METABOLIC CLEARANCE RATE AND PRODUCTION RATE OF OESTRADIOL IN CONSCIOUS RABBITS I. S. FRASER, J. R. G. CHALLIS AND G. D. THORBURN University of Oxford, Nujfield Institute for Medical Research, and John Radcliffe Nuffield Department of Obstetrics and Gynaecology, Headley Way, Headington,
Hospital, Oxford
(Received 20 May 1975) SUMMARY
A continuous isotope infusion technique was used to measure the metabolic clearance rate (MCRB) and production rate (PRB) of oestradiol-17\g=b\ in a group of six non-pregnant conscious Chinchilla rabbits. The mean MCRB of oestradiol was 162 ml/min (0\m=.\0791/ min/weight0\m=.\75), and at equilibrium a significant proportion of the radioactivity in the circulation was present as oestrone (conversion ratio oestradiol to oestrone, 27\m=.\6%). The mean PRB of oestradiol was 4\m=.\3ng/min, which is equivalent to about 5 \g=m\g/day. Some day-to-day variation was seen in the endogenous concentrations of oestradiol, oestrone and progesterone although in general the levels of the two oestrogens were less than 30\p=n-\40pg/ml, and progesterone levels were below 300 pg/ml apart from short peaks up to 600 pg/ml. These values were compared with previous measurements of the ovarian secretion rate of oestradiol measured directly in anaesthetized animals.
INTRODUCTION
Although the rabbit has been used extensively as an experimental model for studying the actions of steroid hormones, there is little information concerning the endogenous production rates of steroids in the conscious animal and the variation in this from day to day. Previous workers have measured the ovarian secretion rates of progesterone, oestrone and oestradiol by anaesthetized rabbits in various reproductive states (Eaton & Hilliard, 1971 ; Hilliard & Eaton, 1971 ; Shaikh & Harper, 1972; Hilliard, Scaramuzzi, Pang, Penardi & Sawyer, 1974). In the present study the production rate of oestradiol was measured using tracer isotope kinetic techniques in conscious non-pregnant rabbits. These determinations are compared with previous direct measurements of ovarian oestradiol secretion rates. MATERIALS AND METHODS
Animals Six non-pregnant, sexually mature Chinchilla rabbits (body weights 2-1-3-2 kg) were used in this study. The animals were caged singly with food and water available ad libitum. Steroids [2,4,6,7-3H]Oestradiol-17/? (85 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, and was purified by Celite column chromatography (Abraham, Tulchinsky & Korenman, 1970). On the day before an infusion experiment, the purity of the [3H]oestradiol was checked by thin-layer chromatography (t.l.c.) in the solvent system cyclohexane : ethyl
(1:1, v/v). The radioactivity was located as a single peak using a DünnschichtScanner II. Other radioactive and non-radioactive oestrogens were obtained and stored as described by Challis, Harrison & Heap (19736). Chemicals All solvents were of nanograde quality (Mallinckrodt Co.), except cyclohexane which was of AR grade (BDH Chemicals Ltd). Thin-layer chromatography was performed using precoated plates, Kieselgel 60 F254 (Merck) of 0-25 mm thickness. The plates were washed once in ethyl acetate before sample application. acetate
Infusion procedure Three to four days before an isotope infusion, the infusion and sampling catheters were implanted into the rabbits under sodium pentobarbitone (30 mg/kg) anaesthesia, supple¬ mented with skin analgesia using 1 % xylocaine. A teflon catheter (0-56 mm i.d. 1-10 mm o.d. Becton Dickinson & Co., Rutherford, New Jersey, U.S.A.) for infusion was implanted into the lower inferior vena cava via the femoral vein, and a vinyl or Polythene catheter (Portex Ltd; 1-27-1-4 mm o.d. 0-63-0-86 mm i.d.) for sampling was inserted into a femoral artery. The catheters were located subcutaneously, and exteriorized at the back of the neck. They were flushed daily with sterile heparin-saline (5 i.u./ml) and heat-sealed. For the measurement of metabolic clearance rate (MCRB), [3H]oestradiol-17/# (20/¿Ci) in sterile 0-9 % NaCl solution containing 6 % ethanol was infused into the vena cava at a constant rate (3-3 ml/h; approx. 0-06 /¿Ci/min, see Table 1). The infusion was maintained using a glass syringe mounted on a Braun infusion pump (B. Braun, Melsungen, Germany; Perfusor pump type 871104). The isotope infusion rate was equivalent to an oestradiol secretion rate of about 0-2 ng/min, which was less than 10 % of the endogenous production rate determined subsequently. During the course of a 4 h infusion, the fully conscious rabbits were restrained in wicker baskets, and their movements were partially restricted by the use of cotton padding. The animals remained generally calm throughout, although all made regular spontaneous move¬ ments and occasional attempts to turn round. Two animals began to hyperventilate at the end of the experiment and this may have been due to the heat of the laboratory. The animals had become used to frequent handling during the previous week. Five experiments were started at approximately 12.00 h; one experiment (rabbit no. 2) was started at 17.46 h. One blood sample (3-5 ml), for the determination of the endogenous oestradiol con¬ centration, was taken immediately before the start of the infusion. Three further samples of about 5 ml were taken at 15 min intervals, beginning 160-225 (mean 185) min after the start of the isotope infusion. The total amount of blood drawn was therefore less than 10 % of the total blood volume of the smallest rabbit. All samples of blood were drawn into heparinized syringes, and transferred to chilled glass vessels containing a known volume of distilled water. The samples were mixed, reweighed, and then stored at 15°C until
analysis.
