- Email: [email protected]
Changes in plasma levels of estrone sulfate and estrone in the pregnant ewe around parturition
855
CHANGES IN PLASMA LEVELS OF ESTRONE SULFATE AND ESTRONE IN THE PREGNANT EWE AROUND PARTURITION
Charles P.W. Tsang Reproductive Physiology Section, Animal Research Institute, Ottawa, Ontario, Canada, KIA OC6
Received:
3/21/74
ABSTRACT A method for the extraction, separation and measurement of estrone sulfate and estrone in a single plasma sample is described. The method has been applied to the determination of plasma levels of estrone sulfate and estrone in pregnant ewes over the period 60 hr before to 20 hr after parturition. The study revealed that the plasma levels of estrone sulfate and estrone began to increase about 40 hr before parturition, reached a peak at parturition and then declined rapidly to levels below the sensitivity of the method by 15 hr postpartum. The peak level of estrone sulfate recorded at parturition was 103 pmof (38 ng) per ml of plasma which was approximately 30 times greater than the corresponding peak level of estrone. INTRODUCTION The development of radioimmunoassay techniques for the determination of unconjugated estrogens has resulted in the measurement of plasma estrogens and establishment of plasma estrogen profiles in a variety of animal species during pregnancy, e.g., sheep (l-4), goat (2, 13), cow (5); guinea pig (6) and rat (7).
In contrast, little
information is currently available on the changes of plasma levels of estrogen conjugates during various physiological states.
In those
instances in which plasma estrogen conjugates, and in particular
Contribution No. 529, Animal Research Institute
VoZwne 23, Number 6
STEROIDS
June, 2974
STEROIDS
plasma estrogen sulfates, have been determined, the sulfate conjugates appear to be present in quantities considerably greater than the corresponding unconjugated estrogen (8-11). In an attempt to establish the relative amounts of estrogens and estrogen sulfates-in plasma as well as a possible functional role for circulating estrogen sulfates in domestic animals, a method has been developed for the extraction, separation and measurement of estrogen sulfates and unconjugated estrogens in a single plasma sample. Initially, the method has been applied to the measurement of plasma estrone sulfate
1
and estrone around parturition in the ewe. MATERIALS AND METHODS
Solvent and reagents All solvents were of spectroquality grade and were used as obtained, Estrone-6, 7-3H sulfate, ammonium salt (S.A. 40 Ci/mmol), estrone-2,4,6,7-3H (S.A. 104 Ci/mmol) and estradiol-17B-2,4,6,7-3H (S,A. 105 Ci/mmol) were obtained from New England Nuclear Corporation, Boston Mass., U.S.A. Stock solutions containing 100 pCi/ml were made up and stored at -20°C in absolute methanol to reduce the effect of self-irradiation. Crystalline estrone-3-sulfate, sodium salt, crystalline estrone and crystalline estradiol-178 were purchased from Sigma Chemical Co., St. Louis, MO., U.S.A. Standard solutions were prepared in absolute methanol. The purity of both radioactive and non-radioactive steroids was checked by thin-layer chromatography before use. Sulfatase Type III (Sigma Chemical Co.) for the hydrolysis of estrone sulfate had very low f3-glucuronidase activity. A stock solution was prepared by dissolving the enzyme preparation in 0.2 M acetate buffer, pH 5.0, at a concentration of 2 mg/ml buffer. This solution could be stored at -2O'C for at least two months without appreciable loss of activity. For routine hydrolysis 0.1 ml of the stock solution was used per sample. Chromatopraphy Thin-layer chromatography was used to separate estrogen sulfates from free steroids and other steroid conjugates in the sample extracts. Chromatography was carried out on aluminum plates coated with silica
STEROIDS
gel F-254, 25 mm thick (E. Merck, Darmstadt) and the chromatograms developed in the system ethylacetate: ethanol: ammonium hydroxide (5:5:1) as described by Loriaux et al (10). Reference estrone sulfate was run alongside the sample extracts and located by cutting out the strip containing the standard, spraying with 10% sulfuric acid in ethanol and heating at 1lO'C for 10 minutes. The area of silica gel corresponding to estrone sulfate on the sample strips was aspirated into a Pasteur pipette plugged lightly with a dental cotton pellet which had been pre-washed with methanol. The sulfate fraction was eluted from the silica gel with 2 x 2 ml methanol into a 15 ml stoppered centrifuge tube. Sephadex LH-20 columns were used to separate estrone from the isomeric estradiols in the plasma extracts. Columns were prepared by gently pouring 1 g Sephadex LH-20 (Pharmacia, Uppsala, Sweden) through a funnel into 10 ml disposable Pyrex pipettes (ID 0.9 mm approx.) containing a small glass bead. The columns were washed initially with 10 ml methanol and then allowed to swell overnight in 7.0 ml methanol. Before use, the methanol was allowed to drain from the column which was then washed with 20 ml of the eluting solvent. The sample was applied to the column and the estrone eluted from the column as described below. After use, the columns were washed with 20 ml methanol and maintained in this solvent until the next run. Columns prepared in this manner were found to have uniform elution properties, could be used for several months and gave clean blanks. Antiserum for radioimmunoassay Sheep antiserum to estradiol-178 hemisuccinyl BSA was kindly provided by Dr. B.V. Caldwell, Department of Obstetrics & Gynecology, Department of Medicine, Yale University, Connecticut, U.S.A. The antiserum was diluted 1:lOO with Buffer A, prepared by dissolving NaH2P04, 4H20, 5.38 g; Na2HP04, 7H 0, 16.35 g; NaCl, 9.0 g; NaN 1 g; gelatin 1 g in 1 liter of distille 3 water, and stored at -2O'C ?n 0.5 ml aliquots. For the assay, an aliquot of the antiserum was diluted further with the same buffer to give a final dilution of 1:32,000. Dextran-coated charcoal Dextran T80 (Pharmacia, Uppsala, Sweden), 50 mg and 500 mg Norit A charcoal were suspended in 100 ml Buffer A. The mixture was kept in ice and maintained in suspension by stirring continuously with a magnetic stirrer. Measurement of radioactivity Because of differences in solubility between estrone and estrone sulfate, quantitation of radioactivity present in the samples was carried out using two scintillation solutions. (a) Standard 3H labelled estrone and benzene extracts from plasma were counted in 10 ml of a scintillation fluid which contained 4 g
