RADIOIMMUNOASSAY OF PLASMA ETHINYLESTRADIOL IN THE PRESENCE OF CIRCULATING NORETHINDRONE

Transcription

1 RADIOIMMUNOASSAY OF PLASMA ETHINYLESTRADIOL IN THE PRESENCE OF CIRCULATING NORETHINDRONE Frank 2. Stanczyk, Judith A. Gale, Uwe Goebelsmann, Clint Nerenberg and Shaikh Matin Department of Obstetrics and Gynecology University of Southern California School of Medicine and Los Angeles County-University of Southern California Medical Center Women's Hospital Los Angeles, California and Department of Analytical and Metabolic Chemistry Syntex Research Palo Alto, California ABSTRACT A reliable radioimmunoassay (RIA) for the measurement of ethinylestradiol (EE2) in plasma after the separation of norethindrone (NET) by column chromatography has been developed. The procedure involves diethyl ether extraction, followed by celite column partition chromatography to separate NET from EE2. An antiserum against ethinylestradiol-7-(3-thiopropionic acid)-bovine serum albumin conjugate was employed. Tritiated EE2 was used as the radioligand. The separation of bound from free 3H-EE2 was carried out with dextran-coated charcoal. This RIA procedure was used to measure plasma EE2 at specified time intervals in three women following oral administration of a tablet containing EE2 (0.12 mg) and NET (1.0 mg). The results show that EE2 is rapidly absorbed after oral administration. Peak concentrations of plasma EE2 were reached within 1.0 to 1.5 hours and then declined biphasically. The EE2 a phase half-life values in three subjects were 1.5, 2.8 and 3.8 hours. By comparison, the half-life values for the 8 phase for the same three women were 6.2, 12.3 and 14.5 hours, respectively. Plasma samples were also analyzed for NET by RIA. The half-life values for the B phase for EE2 and NET in the three subjects were similar. Accepted for publication September 16, 1980 NOVEMBER 1980VOL.22NO.5 457

2 The wide usage of oral contraceptives and the implication that it is the estrogenic component which is the major cause of adverse metabolic effects (1) makes the knowledge of pharmacokinetics of these synthetic estrogens essential. In recent years, several radioimmunoassay (RIA) methods for the measurement of plasma ethinylestradiol (EE2) have been reported (2-8). These methods employed tritiated EE2 as the radioligand and an antiserum produced against either an ethinylestradiol-6-(o-carboxymethyl)oxime-bovine serum albumin, ethinylestradiol-3- hemisuccinate-bovine serum albumin or ethinylestradiol-7- (3-thiopropionic acid)-bovine serum albumin conjugate. Only minor cross-reactions (approximately 1%) were reported with norethindrone (NET). In women taking oral contraceptives containing NET, the NET plasma concentrations can be as high as 5 to 10 ng/ml. At these levels,cross-reaction could be significant and therefore lead to falsely elevated EE2 measurements. We developed an RIA method for the measurement of EE2 in plasma after removal of NET by column chromatography. The applicability of this method to the measurement of plasma levels of EE2 was established. Subjects MATERIALS AND METHODS Three women were selected for this study on the criteria that they be in good general health, under 50 years of age, and that they had not received oral contraceptive steroids for at least 3 weeks prior to the start of the study. Dosing was carried out during days 6 to 24 of the menstrual cycle. At the start of the study, predose antecubital venous blood samples were taken from the three volunteers at 8 a.m. after a 12-hour fast. Each subject then received a dose of 2 tablets, each containing 0.06 mg of EE2 and 0.5 mg of NET. The tablets were followed by 240 ml of water and the subjects continued fasting for an additional 2 hours. Following this, normal meals were resumed and water was allowed ad libitum for the remainder of the study. After drug adznistration, antecubital venous blood samples were drawn at the following times: 0.5, 1.0, 1.5, 3.0, 6.0, 12.0 and 24.0 hours. Samples were centrifuged and the plasma separated and stored at -150C until it was analyzed. Radioimmunoassay of Ethinylestradiol Unlabeled EE2 was purchased from Steraloids, Inc. (Wilton, N.H.) and was recrystallized twice from mixtures of methanol, acetone and hexane. [6,7-3H]-EE2 with a specific activity of 55 Ci/mmole was obtained from New England Nuclear Corporation (Boston, Mass.) and checked for radiochemical purity by subjecting an aliquot to paper 458 NOVEMBER 1980 VOL. 22 NO. 5