For the calculation of infusion rate, triplicate timed collections of the infusion catheter before and after an infusion experiment. from the residual infúsate (Longcope, Layne & Tait, 1968).
—
taken from the end Samples were also taken were
Daily blood sampling Samples of blood (1-3 ml) were obtained regularly from three of the rabbits during a period of 9-17 days after the animals had been used in an MCR experiment. The samples were taken from an ear vein by venipuncture into heparinized syringes, and frozen as whole blood until analysis.
Measurement of endogenous steroid concentrations The concentrations of oestradiol-17ß, oestrone and progesterone were determined in 1-2 ml aliquots of the pre-infusion and daily blood samples by radioimmunoassay (Challis, Davies & Ryan, 1973 a).
Analysis of radioactivity Haemolysed blood was thawed slowly overnight at 4 °C. Samples (4-5 ml) were added to 4 dr vials (Camlab, Cambridge), containing 50 ng each of oestradiol and oestrone in 50 /¿l ethanol for recovery estimations. The samples were mixed gently and extracted with 3 6 ml fresh diethyl ether (Mallinckrodt). Emulsions, if they formed, were broken by centrifugation. The pooled extracts were dried, and applied to t.l.c. plates as described previously (Challis et al. 19736). Chromatograms were developed in the system ethyl acetate : cyclohexane (1:1, v/v) and the oestrone and oestradiol- 17/Î zones were located by scanning wellseparated lanes containing tritiated marker oestrogens. Blank lanes were run either side of the markers. This system permits a good separation of oestrone (Rr, 0-68) from oestradiol17/? (RF, 0-52) with oestradiol-17a occupying the intermediate area. The oestrone and oestradiol-17/? zones were eluted (Challis et al. 19736), dried, and redissolved in 1 ml isooctane for Celite chromatography (Abraham et al. 1970; Challis et al. 1973 ). The oestrone and oestradiol fractions were then dried and redissolved in 2-0 ml ethanol. Aliquots were taken for liquid scintillation counting and for radioimmunoassay to determine methodo¬ logical losses. In a preliminary experiment, 0-5 ml aliquots of the blood samples taken at equilibrium from two rabbits were pooled, extracted with diethyl ether and subjected to t.l.c. The chromatogram lane was divided into 1 cm bands which were eluted and counted separately. More than 90 % of the radioactivity was located in the zones corresponding to oestradiol17/? and oestrone. Negligible radioactivity was found in the zones corresponding to oestra¬ diol-17a
or
16-oxo oestradiol.
Calculations The metabolic clearance rate (MCRB) and total production rate (PRB; Tait & Burstein, 1964) of oestradiol measured in whole blood was calculated as described by Baird & Fraser (1974). The conversion ratio of oestradiol to oestrone (CBB) was calculated according to Longcope et al. (1968). RESULTS
Evidence for radiochemical purity of the isolated oestrogens batch of one analyses, samples from fractions isolated as oestradiol-17/? or oestrone were pooled separately. Approximately 150 d.p.m. of [14C]oestradiol-17/? and 100 d.p.m. of [14C]oestrone were added to the respective pools and the [3H] : [UC] ratios of each were determined. The samples were acetylated at 50 °C for 1 h, and the acetates chromatographed in the t.l.c. system cyclohexane : ethyl acetate (2:1, v/v) which separates oestrone monoacetate (RF, 0-54) from oestradiol diacetate (RF, 0-72). The ratio of [3H]:[14C] in oestradiol was 3-53 and in oestradiol diacetate 3-31. The ratio of [3H]:[14C] in oestrone was 0-22 and in oestrone monoacetate 0T9, thereby indicating that radiochemical purity for both steroids had been achieved. Evidence for isotopie equilibrium Table 1 shows the concentrations of radioactivity isolated as oestradiol-17/? and oestrone in individual samples of blood taken at equilibrium (mean sample times 185,200 and 215 min from the start of the infusion). Individual values for each animal were calculated as a In
mean, and related to the time of sampling. When these normalized data analysed by regression analysis, the slopes for both oestradiol and oestrone were not significantly different from zero (regression equations: oestradiol, y 101—0-02*; oestrone, y 108-OT38.x) and it was considered that isotopie steady state had been achieved for both precursor and product steroids. The mean coefficient of variation for the
percentage of the were
—
=
oestradiol equilibrium values was 13-9 ± 1-9 (s.e.m.) %. For oestrone, reasonable agreement between replicates was found for rabbits 1, 3, 5 and 6, but large variations were found for rabbits 2 and 4. The mean coefficient of variation for all animals was 30-1 + 10-3 %. Table 1. Concentration of radioactivity in blood samples taken
as
at
oestradiol-17ß and oestrone equilibrium
Infusion Rabbit
Oestradiol-17/? (d.p.m./ml blood)
rate
,-'-,
no.
(/¿Ci/min)
185 min
200 min
215min
Mean
00565
1217 460 833 689 1411 1272
1249 541 1013 615 1720 967
1446
1304 491
00580
00674 00671 00688 00616
*, Final count No sample.