857
STEROIDS
Omniflour (New England Nuclear) dissolved in 1 liter toluene. (b) Standard 3H labelled estrone sulfate and estrone derived from hydrolysis of extracts containing the sulfate fraction were counted in 10 ml of a solution which contained 8 g Omniflour and 100 mg naphthalene dissolved in 1 liter dioxane +,0.5 ml buffer A. Samples were counted in a Beckman Model LS-250 Scintillation Spectrometer. The efficiency of counting 3H under conditions (a) and (b) was 65% and 40% respectively. All samples were counted to an error of + 2%. Plasma Samples For estimation of blanks and recovery experiments blood was obtained by jugular venous puncture from castrate male lambs. Immediately after collection into heparinized vaccutainer tubes, the blood was cooled in ice and centrifuged at 4'C within 1 hr. The plasma was removed and stored in 3 ml aliquots at -20°C. Pregnancy blood samples were obtained from eight Western ewes near term as described previously (5). Briefly, blood samples were collected every 6 hr from 96 hr before to 72 after parturition. Plasma samples were prepared as described for the castrate male plasma. The samples were kept frozen for fourteen months before being assayed and were thawed and re-frozen occasionally in connection with other studies. For the assay, samples from 4 ewes giving birth to single lambs and from 4 ewes giving birth to twins were separated into 2 groups and a pooled sample was made for every 6 hr collection period within each group. Thus every pooled sample contained equal aliquots from 4 ewes. In addition, assays were carried out on individual samples taken at 30-36 hr and 6-12 hr before parturition to estimate individual variations and to determine whether a statistically significant difference existed between the single and twin lamb groups. Internal standards Approximately 2000 cpm of 3H labelled estrone sulfate (equivalent to 5.4 x 10-2 pmol or 20 pg) and 1000 cpm 3H labelled estrone (equivalent to 7.4 x 10-3 pmol or 2 pg) were added to all plasma samples to correct for procedural losses. Extraction of unconiupated estrogens from plasma. To plasma samples (0.2 - 1.0 ml) in 45 ml stoppered centrifuge tubes was added 2 ml of 0.5 M sodium bicarbonate (pH 8.0) and the contents mixed with a Vortex mixer. Benzene (5 ml) was added and the free estrogens extracted by mixing the contents for 2 min. The tube was then centrifuged at 1500 rpm for 5 min, the benzene phase transferred to 5 ml disposable siliconized culture tubes and evaporated to dryness under nitrogen.
STEROIDS
Extraction and separation of estrogen sulfates The sulfate conjugates were similarly extracted from the aqueous phase remaining in the centrifuge tube by extraction with 2 x 5 ml of tetrahydrofuran: ethyl acetate (1:l v/v). However, the centrifugation time after each extraction was extended to lo-20 min, particularly when an emulsion formed. The organic phases were transferred to 5 ml siliconized culture tubes, evaporated to dryness under nitrogen and the dry extract applied quantitatively to a thin-layer plate with 3 x 0.025 ml absolute methanol. The sulfate fraction was separated and eluted from the plate as described above. Hydrolysis of the sulfate fraction After removal of the methanol under nitrogen, the dry extract was dissolved in 0.5 ml of 0.2 M sodium acetate buffer, pH 5.0, containing 200 ug sulfatase Type III and allowed to incubate at 37'C overnight with constant shaking in a water bath. On completion of hydrolysis, the free steroids liberated were extracted with 2 x 2 ml benzene, the organic phases transferred to siliconized culture tubes and evaporated to dryness under nitrogen. Separation of estrone from the isomeric estradiols Dry benzene extracts, whether derived from initial extraction of the plasma sample or from hydrolysis of the sulfate fraction, were dissolved in 0.2 ml of iso-octane: benzene: methanol (62:20:1% v/v/v) and applied to Sephadex LH-20 columns equilibrated in the same solvent. Two additional rinses each of 0.1 ml column solvent were similarly applied to the column. After the sample had entered the column, the column was eluted with 4 ml of the same solvent mixture, which was discarded. The estrone fraction was then eluted from the column into siliconized tubes with 4 ml of the column solvent. If required, the estradiol fraction could be eluted from the column with a further 4.5 ml column solvent, by discarding the first 0.5 ml and collecting the next 4.0 ml. Estimation of procedural losses The estrone fractions obtained after Sephadex LH-20 chromatography were evaporated to dryness under nitrogen and the dry residues dissolved ;n 1.0 ml absolute methanol. An aliquot of 0.2 ml was taken for counting to estimate procedural losses. The remainder of & each sample, or an aliquot (depending on the expected concentration in the sample), was dried down under nitrogen for determination of estrogen content. Radioimmunoassay procedure To each tube to be assayed was added 0.5 ml of a buffer A containing 12,000 cpm of 3H labelled estradiol-176 (equivalent to 9.2 x 10-2 pmol or 25 pg) and the tubes gently mixed and incubated at 37°C for 10 min to ensure 'complete solution of the estrogen. Then 0.1 ml of a 1:32,000 dilution of the antiserum in buffer A was added, the tubes
STEROIDS
again mixed and left at 4°C overnight. The unbound estrogen was absorbed by addition of 0.5 ml dextran coated charcoal suspension to each tube kept in an ice/water bath. The tubes were allowed to stand for 5 min, mixed.for 10 set, allowed to stand for a further 5 min and finally centrifuged for 2.5 min at 1500 rpm and 4°C. The supernatant from each tube was transferred to counting vials and the radioactivity determined. A standard curve for estrone was obtained by treating tubes containing O-200 pg (equivalent to O-O.54 pmol) in a manner similar to that for the unknown samples. The tube containing zero picograms of estrone served to establish the assay blank. RESULTS For clarity and ease of comparison, the values for estrone and estrone sulfate are expressed in terms of pmol per ml with equivalent values in pg per ml in parentheses wherever convenient.
