn in ice water. During the succeeding procedure. the tubes were handled in series of eight. in order to keep the time schedule approximately constant. To each tube. still kept in ice water. was added I ml of a Dextran-coated charcoal suspension. The mixture was prepared as earlier described [X] and contained 002j’,, charcoal and 0~006?‘,, Dextran in borate buffer. During USC. the charcoal was maintained in suspension by a magnetic stirrer. The tubes were then shaken in ice water for ahout 2 min. and exactly 3 min after addition of the charcoa I suspension wcrc centrifLlgcd for 3 min at 2000 rcvmin. One ml of the supernatant was pipetted into counting vials and mi\cd with IO ml scintillation lluid (Ilnisolve. Koch -Light Laboratories Ltd.. C’nlnbrook. Bucks.. England). The samptcs wcrc counted for IO min in a Packard TriCarb Scintillation spcctromoter.
Pcriphcrat blood was taken 1~~ venipuncture and collected in hcparinized tubes. After centrifugation. the ptasma \V;IS processed immediatetg or stored at _ 20 c‘. Usually 0.25 ml plasma diluted I : I with water was extracted with 5 ml diethyl ether. To correct for experimental losses. [3H]-progesterone corresponding to 2000 d.p.m. was added as an internal standard. Heforc extraction. t/IO vol. of I N NaOH was also added to the plasma. The ether extraction was carried out by use of a Vortex mixer. After the extract had been washed with I/‘10 vol. of 0.1 N acetic acid and l!‘lO vol. of water. it was evaporated to dryness and the residue dissolved in 0.5 ml methanol for further purification. For isolation and identification of 5r-pregnane-3.20dionr h> pas liquid chromatograph> and mass spectrometry. ;I 175 ml pool of late pregnancy plasma was extracted with three portions of 500ml ethyl ether. after the addition of I.!IO vol. of 1 N NaOH. The ether extract was washed with I :I0 vol. of 0.1 N acetic acid
and I,’IO vol. of water. After evaporation. the rcsiduc MX dissolved in a small volume of methanol and suhjutted to thin layer chromatograph!.
For the purification of the plasma extracts. the fotlowing thin taqcr systems were used: (I ) cyclohexane ethyl acetate (X:2 v’v) (the chromatography was repeated once after drying the plate at room tcmpcraturo); (2) toluenc methytal methanol (X7: IO: 3 by vol.). Progesterone and Sr-prcgnanedionc were isolated by chrolnatograph\ in system I 1tlic localization being made bq simultaneous chromatography of the authcntic standards. Following clution \vith methanol. the progcstcrone and fix-pregnanedione fractions were chromatographcd separatctj in system 2. The eluates containing progcsteronc and 5x-pregnanedionc wcrc then subjected to competitive protein binding assay. Correction for procedural tosses were done on the basis of the recovery of [-‘HI-progesterone in the linal cluatc. The gas cht-omatograpll) mash spectromotry was carried out using a Varian MAT 111 svstcm. A 3”,, OV75 on Gas Chrom Q (100 120 mesh) glass column (0.2 x I50 cm) was emplo\cd at a cotu mn temperature of 270 C’.
KWL’LTS
E.~iturcrior~. The extracting capacity of diRerent sotvents was studied in experiments with [3H]-progcsterone added in known amounts to plasma samples. With ethyl ether. rl-hexane and dichtorome(hane 96 & 3.X (per cent _t SD.; tight assays). 84 k 2.0 (four assays) and 4X.1 & 4.3 (IO assays) respectively were oxtractcd. Hence ethyl cthcr was chosen as extraction solvent. S~JCL$C~!_I~. Thin layer chromatography carried out in system 2 and scanning of the chromatogram with rcspcct to protein binding showed that the plasma cxtrncts contained one fraction, different from progcsterone. but with a very significant displacement effect towards this steroid. On chromatography with authentic standards. it could be shown that the fraction had a relative R,--value which corrcspondrd to pregnaneX17-dionc. However, the ix and S/j isomers of this compound could not bc separated by the system used. In an investigation of the ability of the two pregnancdioncs to compete for binding sites on the progesterone-binding plasma protein. Sz-pregnanedionc proved to reduce binding of [-‘HI-progesterone to the same culent as progcsteronc itself (Fig. I ). On the other
5rx-Pregnanedione in pregnancy plasma
Steroid, ng Fig. 1. The ability of the two pregnanediones to compete with progesterone for binding sites on the progesteronebinding protein of plasma from pregnant guinea-pigs. P: Ptoeesterone. 5r-DHP: 5a-Pregnane-3.20-dione. SB-DHP: 5j-Fregnane-3,20-dione. Absci&: Steroid, ng. drdinate: C3H]-Progesterone bound, c.p.m. Binding assays carried out as described in text, except that guinea-pig plasma was diluted 1:5000.