Oestrone
(d.p.m./ml blood)
,—
736
861
456 1719 1379
587 1617 1206
—,
185 min
200 min
215 min
Mean
343 308 208 360 370
304
324
324 158 412 239
275 218 420 219
108 294 138 478 198
*
too low for accurate determination.
—,
Table 2. Metabolic clearance
rate
oestradiol-17ß in Rabbit No. 1 2 3 4 5 6
Body wt (kg) 213 301 3-20 2-40 2-80 2-45
(MCR%ß) and production rate the non-pregnant rabbit
Isotope concentration (d.p.m./ml blood)
MCR!**
—*-
Oestradiol Oestrone 1304 491 861 587 1617 1206
ml/min 95-3 259-7
324
Endogenous 17-6
Mean
172-3 251-3 93-6 112-4 1641
44-7 86-2 53-8 104-7 33-4 45-9 61-5
260 43 0 200 210 241
+ S.E.M.
±31-2
±11-3
±40
275 218 420 219
product steady
Not calculated
PR!"
oestradiol
ml/min/kg (pg/ml) 170
state not achieved
(PRlß) of
(ng/min)
(%)
1-67 4-40 4-48 10-81 1-87 2-36 4-3 + 1-4
24-8 31-9 37-1 260 18-2 27-6
±3-2
(Table 1).
-
Metabolic clearance rate and production rate of oestradiol-17ß variation was found between individual rabbits in the value for MCRB (range Appreciable 93-260 ml/min; mean 164 ml/min, Table 2). This variation was still present after correcting for body weight. The mean MCRB after correcting for the mean body weight075 (W0-75; Kleiber, 1965) was 0-079 l/min/W075. The average endogenous concentration of oestradiol in the pre-infusion sample was 24 pg/ml whole blood, and the mean PRB of oestradiol was 4-3 ng/min (range 1-6-10-8 ng/ min). In rabbit no. 4 (PRB of oestradiol 10-8 ng/min), the endogenous oestradiol in a sample taken 24 h earlier was 14 pg/ml, which, with a similar MCR, would result in a calculated PRB of 3-5 ng/min. There was a significant conversion of oestradiol to oestrone (mean CBB 27-6 %). =
Endogenous steroid levels The concentrations of oestradiol, oestrone and progesterone in daily samples of blood from three rabbits are shown in Fig. 1. The mean concentration of oestradiol was about 20 pg/ml, and did not vary in any consistent manner. The oestrone concentration was generally ( ) Rabbit
800
-
600
-
400
-
200
-
3
160
120
80 40
0200
(b) Rabbit 2 800
f -
g 600
120
a
400
80
200
40
| •o
1
§
(c) Rabbit 5 400
-
200
-
Time
80 40
(days)
Fig. 1. Daily changes in peripheral blood concentrations of oestrone, oestradiol and progesterone in non-pregnant rabbits. I, Infusion day; , progesterone; O, oestrone; , oestradiol. than oestradiol, and in two of the rabbits (nos 2 and 3) peaks of ^ 100 pg/ml, of short duration and not associated with high oestradiol peaks, were found. The concentra¬ tion of progesterone was less than 300 pg/ml in most samples.
higher
There was a tendency for the concentrations of oestrone and oestradiol to change in similar fashion, and there was a significant correlation between the two oestrogens when the values for all three rabbits were considered together (r 0-367, 43, < 005). Progesterone was not correlated with either oestradiol or oestrone, except in rabbit no. 2, where it was marginally so ( < 0 5). =
=
DISCUSSION
validity of the measurements reported in this paper depends to a large extent on establishing a steady state during the isotope infusions (see discussion by Hembree, Bardin & Lipsett, 1969; Longcope & Tait, 1971). The equilibrium values for the isotope concentra¬ tions indicate that this requirement had been met for the precursor steroid, oestradiol, and in most instances for the product steroid, oestrone. Variation in oestrone did not allow calculation of the conversion ratios in all instances. The coefficients of variation of the equilibrium isotope values (13 % for oestradiol and 30 % for oestrone) were satisfactory, considering that these experiments were performed on conscious animals that were able to adjust their posture throughout the infusion. Similar values have been found in experiments on conscious sheep (Challis et al. 19736) and only a marginal improvement has been achieved in studies in which human subjects have been fasted and strictly standardized with respect to position (see Baird & Fraser, 1974). The mean MCRB in this study was 164 ml/min. A slightly lower value (approx. 70100 ml/min) may be calculated from the data of Crocker & Thomas (1973), although in that study the rabbits had been anaesthetized, a procedure which may provoke a reduction in MCR (Baird & Fraser, 1974). Cardiac output in non-pregnant Chinchilla rabbits is approx. 400-500 ml/min (AbdulKarim & Bruce, 1973; I. S. Fraser, unpublished observations). Assuming that liver (total splanchnic) blood flow is about 20 % of cardiac output (Warren & Ledingham, 1974), it is apparent that even if the hepatic metabolism of oestradiol approaches 100 %, there must be appreciable extrahepatic metabolism and uptake of the steroid. Possible sites of oestradiol uptake have been demonstrated in the reproductive tract and the brain of the rabbit both by studies in vivo and in vitro (Korenman, 1968; Crocker & Thomas, 1973; Russell & Thomas, 1973), and the present experiments have demonstrated a high peripheral conversion to oestrone. Of interest is the finding that when MCRB is corrected by body weight075 (Kleiber, 1965), similar values are found for the rabbit (0-079 1/min/ weight075) as in man (0-074; Longcope et al. 1968; Baird & Fraser, 1974) and the rat (0-075; de Hertogh, Ekka, Vanderheyden & Hoet, 1970). These values are appreciably less than in the sheep (0-130 l/min/W075; Challis et al. 19736), and could be related to factors such as differences in plasma protein binding of oestrogens between these The
species.