The molecular
weights of estrone and estrone sulfate (sodium salt) have been taken as 270 and 372 respectively. Evaluation of the method Recovery:
The recovery for added 3H labelled estrone after extraction
and chromatography was 75 2 5% (S.D,; n = 40) and for added 3H labelled estrone sulfate after extraction, hydrolysis and chromatography was 46 + 6% (S.D.; n = 40). Accuracy:
Table I shows the degree of accuracy in measuring a
relatively large number of known amounts of estrone sulfate added to 1 ml of castrate male lamb plasma.
All values given are net values,
after correction for the blank and procedural losses.
At the lower
range there was an overestimation of about 14%, which was not evident at levels >5.38 pmol (2000 pg).
A linear relationship was obtained
between the quantity of estrone sulfate added and that estimated. Linear regression analysis established a regression equation of y = x + 0.14 (where x = pmol added and y = pmol recovered) and a coefficient of correlation approximately equal to unity.
STEROIDS
861
Table I Accuracy of estimating different levels of estrone sulfate ElS added to 1 ml plasma pmol/ml (pg/ml) Plasma blanka 0.54 1.34 2.69 5.38 53.80
(200) (500) (1000) (2000) (20,000)
Estimated value
Accuracy %
bnolhl)
of added value
0.10 t 0.02b (5)c 0.60 1.58 3.06 5.42 54.5
+ 0.10 + 0.16 + 0.46 f. 0.62 + 6.31
(14) (16) (26) (21) (40)
111 117 114 101 101
+ 19 + 12 + 17 + 12 f. 12
Estrone sulfate (ElS) was added to 1 ml of castrate male lamb plasma and extracted, separated, hydrolysed and measured as estone by radioimmunoassay as described in the text. aThe values shown are those obtained when measured against the assay blank. b Standard deviation. C
Number of determinations.
Table II Intra- and inter-assay precision of the method. Replicate assays each in quadruplicate, of the same sample (pmoles + S.D,)
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
58.6 + 4.49 55.0 -7 2.92 53.6 T 3.71 55.8 T 1.23 62.4 T 3.22 49.0 T 0.22 62.9 T 5.12 52.8 T 1.76 48.9 +. 3.44 45.7 2 5.29
Mean f. S.D. 54.5 2 5.70
Intra-assay coefficient of variation (%) 7.7 5.3 6.9 5.2 2.2 0.4 8.2 3.3 7.0 11.6 5.8 + 3.2
STEROIDS
Precision:
The inter- and intra-assay precision was estimated by
assaying 10 replicates from a plasma pool at weekly intervals, every assay being carried out in quadruplicate. in Table II.
The data are summarized
The intra-assay coefficient of variation ranged from
0 ..4- 11.6% with a mean of 5.8%.
The inter-assay precision, as
estimated from the mean of replicate assays, gave a coefficient of variation of 10.5%. Specificity:
The antiserum has a slightly higher affinity for
estradiol-178 than foe estrone.
However, these compounds were
separated from each other by Sephadex LH-20 chromatography prior to assay.
Previous studies have also established that the antiserum has
little cross reactivity with several naturally occuring Cl9 and C21 steroids (10).
Furthermore, the sulfate fraction extracted from
plasma was separated initially from other steroid conjugates and free steroids by thin-layer chromatography and then hydrolyzed by a sulfatase preparation having very low S-glucuronidase activity. the experimental conditions only 4% of an equimolar quantity
Under
or
estrone glucuronide was hydrolyzed compared to 80% of the corresponding sulfate.
Further evidence of high specificity is indicated by
the low values obtained for the castrate male lamb plasma (see below) which were not distinguishable from a bicarbonate buffer blank. Sensitivity:
The sensitivity of the assay is dependent on the detec-
tion limit of the antibody system, the plasma blank and the procedural losses.
Under the present conditions the sensitivity of the assay
has a practical detection limit of 1.85 x 10
-2
pm01 (5 pg) for estrone.
When assaying 1 ml of pl&.ma from castrate male lambs a mean value of 0.1 pmol (37 pg) of estrone sulfate was obtained when measured
STEROIDS
against the assay blank.
863
In practice, it has been found that to
obtain an accuracy of > 85% the minimum amount of estrone sulfate present in the plasma should be twice the value obtained for castrate male lamb plasma.
Taking
into consideration an overall recovery of
estrone sulfate of 45% and the fact that only 80% of the sample is available for assay (the remainder being used for estimation
of
procedural losses) the operational limit of sensitivity of the method is approximately 0.54 pm01 (200 pg) of estrone sulfate per ml of plasma. Changes in plasma levels of estrone sulfate and estrone around parturition in ewes. The changes in plasma levels of estrone sulfate and estrone in pooled plasma samples taken from two groups of ewes bearing single or twin lambs at 6 hr periods from 60 - 66 hr before parturition to 12 - 18 hr after parturition are shown in Figs. 1 6 2.
It can be
seen that measurable levels of estrone sulfate and estrone were present in the plasma of both groups at 60 - 66 hr before parturition. A rapid increase in plasma concentration of estrone sulfate began about 30 - 36 hr before parturition and reached a maximum at the time of parturition.
At this time the plasma level of estrone sulfate
was 103 pmol per ml (38320 pg) and 62 pmol per ml (23060 pg) for the twin and single lamb groups respectively.
The profile of changes
in plasma estrone was similar to that observed for plasma estrone sulfate except that the corresponding peak values were 3.9 pmol per ml (1050 pg) and 2.7 pmol per ml (730 pg).
The plasma levels of
both estrone sulfate and estrone declined rapidly after parturition and by 12 - 18 hr post partum the levels were below the sensitivity of
STEROIDS
864
SINGLE TWIN
HOURS CHANGES
FIG 1
AROUND
IN
TO
P
PARTURITION
PLASMA
ESTRONE
PARTURITION
IN
SULPHATE
CONCENTRATION
EWES
;4 E a 2 03 cc =; w 2
I
HOURS FIG
2.
CHANGES PARTURITION
IN
TO
PLASMA IN
PARTURITION
ESTRONk
CONCENTRATION
P
AROUND
EWES
NOTE: The times shown on the abscissae represent the mean of the 6 hr time interval, over which individual samples were collected. The value at each point is the mean of two or more determinations.