hand, the binding affinity of the isomeric 5@ompound was found to be considerably lower. Furthermore, the b-isomer, if present, could be expected to be eliminated, at least partly, by the thin layer chromatography in system I (Fig. 2). Sensiriuity. On the basis of duplicate assays of standards in the range between 0.1 and 2 ng, the lowest amounts of progesterone and Sa-pregnanedione which could be distinguished from 0 with 95’:; confidence were calculated to be 0.20 and 0.15 ng respectively. Accuracy. Accuracy was tested by adding known amounts of progesterone and 5z-pregnanedione to
P
23
plasma and performing the assays on samples with and without steroid addition. On addition of lOOng/ml steroid to different samples of a pool of pregnancy plasma. the mean recoveries were 94.7 and 86.0% for and Sa-pregnanedione respectively progesterone (Table 1). Precision. In duplicate measurements in plasma samples selected at random. the coefficient of variation was calculated to be l25:/, for progesterone and 1X.4”;, for 5~-preg~dnedione. ~~~~~~?z~~u~~or~ o~p~~~~st~r~~1~ and Sa-prr~rmr?rdiotr(‘. Making use of the presented method. simultaneous determinations of progesterone and 5r-pregnane-3,20dione were carried out in a series of plasma samples from normal pregnant women. The results are shown in Table 2. The samples were taken during the last trimester. from 192 to 294 days after the last menstruation. The average progesterone values rose from 62.4 to 167.9 ng/ml during the period of examination. The results for Sr-pregnanedione were about 35:~:;of the progesterone values, rising from an average of 19.6 to 569 ngjml, however, with considerable individ~l variations. There WdS a positive correlation (1.= 0.69) between the values for progesterone and Sr-pregnanedione. ~tlent$cutior~ of‘ Sr-ple(lnarle-3,~O-rliorIr. Following extraction of late pregnancy plasma and isolation of ~~-pregnane-3.20-dione, the isolated compound was subjected to gas chromatography and mass spectrometry. On gas chromatography. the compound gave a peak with a retention time of 3.5 min. The peak showed a mass spectrum with prominent fragment ions at the following m/e values: 316 (M+). 298, 283. 212.269. 258 and 231. An authentic standard run under the same conditions, gave a peak with identical retention time and m/e values.
SC4DHP
5BDWP
Fig. 2. Chromatography of authentic standards of progesterone (P), 5r-pregnane-3,17-dione (5x-DHP) and 5&pregnane-3.17-dione (5/SDHP) on a silica gel thin layer plate in the system cyclohexane-ethyl acetate (8:2 v/v). Recbromatography in the same system on the same plate. Localization of spots were carried out by spraying with ethanolic sulphuric acid (SO’lQ.
74
K. F. Table I. Determination plasma from pregnant
Steroid
and A. Htssrsb\
of progesterone and 5r-prcgnane-i.20-dlotlc women before and after addition of ;I known respective steroids
None IO0 ng’ml 01
4
s per cent
The
progesterone-binding plasma protein from guinea-pigs has been shown to have a binding attinity for j,-pregnane-.i.20-dione which is equal to the affinity for progesterone. When measuring progestcrone in plasma by means of this protein without chromatographic purification [6.9], an overestimation can therefore be cxpccted if considerable amounts of Sr-pregnanedione are present and this steroid is extracted with progesterone. Actually. it has been shown (unpublished observations) that Sr-pregnancdione is extracted at about the same rate as progesterone, not only with ethyl ether. but with petroleum ether as well. The high binding affinities of the guinea-pig plasma protein for progesterone and 5z-pregnanedione [3] makes a dilute solution of the protein a very useful reagent for competitive protein binding assay of both steroids. Based on an assay procedure which in principlc is similar to that described by Pichon and Milgrom[7], a method has been developed for the simultancous determination of progesterone and %pregnane-3.X-dione in human pregnancy plasma. To our knowledge. this is the first time that .5x-prcgnancdionc pregnant
Table
2. Simultaneous
Weeks after last menstrual period 27 30 22 25
111‘I p~u’l 01 amount 01 1hc
Number of UalyseS
added
each steroid Mean recovery.