In the present study the mean production rate of oestradiol measured in conscious rabbits about 4 ng/min. This agrees with estimates of oestrogen production rates based on a biological end point, the uterine synthesis of actomyosin (Michael & Schofield, 1967). Direct measurements of ovarian oestradiol secretion rates in anaesthetized animals vary, but in general range between 5 and 50% of the present values (Eaton & Hilliard, 1971; Shaikh & Harper, 1972; Hilliard et al. 1974). Several factors could contribute to the apparent difference between the total oestradiol production rates measured in conscious rabbits and the direct measurements of ovarian oestradiol secretion rates made in the anaesthetized animal. Gonadotrophin and ovarian steroid secretion may be suppressed in the pento¬ barbitone anaesthetized rabbit although clear information on this is lacking. Vascular spasm is very easily provoked by manipulation and may have altered ovarian blood flow in direct cannulation experiments. was
identified as the major site of oestradiol production by experi¬ of the follicles or oophorectomy (Hilliard et al. 1974), and the adrenal does not appear to contribute significantly to levels of oestradiol in the circula¬ tion. It is possible that some oestradiol arises by peripheral conversion from oestrone, and this could be of some importance in the situation shown in Fig. 1 and 6 where high peaks of oestrone have occurred independently of high peaks of oestradiol. The origin of these oestrone peaks is uncertain. They may be of adrenal origin although the timing of the peaks does not clearly suggest a stress factor related to the infusions. Low levels of pro¬ gesterone persist after oophorectomy (Thau & Lanman, 1974) and there may also be an adrenal contribution to the levels of this hormone in blood. The rabbit is a reflex ovulator with a group of ovarian follicles apparently in a state of preovulatory readiness at all times. It is not known how the steroid production of these follicles varies and there is no clear pattern from the three animals followed over 9-17 days in this study. However, the present study has indicated that the production rate of oestradiol in the non-pregnant rabbit is unlikely to exceed 5-10/¿g/24h and experiments in which greater amounts of exogenous steroid have been used to produce biological responses should perhaps be interpreted with appropriate reservations. The Graafian follicles
ments
We
were
involving thermocautery
are
grateful
to Miss Christine Forster for her excellent technical assistance. The work
supported by grants from the M.R.C, and I.S.F. was in receipt of an M.R.C. Clinical Research Fellowship. G.D.T. is an external scientific staff member of the M.R.C.
was
REFERENCES
Abdul-Karim, R. W. & Bruce, N. (1973). Blood flow to the ovary and corpus luteum at different stages of gestation in the rabbit. Fertility and Sterility 24, 44-47. Abraham, G. E., Tulchinsky, D. & Korenman, S. G. (1970). Chromatographie purification of estradiol-17/? for use in radioligand assay. Biochemical Medicine 3, 365-368. Baird, D. T. & Fraser, I. S. (1974). Blood production and ovarian secretion rates of estradiol-17/? and estrone in women throughout the menstrual cycle. Journal of Clinical Endocrinology and Metabolism 38, 1009-1017.
Challis, J. R. G., Davies, I. J. & Ryan, K. J. (1973a). The concentration of progesterone, estrone and estradiol-17/? in the plasma of pregnant rabbits. Endocrinology 93, 971-976. Challis, J. R. G., Harrison, F. A. & Heap, R. B. (19736). The kinetics of oestradiol-17/? metabolism in the sheep. Journal of Endocrinology 57, 97-110. Crocker, J. M. & Thomas, G. H. (1973). Uptake and interconversion of tritiated oestrone and oestradiol17/? in the intact rabbit in vivo. Journal of Endocrinology 56, 213-218. Eaton, L. W. & Hilliard, J. (1971). Estradiol-lip, progesterone and 20a-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma. 1. Steroid secretion from paired ovaries with and without corpora lutea; effect of LH. Endocrinology 89, 105-111. Hembree, W. C, Bardin, C. W. & Lipsett, M. B. (1969). A study of estrogen metabolic clearance rates and transfer factors. Journal of Clinical Investigation 48, 1809-1819. de Hertogh, R., Ekka, E., Vanderheyden, I. & Hoet, J. J. (1970). Metabolic clearance rates and interconversion factors of estrone and estradiol-17/? in the immature and adult female rat. Endocrinology 87, 874-880.
Hilliard, J. & Eaton, L. W. (1971). Estradiol-17/?, progesterone and 20a-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma. 2. From mating through implantation. Endocrinology 89, 522-527. Hilliard, J., Scaramuzzi, R. J., Pang, C, Penardi, R. & Sawyer, C. H. (1974). Testosterone secretion by rabbit ovary in vivo. Endocrinology 94, 267-271. Kleiber, M. (1965). Respiratory exchange and metabolic rate. In Handbook ofphysiology, Section 3, Respira¬ tion, Vol. II, pp. 927-938. Eds W. O. Fenn & H. Rahn. Baltimore: Williams and Wilkins. Korenman, S. G. (1968). Radio-ligand binding assay of specific estrogens using a soluble uterine macromolecule. Journal of Clinical Endocrinology and Metabolism 28, 127-130. Longcope, C, Layne, D. S. & Tait, J. F. (1968). Metabolic clearance rates and interconversions of estrone and 17/ì-estradiol in normal males and females. Journal of Clinical Investigation 47, 93-106. Longcope, C. & Tait, J. F. (1971). Validity of metabolic clearance and interconversion rates of estrone and 17/?-estradiol in normal adults. Journal of Clinical Endocrinology and Metabolism 32, 481-490.