STEROIDS
the assay.
865
It is of interest that before parturition a consistent
difference was observed in the level of estrone sulfate between the two groups.
To determine if this difference might be significant,
individual samples from the two groups at 30 - 36 hr and 6 - 12 hr before parturition were assayed.
The results are presented in Table
III. Table III Estrone sulfate levels (pmol/ml) in maternal plasma of individual ewes bearing single and twin lambs at 30 - 36 hr and 6 - 12 hr before parturition. Group
Ewe No.
Time Before Parturition 30 - 36 hr
Single Lambs
B-21 R-35 Y-39 Y-40
17.3 20.4 8.7 15.5 Mean + S.D.
Twin Lambs
B-34 G-34 G-35 R-36
15.5 + 4.95 (14.7)a 35.6 33.1 13.1 31.1
Mean + S.D.
28.2 + 10.3 (32.1)
6 - 12 hr 61.4 75.3 47.4 46.6 57.7 + 13.6 (58.20) 113.0 123.0 48.1 84.5 92.1 t 33.6 (85.7)
a
Figures in parentheses represent values obtained from pooled plasma samples. The difference between single and twin lamb groups is not significant (P > 0.05). Although the differences did not appear to be significant, because of the small number of animals in each group and the wide variation in individual plasma levels, it should be noted that the values obtained for the pooled samples agree well with the mean of the corresponding individual samples.
No consistent or significant difference was
STEROIDS
866
observed in the estrone concentration between the twin and single lamb groups. In preliminary investigations the estradiol fraction derived from the conjugate extracts was assayed.
The results indicated that the
amounts present were too low to warrant further investigation. DISCUSSION The present method has been developed to measure the level of free and sulfate conjugated estrogens in a single plasma sample. The extraction and separation of the sulfate conjugate is, in et al (10) with cerain principle, similar to that described by Loriaux -modifications.
In preliminary studies, tetranydrofuran was used to
extract the sulfate conjugate from plasma.
The extracts were found to
contain a high lipid content which caused difficulty in subsequent evaporation and application of the extract to thin-layer plates. Subsequently, tetrahydrofuran: ethylacetate (l:lv/v) was found to provide the most desirable results in terms of efficiency and purity of extraction and ease of evaporation. The present results show that the plasma estrone concentration at parturition is 20-30 times lower than that of plasma estrone sulfate and accounts for only 4-5% of the total plasma estrone content (unconjugated and sulfate conjugate).
III
contrast, the concentrations
of the sulfate conjugates of estradiol-17a and -1713 do not appear to follow this pattern.
The quantitative relationships between the plasma
levels of estrone and estrone sulfate calls for very careful handling of the blood samples prior to assay, if the measurement of unconjugated estrone is to be meaningful.
It has been observed in the present
instance that the storage of tne plasma samples for 12 months or more
STEROIDS
with occasional thawing and refreezing has resulted in a 2-4 fold increase in the plasma level of estrone.
This increase can be
accounted for by a 2-3% hydrolysis of the estrone sulfate present. It has been established that the principal circulating unconjugated estrogen in sheep plasma during pregnancy and at parturition is estrone (2,4,12).
In addition, Bedford -et al (12) have established,
in 3 out of 4 sheep, that immediately before parturition there is good agreement between the uterine secretion rate of estrone and its estimated production rate.
The close correlation between these two
kinetic parameters strongly suggests that the uterus is the principal source of unconjugated estrone during late pregnancy. Although the source of the very high plasma levels of estrone sulfate at parturition is not known, it is significant that the rise and fall in the plasma levels of estrone and estrone sulfate parallel each other very closely.
The rapid fall in plasma estrone sulfate
to non-detectable levels within 15 hr after parturition suggests that the feto-placental endocrine system may be an important source of estrone sulfate. ACKNOWLEDGEMENTS The author wishes to express his thanks to Mrs. Ann Burnett for her excellent technical assistance, to Dr. B.V. Caldwell for the antiserum and advice, and to Dr. L. Ainsworth and Dr. F.A. Vandenheuvel, Reproductive Physiology Section, Animal Research Institute, for their interest and constructive criticism. REFERENCES 1. 2. 3. 4.
Challis, J.R.G,, NATURE, LOND. 229, 208 (1971). Thorburn, G.D., Nicol, D.H., Bassett, J.M. Shutt, D.A. and Cox, R.I., J. REPROD. FERT., Suppl. 16, 61 (1972). Liggins, G.C., G&eves, S.A., Kendall, J.Z., and Know, B.S., J. REPROD. FERT., Suppl. 16, 85 (1972). Robertson, H.A. and Smeaton, T.C., J. REPROD. FERT., 35, 761 (1973).
STEROIDS
868
5. 6. 7. 8. 9. 10. 11.
12. 13.
1
Robertson, H.A., J. REPROD. FERT., 36, In Press. Challis, J.R.G., Heap, R.B. and Illingworth, D.V., J. ENDOCR. 51, 1333 (1971). Yoshinaga, K., Hawkins, R.A., and Stocker, J.F., ENDOCRINOLOGY 85, 103 (1969). Findlay, J.K., and Cox, R.I., J. ENDOCR. 46, 281 (1970). Purdy, R.H., Engel, L.L. and Oncley, J.L., J. BIOL. CHEM. 236 (1961). Loriaux, D.L., Ruder, H.J. and Lipsett, M.B., STEROIDS, 18, 463 (1971). Loriaux, D.L., Ruder, H.J., Knab, D.R. and Lipsett, M.B., 3. CLIN. ENDOCRINOL. METAB. 35, 887 (1972). Bedford, C.C., Challis, J.R.G., Harrison, F.A. and Heap, R.B., J. REPROD. FERT., Suppl. 16, 1 (1972). Challis, J.R.G. and Linzell, J.L., J. REPROD. FERT., 26, 401 (1971).