SI’OA
dctermmation
has been detected and dctcrmmcd m human plasma, the amount in pregnant women being at the level of about 35”,, of the progesterone concentration. On the basis of the chemical similarities between 5-/pregnanedione and dihydrotestostoronc. as well as their protein interactions [IO]. it is tempting to consider the possibility that Sr-pregnanedione may be involved in the molecular mechanism of action of progcsteronc in analogy w)ith tcstosteronc and dihydrotestosterone. In this context it is interesting to note that the conversion of progesterone to Sr-prcgnanc3,20-dione has recently been demonstrated in human myometrium [I I] and that 5x-prcgnanedione is bound identically with progesterone to the uterine c~tosol of guinea-pigs [ 121.To what extent and in what way the relatively high concentrations of %pregnanedione which have been detected in circulating plasma of pregnant women may be involved in or interfere with biological progesterone activity is for the time being an open question. Arkrlo~/[,tl~~,)~~,~l~
The author, wish to thank Profcasor R. R. Scheline and Mr. E. Solheim M.Sc.. Department of Pharmacology. Univcl-sit) Bcrgcn. for their euxllcnt help with the gas-chromatographic maaa-\pcctrometrlc analysis.
of
of 5x-pregnane-3.X-dionc of normal pregnant women
and progcsteronc
111the plasma
Steroid concentration. ng;ml (mean * S.I‘.M.) Number of determinations
2’) 31 34 37
x x Y x
3x 39 4% 42
10 9 5
5,-Pregn~lticdionc 19.6 23.7 36.8 37.3
* * * _t
3.9 5.3 6.7 6.1
39.3 * x.9 47.6 i 7. I 56.9 _+ 13.1
Progcstcronc 67.4 2 7.’ 65.2 * x.7 Y7.i + II.1 I IO..,i 15.7 I I6-7 f x.0 IX.0 * I30 167.9 j: 5.0
5~-~regnaned~~ne in pregnancy plasma REFERENCES
t. Lea 0. A. and Stsa K. F.: _I. steroid Bj~c~e~~.3 (1972) 409-4 19. 7. Lea 0. A. and Sttra K. F.: Errrrptu Med. Int. Congr. Series Nu. 256 (1972) 18. 3. Lea 0. A.: B~o~hil~~, bj~p~~s. Acta 317 (1973) 351-363. 4. Diamond M., Rust N. and Westphal U.: Ertdocrirroloy~ 84(1969) 1143~1151. 5. Allouch P. and Milgrom E.: E,YCYI.~&I .Med. Int. Congr. Series /Vo. 256 ( 1952) 70’7. 6. Attal J. and Engels J.-A.: J. Emfocr. SO (1971) 703-704.
25
7. Pichon M.-F. and Milgrom E.: Steroids 21 (1973) 335 346. 8. Stoa K. F.. 3esxsen A. and 3jornsen B. A.: III ~?rrn Procedures with Radioisotopes in Mrdicine. ht. Atomic Energy Agency, Vienna (1970) pp. 417.-426. 9. Lea 0. A.: Biochim. hinphys Acta 322 (1973) G--74. 10. Hansson V., Larsen J. and Reusch E.: Steroids20 (19X?) 555514. I I. Sauer H. D. and Lisboa B. P.: ilcrcl c>&u.. Copurlr. Suilnl. 177 II9731 222. 12. J&e U., i(on&la K.. Rajakoski E., Tanhuanpll E. and Vihko R.: Acru rr~rlou.. Copr1111.Sup/)I. 177 (1973) 249.