Michael, C. . & Schofield, . M. (1967). The influence of hormones on myometrial development. Acta Endocrinologica Suppl. 119, 232. Russell, S. L. & Thomas, G. H. (1973). The interconversion of oestrone and oestradiol-17/? and the con¬ centration of oestradiol- 17/? receptors in cultured rabbit uterus. Journal of Endocrinology 56, 203-211. Shaikh, A. A. & Harper, M. J. K. (1972). Ovarian steroid secretion in estrous, mated and HCG-treated rabbits determined by concurrent cannulation of both ovarian veins. Biology of Reproduction 1, 387-397. Tait, J. F. & Burstein, S. (1964). In vivo studies of steroid dynamics in man. In The hormones, Vol. 5, pp. 441-557. Eds G. Pincus, K. V. Thimann & E. G. Astwood. New York: Academic Press. Thau, R. & Lanman, J. T. (1974). Evaluation of progesterone synthesis in rabbit placentas. Endocrinology 94, 925-926. Warren, D. J. & Ledingham, J. G. G. (1974). Measurement of cardiac output distribution using microspheres. Some practical and theoretical considerations. Cardiovascular Research 8, 570-581.
Hospital, Oxford
(Received 20 May 1975) SUMMARY
A continuous isotope infusion technique was used to measure the metabolic clearance rate (MCRB) and production rate (PRB) of oestradiol-17\g=b\ in a group of six non-pregnant conscious Chinchilla rabbits. The mean MCRB of oestradiol was 162 ml/min (0\m=.\0791/ min/weight0\m=.\75), and at equilibrium a significant proportion of the radioactivity in the circulation was present as oestrone (conversion ratio oestradiol to oestrone, 27\m=.\6%). The mean PRB of oestradiol was 4\m=.\3ng/min, which is equivalent to about 5 \g=m\g/day. Some day-to-day variation was seen in the endogenous concentrations of oestradiol, oestrone and progesterone although in general the levels of the two oestrogens were less than 30\p=n-\40pg/ml, and progesterone levels were below 300 pg/ml apart from short peaks up to 600 pg/ml. These values were compared with previous measurements of the ovarian secretion rate of oestradiol measured directly in anaesthetized animals.
INTRODUCTION
Although the rabbit has been used extensively as an experimental model for studying the actions of steroid hormones, there is little information concerning the endogenous production rates of steroids in the conscious animal and the variation in this from day to day. Previous workers have measured the ovarian secretion rates of progesterone, oestrone and oestradiol by anaesthetized rabbits in various reproductive states (Eaton & Hilliard, 1971 ; Hilliard & Eaton, 1971 ; Shaikh & Harper, 1972; Hilliard, Scaramuzzi, Pang, Penardi & Sawyer, 1974). In the present study the production rate of oestradiol was measured using tracer isotope kinetic techniques in conscious non-pregnant rabbits. These determinations are compared with previous direct measurements of ovarian oestradiol secretion rates. MATERIALS AND METHODS
Animals Six non-pregnant, sexually mature Chinchilla rabbits (body weights 2-1-3-2 kg) were used in this study. The animals were caged singly with food and water available ad libitum. Steroids [2,4,6,7-3H]Oestradiol-17/? (85 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, and was purified by Celite column chromatography (Abraham, Tulchinsky & Korenman, 1970). On the day before an infusion experiment, the purity of the [3H]oestradiol was checked by thin-layer chromatography (t.l.c.) in the solvent system cyclohexane : ethyl
(1:1, v/v). The radioactivity was located as a single peak using a DünnschichtScanner II. Other radioactive and non-radioactive oestrogens were obtained and stored as described by Challis, Harrison & Heap (19736). Chemicals All solvents were of nanograde quality (Mallinckrodt Co.), except cyclohexane which was of AR grade (BDH Chemicals Ltd). Thin-layer chromatography was performed using precoated plates, Kieselgel 60 F254 (Merck) of 0-25 mm thickness. The plates were washed once in ethyl acetate before sample application. acetate
Infusion procedure Three to four days before an isotope infusion, the infusion and sampling catheters were implanted into the rabbits under sodium pentobarbitone (30 mg/kg) anaesthesia, supple¬ mented with skin analgesia using 1 % xylocaine. A teflon catheter (0-56 mm i.d. 1-10 mm o.d. Becton Dickinson & Co., Rutherford, New Jersey, U.S.A.) for infusion was implanted into the lower inferior vena cava via the femoral vein, and a vinyl or Polythene catheter (Portex Ltd; 1-27-1-4 mm o.d. 0-63-0-86 mm i.d.) for sampling was inserted into a femoral artery. The catheters were located subcutaneously, and exteriorized at the back of the neck. They were flushed daily with sterile heparin-saline (5 i.u./ml) and heat-sealed. For the measurement of metabolic clearance rate (MCRB), [3H]oestradiol-17/# (20/¿Ci) in sterile 0-9 % NaCl solution containing 6 % ethanol was infused into the vena cava at a constant rate (3-3 ml/h; approx. 0-06 /¿Ci/min, see Table 1). The infusion was maintained using a glass syringe mounted on a Braun infusion pump (B. Braun, Melsungen, Germany; Perfusor pump type 871104). The isotope infusion rate was equivalent to an oestradiol secretion rate of about 0-2 ng/min, which was less than 10 % of the endogenous production rate determined subsequently. During the course of a 4 h infusion, the fully conscious rabbits were restrained in wicker baskets, and their movements were partially restricted by the use of cotton padding. The animals remained generally calm throughout, although all made regular spontaneous move¬ ments and occasional attempts to turn round. Two animals began to hyperventilate at the end of the experiment and this may have been due to the heat of the laboratory. The animals had become used to frequent handling during the previous week. Five experiments were started at approximately 12.00 h; one experiment (rabbit no. 2) was started at 17.46 h. One blood sample (3-5 ml), for the determination of the endogenous oestradiol con¬ centration, was taken immediately before the start of the infusion. Three further samples of about 5 ml were taken at 15 min intervals, beginning 160-225 (mean 185) min after the start of the isotope infusion. The total amount of blood drawn was therefore less than 10 % of the total blood volume of the smallest rabbit. All samples of blood were drawn into heparinized syringes, and transferred to chilled glass vessels containing a known volume of distilled water. The samples were mixed, reweighed, and then stored at 15°C until
analysis.