Systematic names of steroids used in this study are: ESTRONE = 3-hydroxy-1,3,5(10)-estratrien-17-one ESTRONE SULFATE = 17-oxo-1,3,5(10)-estratrien-3-yl sulfate ESTRADIOL-17B = 1,3,5(10)-estratriene-3, 17B-diol ESTRADIOL-17a = 1,3,5(10)-estratriene-3, 17a-diol
CHANGES IN PLASMA LEVELS OF ESTRONE SULFATE AND ESTRONE IN THE PREGNANT EWE AROUND PARTURITION
Charles P.W. Tsang Reproductive Physiology Section, Animal Research Institute, Ottawa, Ontario, Canada, KIA OC6
Received:
3/21/74
ABSTRACT A method for the extraction, separation and measurement of estrone sulfate and estrone in a single plasma sample is described. The method has been applied to the determination of plasma levels of estrone sulfate and estrone in pregnant ewes over the period 60 hr before to 20 hr after parturition. The study revealed that the plasma levels of estrone sulfate and estrone began to increase about 40 hr before parturition, reached a peak at parturition and then declined rapidly to levels below the sensitivity of the method by 15 hr postpartum. The peak level of estrone sulfate recorded at parturition was 103 pmof (38 ng) per ml of plasma which was approximately 30 times greater than the corresponding peak level of estrone. INTRODUCTION The development of radioimmunoassay techniques for the determination of unconjugated estrogens has resulted in the measurement of plasma estrogens and establishment of plasma estrogen profiles in a variety of animal species during pregnancy, e.g., sheep (l-4), goat (2, 13), cow (5); guinea pig (6) and rat (7).
In contrast, little
information is currently available on the changes of plasma levels of estrogen conjugates during various physiological states.
In those
instances in which plasma estrogen conjugates, and in particular
Contribution No. 529, Animal Research Institute
VoZwne 23, Number 6
STEROIDS
June, 2974
STEROIDS
plasma estrogen sulfates, have been determined, the sulfate conjugates appear to be present in quantities considerably greater than the corresponding unconjugated estrogen (8-11). In an attempt to establish the relative amounts of estrogens and estrogen sulfates-in plasma as well as a possible functional role for circulating estrogen sulfates in domestic animals, a method has been developed for the extraction, separation and measurement of estrogen sulfates and unconjugated estrogens in a single plasma sample. Initially, the method has been applied to the measurement of plasma estrone sulfate
1
and estrone around parturition in the ewe. MATERIALS AND METHODS
Solvent and reagents All solvents were of spectroquality grade and were used as obtained, Estrone-6, 7-3H sulfate, ammonium salt (S.A. 40 Ci/mmol), estrone-2,4,6,7-3H (S.A. 104 Ci/mmol) and estradiol-17B-2,4,6,7-3H (S,A. 105 Ci/mmol) were obtained from New England Nuclear Corporation, Boston Mass., U.S.A. Stock solutions containing 100 pCi/ml were made up and stored at -20°C in absolute methanol to reduce the effect of self-irradiation. Crystalline estrone-3-sulfate, sodium salt, crystalline estrone and crystalline estradiol-178 were purchased from Sigma Chemical Co., St. Louis, MO., U.S.A. Standard solutions were prepared in absolute methanol. The purity of both radioactive and non-radioactive steroids was checked by thin-layer chromatography before use. Sulfatase Type III (Sigma Chemical Co.) for the hydrolysis of estrone sulfate had very low f3-glucuronidase activity. A stock solution was prepared by dissolving the enzyme preparation in 0.2 M acetate buffer, pH 5.0, at a concentration of 2 mg/ml buffer. This solution could be stored at -2O'C for at least two months without appreciable loss of activity. For routine hydrolysis 0.1 ml of the stock solution was used per sample. Chromatopraphy Thin-layer chromatography was used to separate estrogen sulfates from free steroids and other steroid conjugates in the sample extracts. Chromatography was carried out on aluminum plates coated with silica
STEROIDS
gel F-254, 25 mm thick (E. Merck, Darmstadt) and the chromatograms developed in the system ethylacetate: ethanol: ammonium hydroxide (5:5:1) as described by Loriaux et al (10). Reference estrone sulfate was run alongside the sample extracts and located by cutting out the strip containing the standard, spraying with 10% sulfuric acid in ethanol and heating at 1lO'C for 10 minutes. The area of silica gel corresponding to estrone sulfate on the sample strips was aspirated into a Pasteur pipette plugged lightly with a dental cotton pellet which had been pre-washed with methanol. The sulfate fraction was eluted from the silica gel with 2 x 2 ml methanol into a 15 ml stoppered centrifuge tube. Sephadex LH-20 columns were used to separate estrone from the isomeric estradiols in the plasma extracts. Columns were prepared by gently pouring 1 g Sephadex LH-20 (Pharmacia, Uppsala, Sweden) through a funnel into 10 ml disposable Pyrex pipettes (ID 0.9 mm approx.) containing a small glass bead. The columns were washed initially with 10 ml methanol and then allowed to swell overnight in 7.0 ml methanol. Before use, the methanol was allowed to drain from the column which was then washed with 20 ml of the eluting solvent. The sample was applied to the column and the estrone eluted from the column as described below. After use, the columns were washed with 20 ml methanol and maintained in this solvent until the next run. Columns prepared in this manner were found to have uniform elution properties, could be used for several months and gave clean blanks. Antiserum for radioimmunoassay Sheep antiserum to estradiol-178 hemisuccinyl BSA was kindly provided by Dr. B.V. Caldwell, Department of Obstetrics & Gynecology, Department of Medicine, Yale University, Connecticut, U.S.A. The antiserum was diluted 1:lOO with Buffer A, prepared by dissolving NaH2P04, 4H20, 5.38 g; Na2HP04, 7H 0, 16.35 g; NaCl, 9.0 g; NaN 1 g; gelatin 1 g in 1 liter of distille 3 water, and stored at -2O'C ?n 0.5 ml aliquots. For the assay, an aliquot of the antiserum was diluted further with the same buffer to give a final dilution of 1:32,000. Dextran-coated charcoal Dextran T80 (Pharmacia, Uppsala, Sweden), 50 mg and 500 mg Norit A charcoal were suspended in 100 ml Buffer A. The mixture was kept in ice and maintained in suspension by stirring continuously with a magnetic stirrer. Measurement of radioactivity Because of differences in solubility between estrone and estrone sulfate, quantitation of radioactivity present in the samples was carried out using two scintillation solutions. (a) Standard 3H labelled estrone and benzene extracts from plasma were counted in 10 ml of a scintillation fluid which contained 4 g