STOA
and A. BESSESEN
Hormone Laboratory, University of Bergen, School of Medicine, 5000 Bergen. Norway (Rewired
30 May 1974)
SUMMARY Studies of the progesterone binding plasma protein of the pregnant guinea-pig have revealed an affinity of this protein for 5r-pregnane-3.20-dione which is practically as high as the affinity for progesterone. Using thin layer chromatography and a competitive protein binding assay based on the pregnant guinea-pig plasma protein. determinations of progesterone and 5x-pregnanedione have been carried out in the plasma of a group of normal pregnant women in the last trimester. The mean concentrations of Sa-pregnanedione in plasma were found to vary between 19.6 ng/ml at 27-29 weeks of pregnancy and 56.9 ng/ml at 40-42 weeks. The amount of 5x-pregnanedione averaged 35% of the progesterone concentration, and there was a positive correlation between the concentrations of the two steroids (r = 0.69). Identification of 5x-pregnanedionc has been made by gas chromatography and mass spectrometry.
MATERIALS AND METHODS
INTRODUCTION In the course of research on the interaction between testosterone and different serum proteins, a protein fraction showing an unusually high affinity for testosterone was detected in pregnant guinea-pig serum. Preliminary reports on this protein have been presented [I, 21 and in more recent studies [3] its identity with the progesterone-binding protein found in pregnant guinea-pigs by Diamond it a!.[41 and later described by Allouch and Milgrom [S] has been demonstrated. This protein has been shown to have a considerably higher affinity for progesterone than for testosterone and binds progesterone about 100 times more firmly than cortisol [4]. Obviously, this guinea-pig protein offers a basis for the development of a competitive protein-binding method for the assay of progesterone, and the use of this principle in analysis of human pregnancy plasma has recently been reported [6,7]. The high affinity of this guinea-pig protein for progesterone as compared with other steroids known to be present in pregnancy plasma made it reasonable to consider any chromatographic purification of the plasma extract prior to assay as unnecessary. However, as a consequence of the remarkably high values obtained [6]. it was decided to carry out a study of the specificity of the competitive protein-binding method. This work led to the detection and quantitative assessment of important amounts of 5a-pregnane-3,20-dione in pregnancy plasma.
Steroids
Progesterone, 5a-pregnane-3.20-dione and 5/&preg nane-3,20-dione-20a-hydroxy-4-pregnene-3,2O-dione were obtained from Sigma Chemical Company, St. Louis, Missouri, U.S.A. [ l,2-3H]-Progesterone (S.A. 47.X Cijmmol) was supplied by New England Nuclear, Boston, Mass., U.S.A. Its purity was checked by chromatography before use. Solvrnts and reagents
Diethyl ether, methanol, toluene and methylal (dimethoxymethane) were all Analar grade and were redistilled before use. Before distillation, toluene was washed with concentrated sulphuric acid, then with water until neutral reaction and finally dried over sodium sulphate. Ethyl acetate was washed with 8’2; sodium bicarbonate solution and then twice with saturated calcium chloride solution. After drying with calcium chloride, it was filtered and distilled. Precoated silica gel thin layer plates (Kieselgel F& were obtained from E. Merck. Darmstadt. The plates were washed 4 times with methanol and stored in a desiccator until needed. Preparation
of the binding solution
Plasma was obtained by cardiac puncture of pregnantguinea-pigsapproximately 3 weeks before delivery, using heparinized syringes. The blood was centrifuged 21
at once and the plasma diluted
I : 20 in sodium borate hu tfcr. pH 7.S. OG5 mol ~1.This ~I;~sII~;I stock solution was stored at - 20 C no longer than I month. Immcdiately prior to use. the plasma was diluted with borate huffcr to I : 10000. To this finally diluted solution was added O%“,, (w ‘v) bovine strum albumin and [-‘H Jprogcstcronc in a concentration corresponding to approximately I0’c.p.m.. ml.