For the calculation of infusion rate, triplicate timed collections of the infusion catheter before and after an infusion experiment. from the residual infúsate (Longcope, Layne & Tait, 1968).
—
taken from the end Samples were also taken were
Daily blood sampling Samples of blood (1-3 ml) were obtained regularly from three of the rabbits during a period of 9-17 days after the animals had been used in an MCR experiment. The samples were taken from an ear vein by venipuncture into heparinized syringes, and frozen as whole blood until analysis.
Measurement of endogenous steroid concentrations The concentrations of oestradiol-17ß, oestrone and progesterone were determined in 1-2 ml aliquots of the pre-infusion and daily blood samples by radioimmunoassay (Challis, Davies & Ryan, 1973 a).
Analysis of radioactivity Haemolysed blood was thawed slowly overnight at 4 °C. Samples (4-5 ml) were added to 4 dr vials (Camlab, Cambridge), containing 50 ng each of oestradiol and oestrone in 50 /¿l ethanol for recovery estimations. The samples were mixed gently and extracted with 3 6 ml fresh diethyl ether (Mallinckrodt). Emulsions, if they formed, were broken by centrifugation. The pooled extracts were dried, and applied to t.l.c. plates as described previously (Challis et al. 19736). Chromatograms were developed in the system ethyl acetate : cyclohexane (1:1, v/v) and the oestrone and oestradiol- 17/Î zones were located by scanning wellseparated lanes containing tritiated marker oestrogens. Blank lanes were run either side of the markers. This system permits a good separation of oestrone (Rr, 0-68) from oestradiol17/? (RF, 0-52) with oestradiol-17a occupying the intermediate area. The oestrone and oestradiol-17/? zones were eluted (Challis et al. 19736), dried, and redissolved in 1 ml isooctane for Celite chromatography (Abraham et al. 1970; Challis et al. 1973 ). The oestrone and oestradiol fractions were then dried and redissolved in 2-0 ml ethanol. Aliquots were taken for liquid scintillation counting and for radioimmunoassay to determine methodo¬ logical losses. In a preliminary experiment, 0-5 ml aliquots of the blood samples taken at equilibrium from two rabbits were pooled, extracted with diethyl ether and subjected to t.l.c. The chromatogram lane was divided into 1 cm bands which were eluted and counted separately. More than 90 % of the radioactivity was located in the zones corresponding to oestradiol17/? and oestrone. Negligible radioactivity was found in the zones corresponding to oestra¬ diol-17a
or
16-oxo oestradiol.
Calculations The metabolic clearance rate (MCRB) and total production rate (PRB; Tait & Burstein, 1964) of oestradiol measured in whole blood was calculated as described by Baird & Fraser (1974). The conversion ratio of oestradiol to oestrone (CBB) was calculated according to Longcope et al. (1968). RESULTS
Evidence for radiochemical purity of the isolated oestrogens batch of one analyses, samples from fractions isolated as oestradiol-17/? or oestrone were pooled separately. Approximately 150 d.p.m. of [14C]oestradiol-17/? and 100 d.p.m. of [14C]oestrone were added to the respective pools and the [3H] : [UC] ratios of each were determined. The samples were acetylated at 50 °C for 1 h, and the acetates chromatographed in the t.l.c. system cyclohexane : ethyl acetate (2:1, v/v) which separates oestrone monoacetate (RF, 0-54) from oestradiol diacetate (RF, 0-72). The ratio of [3H]:[14C] in oestradiol was 3-53 and in oestradiol diacetate 3-31. The ratio of [3H]:[14C] in oestrone was 0-22 and in oestrone monoacetate 0T9, thereby indicating that radiochemical purity for both steroids had been achieved. Evidence for isotopie equilibrium Table 1 shows the concentrations of radioactivity isolated as oestradiol-17/? and oestrone in individual samples of blood taken at equilibrium (mean sample times 185,200 and 215 min from the start of the infusion). Individual values for each animal were calculated as a In
mean, and related to the time of sampling. When these normalized data analysed by regression analysis, the slopes for both oestradiol and oestrone were not significantly different from zero (regression equations: oestradiol, y 101—0-02*; oestrone, y 108-OT38.x) and it was considered that isotopie steady state had been achieved for both precursor and product steroids. The mean coefficient of variation for the
percentage of the were
—
=
oestradiol equilibrium values was 13-9 ± 1-9 (s.e.m.) %. For oestrone, reasonable agreement between replicates was found for rabbits 1, 3, 5 and 6, but large variations were found for rabbits 2 and 4. The mean coefficient of variation for all animals was 30-1 + 10-3 %. Table 1. Concentration of radioactivity in blood samples taken
as
at
oestradiol-17ß and oestrone equilibrium
Infusion Rabbit
Oestradiol-17/? (d.p.m./ml blood)
rate
,-'-,
no.