857
STEROIDS
Omniflour (New England Nuclear) dissolved in 1 liter toluene. (b) Standard 3H labelled estrone sulfate and estrone derived from hydrolysis of extracts containing the sulfate fraction were counted in 10 ml of a solution which contained 8 g Omniflour and 100 mg naphthalene dissolved in 1 liter dioxane +,0.5 ml buffer A. Samples were counted in a Beckman Model LS-250 Scintillation Spectrometer. The efficiency of counting 3H under conditions (a) and (b) was 65% and 40% respectively. All samples were counted to an error of + 2%. Plasma Samples For estimation of blanks and recovery experiments blood was obtained by jugular venous puncture from castrate male lambs. Immediately after collection into heparinized vaccutainer tubes, the blood was cooled in ice and centrifuged at 4'C within 1 hr. The plasma was removed and stored in 3 ml aliquots at -20°C. Pregnancy blood samples were obtained from eight Western ewes near term as described previously (5). Briefly, blood samples were collected every 6 hr from 96 hr before to 72 after parturition. Plasma samples were prepared as described for the castrate male plasma. The samples were kept frozen for fourteen months before being assayed and were thawed and re-frozen occasionally in connection with other studies. For the assay, samples from 4 ewes giving birth to single lambs and from 4 ewes giving birth to twins were separated into 2 groups and a pooled sample was made for every 6 hr collection period within each group. Thus every pooled sample contained equal aliquots from 4 ewes. In addition, assays were carried out on individual samples taken at 30-36 hr and 6-12 hr before parturition to estimate individual variations and to determine whether a statistically significant difference existed between the single and twin lamb groups. Internal standards Approximately 2000 cpm of 3H labelled estrone sulfate (equivalent to 5.4 x 10-2 pmol or 20 pg) and 1000 cpm 3H labelled estrone (equivalent to 7.4 x 10-3 pmol or 2 pg) were added to all plasma samples to correct for procedural losses. Extraction of unconiupated estrogens from plasma. To plasma samples (0.2 - 1.0 ml) in 45 ml stoppered centrifuge tubes was added 2 ml of 0.5 M sodium bicarbonate (pH 8.0) and the contents mixed with a Vortex mixer. Benzene (5 ml) was added and the free estrogens extracted by mixing the contents for 2 min. The tube was then centrifuged at 1500 rpm for 5 min, the benzene phase transferred to 5 ml disposable siliconized culture tubes and evaporated to dryness under nitrogen.
STEROIDS
Extraction and separation of estrogen sulfates The sulfate conjugates were similarly extracted from the aqueous phase remaining in the centrifuge tube by extraction with 2 x 5 ml of tetrahydrofuran: ethyl acetate (1:l v/v). However, the centrifugation time after each extraction was extended to lo-20 min, particularly when an emulsion formed. The organic phases were transferred to 5 ml siliconized culture tubes, evaporated to dryness under nitrogen and the dry extract applied quantitatively to a thin-layer plate with 3 x 0.025 ml absolute methanol. The sulfate fraction was separated and eluted from the plate as described above. Hydrolysis of the sulfate fraction After removal of the methanol under nitrogen, the dry extract was dissolved in 0.5 ml of 0.2 M sodium acetate buffer, pH 5.0, containing 200 ug sulfatase Type III and allowed to incubate at 37'C overnight with constant shaking in a water bath. On completion of hydrolysis, the free steroids liberated were extracted with 2 x 2 ml benzene, the organic phases transferred to siliconized culture tubes and evaporated to dryness under nitrogen. Separation of estrone from the isomeric estradiols Dry benzene extracts, whether derived from initial extraction of the plasma sample or from hydrolysis of the sulfate fraction, were dissolved in 0.2 ml of iso-octane: benzene: methanol (62:20:1% v/v/v) and applied to Sephadex LH-20 columns equilibrated in the same solvent. Two additional rinses each of 0.1 ml column solvent were similarly applied to the column. After the sample had entered the column, the column was eluted with 4 ml of the same solvent mixture, which was discarded. The estrone fraction was then eluted from the column into siliconized tubes with 4 ml of the column solvent. If required, the estradiol fraction could be eluted from the column with a further 4.5 ml column solvent, by discarding the first 0.5 ml and collecting the next 4.0 ml. Estimation of procedural losses The estrone fractions obtained after Sephadex LH-20 chromatography were evaporated to dryness under nitrogen and the dry residues dissolved ;n 1.0 ml absolute methanol. An aliquot of 0.2 ml was taken for counting to estimate procedural losses. The remainder of & each sample, or an aliquot (depending on the expected concentration in the sample), was dried down under nitrogen for determination of estrogen content. Radioimmunoassay procedure To each tube to be assayed was added 0.5 ml of a buffer A containing 12,000 cpm of 3H labelled estradiol-176 (equivalent to 9.2 x 10-2 pmol or 25 pg) and the tubes gently mixed and incubated at 37°C for 10 min to ensure 'complete solution of the estrogen. Then 0.1 ml of a 1:32,000 dilution of the antiserum in buffer A was added, the tubes
STEROIDS
again mixed and left at 4°C overnight. The unbound estrogen was absorbed by addition of 0.5 ml dextran coated charcoal suspension to each tube kept in an ice/water bath. The tubes were allowed to stand for 5 min, mixed.for 10 set, allowed to stand for a further 5 min and finally centrifuged for 2.5 min at 1500 rpm and 4°C. The supernatant from each tube was transferred to counting vials and the radioactivity determined. A standard curve for estrone was obtained by treating tubes containing O-200 pg (equivalent to O-O.54 pmol) in a manner similar to that for the unknown samples. The tube containing zero picograms of estrone served to establish the assay blank. RESULTS For clarity and ease of comparison, the values for estrone and estrone sulfate are expressed in terms of pmol per ml with equivalent values in pg per ml in parentheses wherever convenient.