The mcthanotic aliquots containing steroid to be assayed were evaporated to dryness at 45 C and 14 ml of the ditutcd guinea-pig plasma RYIS added to the tubes. The tubes were shaken mechanically for IO min and then kept for at least another IOmjn in ice water. During the succeeding procedure. the tubes were handled in series of eight. in order to keep the time schedule approximately constant. To each tube. still kept in ice water. was added I ml of a Dextran-coated charcoal suspension. The mixture was prepared as earlier described [X] and contained 002j’,, charcoal and 0~006?‘,, Dextran in borate buffer. During USC. the charcoal was maintained in suspension by a magnetic stirrer. The tubes were then shaken in ice water for ahout 2 min. and exactly 3 min after addition of the charcoa I suspension wcrc centrifLlgcd for 3 min at 2000 rcvmin. One ml of the supernatant was pipetted into counting vials and mi\cd with IO ml scintillation lluid (Ilnisolve. Koch -Light Laboratories Ltd.. C’nlnbrook. Bucks.. England). The samptcs wcrc counted for IO min in a Packard TriCarb Scintillation spcctromoter.
Pcriphcrat blood was taken 1~~ venipuncture and collected in hcparinized tubes. After centrifugation. the ptasma \V;IS processed immediatetg or stored at _ 20 c‘. Usually 0.25 ml plasma diluted I : I with water was extracted with 5 ml diethyl ether. To correct for experimental losses. [3H]-progesterone corresponding to 2000 d.p.m. was added as an internal standard. Heforc extraction. t/IO vol. of I N NaOH was also added to the plasma. The ether extraction was carried out by use of a Vortex mixer. After the extract had been washed with I/‘10 vol. of 0.1 N acetic acid and l!‘lO vol. of water. it was evaporated to dryness and the residue dissolved in 0.5 ml methanol for further purification. For isolation and identification of 5r-pregnane-3.20dionr h> pas liquid chromatograph> and mass spectrometry. ;I 175 ml pool of late pregnancy plasma was extracted with three portions of 500ml ethyl ether. after the addition of I.!IO vol. of 1 N NaOH. The ether extract was washed with I :I0 vol. of 0.1 N acetic acid
and I,’IO vol. of water. After evaporation. the rcsiduc MX dissolved in a small volume of methanol and suhjutted to thin layer chromatograph!.
For the purification of the plasma extracts. the fotlowing thin taqcr systems were used: (I ) cyclohexane ethyl acetate (X:2 v’v) (the chromatography was repeated once after drying the plate at room tcmpcraturo); (2) toluenc methytal methanol (X7: IO: 3 by vol.). Progesterone and Sr-prcgnanedionc were isolated by chrolnatograph\ in system I 1tlic localization being made bq simultaneous chromatography of the authcntic standards. Following clution \vith methanol. the progcstcrone and fix-pregnanedione fractions were chromatographcd separatctj in system 2. The eluates containing progcsteronc and 5x-pregnanedionc wcrc then subjected to competitive protein binding assay. Correction for procedural tosses were done on the basis of the recovery of [-‘HI-progesterone in the linal cluatc. The gas cht-omatograpll) mash spectromotry was carried out using a Varian MAT 111 svstcm. A 3”,, OV75 on Gas Chrom Q (100 120 mesh) glass column (0.2 x I50 cm) was emplo\cd at a cotu mn temperature of 270 C’.
KWL’LTS
E.~iturcrior~. The extracting capacity of diRerent sotvents was studied in experiments with [3H]-progcsterone added in known amounts to plasma samples. With ethyl ether. rl-hexane and dichtorome(hane 96 & 3.X (per cent _t SD.; tight assays). 84 k 2.0 (four assays) and 4X.1 & 4.3 (IO assays) respectively were oxtractcd. Hence ethyl cthcr was chosen as extraction solvent. S~JCL$C~!_I~. Thin layer chromatography carried out in system 2 and scanning of the chromatogram with rcspcct to protein binding showed that the plasma cxtrncts contained one fraction, different from progcsterone. but with a very significant displacement effect towards this steroid. On chromatography with authentic standards. it could be shown that the fraction had a relative R,--value which corrcspondrd to pregnaneX17-dionc. However, the ix and S/j isomers of this compound could not bc separated by the system used. In an investigation of the ability of the two pregnancdioncs to compete for binding sites on the progesterone-binding plasma protein. Sz-pregnanedionc proved to reduce binding of [-‘HI-progesterone to the same culent as progcsteronc itself (Fig. I ). On the other
5rx-Pregnanedione in pregnancy plasma
Steroid, ng Fig. 1. The ability of the two pregnanediones to compete with progesterone for binding sites on the progesteronebinding protein of plasma from pregnant guinea-pigs. P: Ptoeesterone. 5r-DHP: 5a-Pregnane-3.20-dione. SB-DHP: 5j-Fregnane-3,20-dione. Absci&: Steroid, ng. drdinate: C3H]-Progesterone bound, c.p.m. Binding assays carried out as described in text, except that guinea-pig plasma was diluted 1:5000.