(/¿Ci/min)
185 min
200 min
215min
Mean
00565
1217 460 833 689 1411 1272
1249 541 1013 615 1720 967
1446
1304 491
00580
00674 00671 00688 00616
*, Final count No sample.
Oestrone
(d.p.m./ml blood)
,—
736
861
456 1719 1379
587 1617 1206
—,
185 min
200 min
215 min
Mean
343 308 208 360 370
304
324
324 158 412 239
275 218 420 219
108 294 138 478 198
*
too low for accurate determination.
—,
Table 2. Metabolic clearance
rate
oestradiol-17ß in Rabbit No. 1 2 3 4 5 6
Body wt (kg) 213 301 3-20 2-40 2-80 2-45
(MCR%ß) and production rate the non-pregnant rabbit
Isotope concentration (d.p.m./ml blood)
MCR!**
—*-
Oestradiol Oestrone 1304 491 861 587 1617 1206
ml/min 95-3 259-7
324
Endogenous 17-6
Mean
172-3 251-3 93-6 112-4 1641
44-7 86-2 53-8 104-7 33-4 45-9 61-5
260 43 0 200 210 241
+ S.E.M.
±31-2
±11-3
±40
275 218 420 219
product steady
Not calculated
PR!"
oestradiol
ml/min/kg (pg/ml) 170
state not achieved
(PRlß) of
(ng/min)
(%)
1-67 4-40 4-48 10-81 1-87 2-36 4-3 + 1-4
24-8 31-9 37-1 260 18-2 27-6
±3-2
(Table 1).
-
Metabolic clearance rate and production rate of oestradiol-17ß variation was found between individual rabbits in the value for MCRB (range Appreciable 93-260 ml/min; mean 164 ml/min, Table 2). This variation was still present after correcting for body weight. The mean MCRB after correcting for the mean body weight075 (W0-75; Kleiber, 1965) was 0-079 l/min/W075. The average endogenous concentration of oestradiol in the pre-infusion sample was 24 pg/ml whole blood, and the mean PRB of oestradiol was 4-3 ng/min (range 1-6-10-8 ng/ min). In rabbit no. 4 (PRB of oestradiol 10-8 ng/min), the endogenous oestradiol in a sample taken 24 h earlier was 14 pg/ml, which, with a similar MCR, would result in a calculated PRB of 3-5 ng/min. There was a significant conversion of oestradiol to oestrone (mean CBB 27-6 %). =
Endogenous steroid levels The concentrations of oestradiol, oestrone and progesterone in daily samples of blood from three rabbits are shown in Fig. 1. The mean concentration of oestradiol was about 20 pg/ml, and did not vary in any consistent manner. The oestrone concentration was generally ( ) Rabbit
800
-
600
-
400
-
200
-
3
160
120
80 40
0200
(b) Rabbit 2 800
f -
g 600
120
a
400
80
200
40
| •o
1
§
(c) Rabbit 5 400
-
200
-
Time
80 40
(days)
Fig. 1. Daily changes in peripheral blood concentrations of oestrone, oestradiol and progesterone in non-pregnant rabbits. I, Infusion day; , progesterone; O, oestrone; , oestradiol. than oestradiol, and in two of the rabbits (nos 2 and 3) peaks of ^ 100 pg/ml, of short duration and not associated with high oestradiol peaks, were found. The concentra¬ tion of progesterone was less than 300 pg/ml in most samples.