The molecular
weights of estrone and estrone sulfate (sodium salt) have been taken as 270 and 372 respectively. Evaluation of the method Recovery:
The recovery for added 3H labelled estrone after extraction
and chromatography was 75 2 5% (S.D,; n = 40) and for added 3H labelled estrone sulfate after extraction, hydrolysis and chromatography was 46 + 6% (S.D.; n = 40). Accuracy:
Table I shows the degree of accuracy in measuring a
relatively large number of known amounts of estrone sulfate added to 1 ml of castrate male lamb plasma.
All values given are net values,
after correction for the blank and procedural losses.
At the lower
range there was an overestimation of about 14%, which was not evident at levels >5.38 pmol (2000 pg).
A linear relationship was obtained
between the quantity of estrone sulfate added and that estimated. Linear regression analysis established a regression equation of y = x + 0.14 (where x = pmol added and y = pmol recovered) and a coefficient of correlation approximately equal to unity.
STEROIDS
861
Table I Accuracy of estimating different levels of estrone sulfate ElS added to 1 ml plasma pmol/ml (pg/ml) Plasma blanka 0.54 1.34 2.69 5.38 53.80
(200) (500) (1000) (2000) (20,000)
Estimated value
Accuracy %
bnolhl)
of added value
0.10 t 0.02b (5)c 0.60 1.58 3.06 5.42 54.5
+ 0.10 + 0.16 + 0.46 f. 0.62 + 6.31
(14) (16) (26) (21) (40)
111 117 114 101 101
+ 19 + 12 + 17 + 12 f. 12
Estrone sulfate (ElS) was added to 1 ml of castrate male lamb plasma and extracted, separated, hydrolysed and measured as estone by radioimmunoassay as described in the text. aThe values shown are those obtained when measured against the assay blank. b Standard deviation. C
Number of determinations.
Table II Intra- and inter-assay precision of the method. Replicate assays each in quadruplicate, of the same sample (pmoles + S.D,)
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
58.6 + 4.49 55.0 -7 2.92 53.6 T 3.71 55.8 T 1.23 62.4 T 3.22 49.0 T 0.22 62.9 T 5.12 52.8 T 1.76 48.9 +. 3.44 45.7 2 5.29
Mean f. S.D. 54.5 2 5.70
Intra-assay coefficient of variation (%) 7.7 5.3 6.9 5.2 2.2 0.4 8.2 3.3 7.0 11.6 5.8 + 3.2
STEROIDS
Precision:
The inter- and intra-assay precision was estimated by
assaying 10 replicates from a plasma pool at weekly intervals, every assay being carried out in quadruplicate. in Table II.
The data are summarized
The intra-assay coefficient of variation ranged from
0 ..4- 11.6% with a mean of 5.8%.
The inter-assay precision, as
estimated from the mean of replicate assays, gave a coefficient of variation of 10.5%. Specificity:
The antiserum has a slightly higher affinity for
estradiol-178 than foe estrone.
However, these compounds were
separated from each other by Sephadex LH-20 chromatography prior to assay.
Previous studies have also established that the antiserum has
little cross reactivity with several naturally occuring Cl9 and C21 steroids (10).
Furthermore, the sulfate fraction extracted from
plasma was separated initially from other steroid conjugates and free steroids by thin-layer chromatography and then hydrolyzed by a sulfatase preparation having very low S-glucuronidase activity. the experimental conditions only 4% of an equimolar quantity
Under
or
estrone glucuronide was hydrolyzed compared to 80% of the corresponding sulfate.
Further evidence of high specificity is indicated by
the low values obtained for the castrate male lamb plasma (see below) which were not distinguishable from a bicarbonate buffer blank. Sensitivity:
The sensitivity of the assay is dependent on the detec-
tion limit of the antibody system, the plasma blank and the procedural losses.
Under the present conditions the sensitivity of the assay
has a practical detection limit of 1.85 x 10
-2
pm01 (5 pg) for estrone.
When assaying 1 ml of pl&.ma from castrate male lambs a mean value of 0.1 pmol (37 pg) of estrone sulfate was obtained when measured
STEROIDS
against the assay blank.
863
In practice, it has been found that to
obtain an accuracy of > 85% the minimum amount of estrone sulfate present in the plasma should be twice the value obtained for castrate male lamb plasma.
Taking
into consideration an overall recovery of
estrone sulfate of 45% and the fact that only 80% of the sample is available for assay (the remainder being used for estimation
of
procedural losses) the operational limit of sensitivity of the method is approximately 0.54 pm01 (200 pg) of estrone sulfate per ml of plasma. Changes in plasma levels of estrone sulfate and estrone around parturition in ewes. The changes in plasma levels of estrone sulfate and estrone in pooled plasma samples taken from two groups of ewes bearing single or twin lambs at 6 hr periods from 60 - 66 hr before parturition to 12 - 18 hr after parturition are shown in Figs. 1 6 2.
It can be
seen that measurable levels of estrone sulfate and estrone were present in the plasma of both groups at 60 - 66 hr before parturition. A rapid increase in plasma concentration of estrone sulfate began about 30 - 36 hr before parturition and reached a maximum at the time of parturition.
At this time the plasma level of estrone sulfate
was 103 pmol per ml (38320 pg) and 62 pmol per ml (23060 pg) for the twin and single lamb groups respectively.
The profile of changes
in plasma estrone was similar to that observed for plasma estrone sulfate except that the corresponding peak values were 3.9 pmol per ml (1050 pg) and 2.7 pmol per ml (730 pg).
The plasma levels of
both estrone sulfate and estrone declined rapidly after parturition and by 12 - 18 hr post partum the levels were below the sensitivity of
STEROIDS
864
SINGLE TWIN
HOURS CHANGES
FIG 1
AROUND
IN
TO
P
PARTURITION
PLASMA
ESTRONE
PARTURITION
IN
SULPHATE
CONCENTRATION
EWES
;4 E a 2 03 cc =; w 2
I
HOURS FIG
2.
CHANGES PARTURITION
IN
TO
PLASMA IN
PARTURITION
ESTRONk
CONCENTRATION
P
AROUND
EWES
NOTE: The times shown on the abscissae represent the mean of the 6 hr time interval, over which individual samples were collected. The value at each point is the mean of two or more determinations.
STEROIDS
the assay.