hand, the binding affinity of the isomeric 5@ompound was found to be considerably lower. Furthermore, the b-isomer, if present, could be expected to be eliminated, at least partly, by the thin layer chromatography in system I (Fig. 2). Sensiriuity. On the basis of duplicate assays of standards in the range between 0.1 and 2 ng, the lowest amounts of progesterone and Sa-pregnanedione which could be distinguished from 0 with 95’:; confidence were calculated to be 0.20 and 0.15 ng respectively. Accuracy. Accuracy was tested by adding known amounts of progesterone and 5z-pregnanedione to
P
23
plasma and performing the assays on samples with and without steroid addition. On addition of lOOng/ml steroid to different samples of a pool of pregnancy plasma. the mean recoveries were 94.7 and 86.0% for and Sa-pregnanedione respectively progesterone (Table 1). Precision. In duplicate measurements in plasma samples selected at random. the coefficient of variation was calculated to be l25:/, for progesterone and 1X.4”;, for 5~-preg~dnedione. ~~~~~~?z~~u~~or~ o~p~~~~st~r~~1~ and Sa-prr~rmr?rdiotr(‘. Making use of the presented method. simultaneous determinations of progesterone and 5r-pregnane-3,20dione were carried out in a series of plasma samples from normal pregnant women. The results are shown in Table 2. The samples were taken during the last trimester. from 192 to 294 days after the last menstruation. The average progesterone values rose from 62.4 to 167.9 ng/ml during the period of examination. The results for Sr-pregnanedione were about 35:~:;of the progesterone values, rising from an average of 19.6 to 569 ngjml, however, with considerable individ~l variations. There WdS a positive correlation (1.= 0.69) between the values for progesterone and Sr-pregnanedione. ~tlent$cutior~ of‘ Sr-ple(lnarle-3,~O-rliorIr. Following extraction of late pregnancy plasma and isolation of ~~-pregnane-3.20-dione, the isolated compound was subjected to gas chromatography and mass spectrometry. On gas chromatography. the compound gave a peak with a retention time of 3.5 min. The peak showed a mass spectrum with prominent fragment ions at the following m/e values: 316 (M+). 298, 283. 212.269. 258 and 231. An authentic standard run under the same conditions, gave a peak with identical retention time and m/e values.
SC4DHP
5BDWP
Fig. 2. Chromatography of authentic standards of progesterone (P), 5r-pregnane-3,17-dione (5x-DHP) and 5&pregnane-3.17-dione (5/SDHP) on a silica gel thin layer plate in the system cyclohexane-ethyl acetate (8:2 v/v). Recbromatography in the same system on the same plate. Localization of spots were carried out by spraying with ethanolic sulphuric acid (SO’lQ.
74
K. F. Table I. Determination plasma from pregnant
Steroid
and A. Htssrsb\
of progesterone and 5r-prcgnane-i.20-dlotlc women before and after addition of ;I known respective steroids
None IO0 ng’ml 01
4
s per cent
The
progesterone-binding plasma protein from guinea-pigs has been shown to have a binding attinity for j,-pregnane-.i.20-dione which is equal to the affinity for progesterone. When measuring progestcrone in plasma by means of this protein without chromatographic purification [6.9], an overestimation can therefore be cxpccted if considerable amounts of Sr-pregnanedione are present and this steroid is extracted with progesterone. Actually. it has been shown (unpublished observations) that Sr-pregnancdione is extracted at about the same rate as progesterone, not only with ethyl ether. but with petroleum ether as well. The high binding affinities of the guinea-pig plasma protein for progesterone and 5z-pregnanedione [3] makes a dilute solution of the protein a very useful reagent for competitive protein binding assay of both steroids. Based on an assay procedure which in principlc is similar to that described by Pichon and Milgrom[7], a method has been developed for the simultancous determination of progesterone and %pregnane-3.X-dione in human pregnancy plasma. To our knowledge. this is the first time that .5x-prcgnancdionc pregnant
Table
2. Simultaneous
Weeks after last menstrual period 27 30 22 25
111‘I p~u’l 01 amount 01 1hc
Number of UalyseS
added
each steroid Mean recovery.