higher
There was a tendency for the concentrations of oestrone and oestradiol to change in similar fashion, and there was a significant correlation between the two oestrogens when the values for all three rabbits were considered together (r 0-367, 43, < 005). Progesterone was not correlated with either oestradiol or oestrone, except in rabbit no. 2, where it was marginally so ( < 0 5). =
=
DISCUSSION
validity of the measurements reported in this paper depends to a large extent on establishing a steady state during the isotope infusions (see discussion by Hembree, Bardin & Lipsett, 1969; Longcope & Tait, 1971). The equilibrium values for the isotope concentra¬ tions indicate that this requirement had been met for the precursor steroid, oestradiol, and in most instances for the product steroid, oestrone. Variation in oestrone did not allow calculation of the conversion ratios in all instances. The coefficients of variation of the equilibrium isotope values (13 % for oestradiol and 30 % for oestrone) were satisfactory, considering that these experiments were performed on conscious animals that were able to adjust their posture throughout the infusion. Similar values have been found in experiments on conscious sheep (Challis et al. 19736) and only a marginal improvement has been achieved in studies in which human subjects have been fasted and strictly standardized with respect to position (see Baird & Fraser, 1974). The mean MCRB in this study was 164 ml/min. A slightly lower value (approx. 70100 ml/min) may be calculated from the data of Crocker & Thomas (1973), although in that study the rabbits had been anaesthetized, a procedure which may provoke a reduction in MCR (Baird & Fraser, 1974). Cardiac output in non-pregnant Chinchilla rabbits is approx. 400-500 ml/min (AbdulKarim & Bruce, 1973; I. S. Fraser, unpublished observations). Assuming that liver (total splanchnic) blood flow is about 20 % of cardiac output (Warren & Ledingham, 1974), it is apparent that even if the hepatic metabolism of oestradiol approaches 100 %, there must be appreciable extrahepatic metabolism and uptake of the steroid. Possible sites of oestradiol uptake have been demonstrated in the reproductive tract and the brain of the rabbit both by studies in vivo and in vitro (Korenman, 1968; Crocker & Thomas, 1973; Russell & Thomas, 1973), and the present experiments have demonstrated a high peripheral conversion to oestrone. Of interest is the finding that when MCRB is corrected by body weight075 (Kleiber, 1965), similar values are found for the rabbit (0-079 1/min/ weight075) as in man (0-074; Longcope et al. 1968; Baird & Fraser, 1974) and the rat (0-075; de Hertogh, Ekka, Vanderheyden & Hoet, 1970). These values are appreciably less than in the sheep (0-130 l/min/W075; Challis et al. 19736), and could be related to factors such as differences in plasma protein binding of oestrogens between these The
species.
In the present study the mean production rate of oestradiol measured in conscious rabbits about 4 ng/min. This agrees with estimates of oestrogen production rates based on a biological end point, the uterine synthesis of actomyosin (Michael & Schofield, 1967). Direct measurements of ovarian oestradiol secretion rates in anaesthetized animals vary, but in general range between 5 and 50% of the present values (Eaton & Hilliard, 1971; Shaikh & Harper, 1972; Hilliard et al. 1974). Several factors could contribute to the apparent difference between the total oestradiol production rates measured in conscious rabbits and the direct measurements of ovarian oestradiol secretion rates made in the anaesthetized animal. Gonadotrophin and ovarian steroid secretion may be suppressed in the pento¬ barbitone anaesthetized rabbit although clear information on this is lacking. Vascular spasm is very easily provoked by manipulation and may have altered ovarian blood flow in direct cannulation experiments. was
identified as the major site of oestradiol production by experi¬ of the follicles or oophorectomy (Hilliard et al. 1974), and the adrenal does not appear to contribute significantly to levels of oestradiol in the circula¬ tion. It is possible that some oestradiol arises by peripheral conversion from oestrone, and this could be of some importance in the situation shown in Fig. 1 and 6 where high peaks of oestrone have occurred independently of high peaks of oestradiol. The origin of these oestrone peaks is uncertain. They may be of adrenal origin although the timing of the peaks does not clearly suggest a stress factor related to the infusions. Low levels of pro¬ gesterone persist after oophorectomy (Thau & Lanman, 1974) and there may also be an adrenal contribution to the levels of this hormone in blood. The rabbit is a reflex ovulator with a group of ovarian follicles apparently in a state of preovulatory readiness at all times. It is not known how the steroid production of these follicles varies and there is no clear pattern from the three animals followed over 9-17 days in this study. However, the present study has indicated that the production rate of oestradiol in the non-pregnant rabbit is unlikely to exceed 5-10/¿g/24h and experiments in which greater amounts of exogenous steroid have been used to produce biological responses should perhaps be interpreted with appropriate reservations. The Graafian follicles
ments
We
were
involving thermocautery
are
grateful
to Miss Christine Forster for her excellent technical assistance. The work
supported by grants from the M.R.C, and I.S.F. was in receipt of an M.R.C. Clinical Research Fellowship. G.D.T. is an external scientific staff member of the M.R.C.
was
REFERENCES
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Challis, J. R. G., Davies, I. J. & Ryan, K. J. (1973a). The concentration of progesterone, estrone and estradiol-17/? in the plasma of pregnant rabbits. Endocrinology 93, 971-976. Challis, J. R. G., Harrison, F. A. & Heap, R. B. (19736). The kinetics of oestradiol-17/? metabolism in the sheep. Journal of Endocrinology 57, 97-110. Crocker, J. M. & Thomas, G. H. (1973). Uptake and interconversion of tritiated oestrone and oestradiol17/? in the intact rabbit in vivo. Journal of Endocrinology 56, 213-218. Eaton, L. W. & Hilliard, J. (1971). Estradiol-lip, progesterone and 20a-hydroxypregn-4-en-3-one in rabbit ovarian venous plasma. 1. Steroid secretion from paired ovaries with and without corpora lutea; effect of LH. Endocrinology 89, 105-111. Hembree, W. C, Bardin, C. W. & Lipsett, M. B. (1969). A study of estrogen metabolic clearance rates and transfer factors. Journal of Clinical Investigation 48, 1809-1819. de Hertogh, R., Ekka, E., Vanderheyden, I. & Hoet, J. J. (1970). Metabolic clearance rates and interconversion factors of estrone and estradiol-17/? in the immature and adult female rat. Endocrinology 87, 874-880.
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