865
It is of interest that before parturition a consistent
difference was observed in the level of estrone sulfate between the two groups.
To determine if this difference might be significant,
individual samples from the two groups at 30 - 36 hr and 6 - 12 hr before parturition were assayed.
The results are presented in Table
III. Table III Estrone sulfate levels (pmol/ml) in maternal plasma of individual ewes bearing single and twin lambs at 30 - 36 hr and 6 - 12 hr before parturition. Group
Ewe No.
Time Before Parturition 30 - 36 hr
Single Lambs
B-21 R-35 Y-39 Y-40
17.3 20.4 8.7 15.5 Mean + S.D.
Twin Lambs
B-34 G-34 G-35 R-36
15.5 + 4.95 (14.7)a 35.6 33.1 13.1 31.1
Mean + S.D.
28.2 + 10.3 (32.1)
6 - 12 hr 61.4 75.3 47.4 46.6 57.7 + 13.6 (58.20) 113.0 123.0 48.1 84.5 92.1 t 33.6 (85.7)
a
Figures in parentheses represent values obtained from pooled plasma samples. The difference between single and twin lamb groups is not significant (P > 0.05). Although the differences did not appear to be significant, because of the small number of animals in each group and the wide variation in individual plasma levels, it should be noted that the values obtained for the pooled samples agree well with the mean of the corresponding individual samples.
No consistent or significant difference was
STEROIDS
866
observed in the estrone concentration between the twin and single lamb groups. In preliminary investigations the estradiol fraction derived from the conjugate extracts was assayed.
The results indicated that the
amounts present were too low to warrant further investigation. DISCUSSION The present method has been developed to measure the level of free and sulfate conjugated estrogens in a single plasma sample. The extraction and separation of the sulfate conjugate is, in et al (10) with cerain principle, similar to that described by Loriaux -modifications.
In preliminary studies, tetranydrofuran was used to
extract the sulfate conjugate from plasma.
The extracts were found to
contain a high lipid content which caused difficulty in subsequent evaporation and application of the extract to thin-layer plates. Subsequently, tetrahydrofuran: ethylacetate (l:lv/v) was found to provide the most desirable results in terms of efficiency and purity of extraction and ease of evaporation. The present results show that the plasma estrone concentration at parturition is 20-30 times lower than that of plasma estrone sulfate and accounts for only 4-5% of the total plasma estrone content (unconjugated and sulfate conjugate).
III
contrast, the concentrations
of the sulfate conjugates of estradiol-17a and -1713 do not appear to follow this pattern.
The quantitative relationships between the plasma
levels of estrone and estrone sulfate calls for very careful handling of the blood samples prior to assay, if the measurement of unconjugated estrone is to be meaningful.
It has been observed in the present
instance that the storage of tne plasma samples for 12 months or more
STEROIDS
with occasional thawing and refreezing has resulted in a 2-4 fold increase in the plasma level of estrone.
This increase can be
accounted for by a 2-3% hydrolysis of the estrone sulfate present. It has been established that the principal circulating unconjugated estrogen in sheep plasma during pregnancy and at parturition is estrone (2,4,12).
In addition, Bedford -et al (12) have established,
in 3 out of 4 sheep, that immediately before parturition there is good agreement between the uterine secretion rate of estrone and its estimated production rate.
The close correlation between these two
kinetic parameters strongly suggests that the uterus is the principal source of unconjugated estrone during late pregnancy. Although the source of the very high plasma levels of estrone sulfate at parturition is not known, it is significant that the rise and fall in the plasma levels of estrone and estrone sulfate parallel each other very closely.
The rapid fall in plasma estrone sulfate
to non-detectable levels within 15 hr after parturition suggests that the feto-placental endocrine system may be an important source of estrone sulfate. ACKNOWLEDGEMENTS The author wishes to express his thanks to Mrs. Ann Burnett for her excellent technical assistance, to Dr. B.V. Caldwell for the antiserum and advice, and to Dr. L. Ainsworth and Dr. F.A. Vandenheuvel, Reproductive Physiology Section, Animal Research Institute, for their interest and constructive criticism. REFERENCES 1. 2. 3. 4.
Challis, J.R.G,, NATURE, LOND. 229, 208 (1971). Thorburn, G.D., Nicol, D.H., Bassett, J.M. Shutt, D.A. and Cox, R.I., J. REPROD. FERT., Suppl. 16, 61 (1972). Liggins, G.C., G&eves, S.A., Kendall, J.Z., and Know, B.S., J. REPROD. FERT., Suppl. 16, 85 (1972). Robertson, H.A. and Smeaton, T.C., J. REPROD. FERT., 35, 761 (1973).
STEROIDS
868
5. 6. 7. 8. 9. 10. 11.
12. 13.
1
Robertson, H.A., J. REPROD. FERT., 36, In Press. Challis, J.R.G., Heap, R.B. and Illingworth, D.V., J. ENDOCR. 51, 1333 (1971). Yoshinaga, K., Hawkins, R.A., and Stocker, J.F., ENDOCRINOLOGY 85, 103 (1969). Findlay, J.K., and Cox, R.I., J. ENDOCR. 46, 281 (1970). Purdy, R.H., Engel, L.L. and Oncley, J.L., J. BIOL. CHEM. 236 (1961). Loriaux, D.L., Ruder, H.J. and Lipsett, M.B., STEROIDS, 18, 463 (1971). Loriaux, D.L., Ruder, H.J., Knab, D.R. and Lipsett, M.B., 3. CLIN. ENDOCRINOL. METAB. 35, 887 (1972). Bedford, C.C., Challis, J.R.G., Harrison, F.A. and Heap, R.B., J. REPROD. FERT., Suppl. 16, 1 (1972). Challis, J.R.G. and Linzell, J.L., J. REPROD. FERT., 26, 401 (1971).
Systematic names of steroids used in this study are: ESTRONE = 3-hydroxy-1,3,5(10)-estratrien-17-one ESTRONE SULFATE = 17-oxo-1,3,5(10)-estratrien-3-yl sulfate ESTRADIOL-17B = 1,3,5(10)-estratriene-3, 17B-diol ESTRADIOL-17a = 1,3,5(10)-estratriene-3, 17a-diol