SI’OA
dctermmation
has been detected and dctcrmmcd m human plasma, the amount in pregnant women being at the level of about 35”,, of the progesterone concentration. On the basis of the chemical similarities between 5-/pregnanedione and dihydrotestostoronc. as well as their protein interactions [IO]. it is tempting to consider the possibility that Sr-pregnanedione may be involved in the molecular mechanism of action of progcsteronc in analogy w)ith tcstosteronc and dihydrotestosterone. In this context it is interesting to note that the conversion of progesterone to Sr-prcgnanc3,20-dione has recently been demonstrated in human myometrium [I I] and that 5x-prcgnanedione is bound identically with progesterone to the uterine c~tosol of guinea-pigs [ 121.To what extent and in what way the relatively high concentrations of %pregnanedione which have been detected in circulating plasma of pregnant women may be involved in or interfere with biological progesterone activity is for the time being an open question. Arkrlo~/[,tl~~,)~~,~l~
The author, wish to thank Profcasor R. R. Scheline and Mr. E. Solheim M.Sc.. Department of Pharmacology. Univcl-sit) Bcrgcn. for their euxllcnt help with the gas-chromatographic maaa-\pcctrometrlc analysis.
of
of 5x-pregnane-3.X-dionc of normal pregnant women
and progcsteronc
111the plasma
Steroid concentration. ng;ml (mean * S.I‘.M.) Number of determinations
2’) 31 34 37
x x Y x
3x 39 4% 42
10 9 5
5,-Pregn~lticdionc 19.6 23.7 36.8 37.3
* * * _t
3.9 5.3 6.7 6.1
39.3 * x.9 47.6 i 7. I 56.9 _+ 13.1
Progcstcronc 67.4 2 7.’ 65.2 * x.7 Y7.i + II.1 I IO..,i 15.7 I I6-7 f x.0 IX.0 * I30 167.9 j: 5.0
5~-~regnaned~~ne in pregnancy plasma REFERENCES
t. Lea 0. A. and Stsa K. F.: _I. steroid Bj~c~e~~.3 (1972) 409-4 19. 7. Lea 0. A. and Sttra K. F.: Errrrptu Med. Int. Congr. Series Nu. 256 (1972) 18. 3. Lea 0. A.: B~o~hil~~, bj~p~~s. Acta 317 (1973) 351-363. 4. Diamond M., Rust N. and Westphal U.: Ertdocrirroloy~ 84(1969) 1143~1151. 5. Allouch P. and Milgrom E.: E,YCYI.~&I .Med. Int. Congr. Series /Vo. 256 ( 1952) 70’7. 6. Attal J. and Engels J.-A.: J. Emfocr. SO (1971) 703-704.
25
7. Pichon M.-F. and Milgrom E.: Steroids 21 (1973) 335 346. 8. Stoa K. F.. 3esxsen A. and 3jornsen B. A.: III ~?rrn Procedures with Radioisotopes in Mrdicine. ht. Atomic Energy Agency, Vienna (1970) pp. 417.-426. 9. Lea 0. A.: Biochim. hinphys Acta 322 (1973) G--74. 10. Hansson V., Larsen J. and Reusch E.: Steroids20 (19X?) 555514. I I. Sauer H. D. and Lisboa B. P.: ilcrcl c>&u.. Copurlr. Suilnl. 177 II9731 222. 12. J&e U., i(on&la K.. Rajakoski E., Tanhuanpll E. and Vihko R.: Acru rr~rlou.. Copr1111.Sup/)I. 177 (1973) 249.