3 partition chromatography using isooctane:t-butanol:methanol:water (10:2:7:1, by volume) for 24 hours. Anti-ethinylestradiol-7-(3-thiopropionic acid)-bovine serum albumin serum (9) was obtained from Research Triangle Institute (Research Triangle Park, N.C.). Extraction and column purification were carried out as follows: A 0.1 ml aliquot of 3H-EE2 (1000 d.p.m.) dissolved in phosphate-gelatin buffer (PGB) was added to 1.0 ml of plasma and incubated for 30 min at 370C. The PGB consisted of 0.15 M sodium chloride, 0.1 M sodium phosphate at ph 7.0, 0.1% (w:v) of Knox unflavored gelatin and 0.1% (w:v) sodium azide. Following incubation the plasma was extracted twice with 5 ml diethyl ether. The ether extracts were pooled and evaporated under nitrogen. The residue was redissolved in 1 ml of 30% benzene in isooctane (v:v). A partition column was prepared using celite (Johns-Manville, Analytical Filter Aid) which was heated overnight in a furnace at 540 C and then cooled. A mixture of 0.5 grams of celite and 0.25 ml of ethylene glycol was poured into a 5 ml disposable glass pipette. Isooctane was added to the column until the celite was covered and the celite was packed with a plunger. Columns were prewashed with 4 ml of isooctane. After loading the samples, the columns were eluted with 5 ml of 30% benzene in isooctane (v:v) followed by 2 ml of benzene. Both these eluates were discarded. The EE2 fraction was then eluted with 4 ml of 5% ethyl acetate in benzene (v:v). The solvent was evaporated and the residue redissolved in 0.7 ml of 7% ethanol in PGB (v:v). Duplicate 0.2 ml aliquots were used for RIA analysis. Also, a 0.2 ml aliquot was counted for 20 minutes in a scintillation counter to assess procedural losses. For thirty-four determinations, the mean recovery was 71% with a standard deviation of - +6%. The EE2 RIA was carried out as follows: A 0.1 ml aliquot of 3H-EE2 (approx. 10,000 d.p.m.) was added to all assay tubes. A 0.1 ml aliquot of antisera (diluted l:lo,ooo) was added to all appropriate assay tubes. Both the above reagents were dissolved in PGB. The RIA tubes were vortexed and incubated for 30 minutes at 370C and then for 20 hours at 4oC. The separation of bound from free 3H-EE2 was carried out by the addition of 0.5 ml of dextran-coated charcoal suspension to the appropriate assay tubes, vortexing and incubation for 15 minutes at 4oc. The charcoal suspension was prepared by adding 500 mg of charcoal and 50 mg of Dextran T-70 to 100 ml of PGB. After completion of the charcoal reaction,all tubes were centrifuged at 4oC. An a-point standard curve ranging from 3.9 to 500 pg was run in duplicate in each assay. A logit transformation (10) was used to construct standard curves and interpolate unknowns on a programmable Hewlett-Packard Model 97 calculator. NOVEMBER 1980 VOL. 22 NO

4 Radioimmunoassay of Norethindrone The measurement of NET in plasma was carried out by a previously described method (11). The antiserum used was produced against a lla-hydroxynorethindrone-ll-hemisuccinyl-bovine serum albumin conjugate. The radioligand was norethindrone-3-(0-carboxymethyl)oximino-[l25i]-iodohistamine. Measurement of the Half-Lives of Ethinylestradiol Norethindrone and Plasma EE2 and NET concentrations measured in the three subjects were plotted on a semilogarithmic scale. The resulting curves were analyzed by the method of residuals and by regression analysis and the half-lives for the 2 elimination phases, t1/2 (a) and t1/2 (S),were computed (12) - RESULTS 1. Reliability of the Ethinylestradiol Radioimmunoassay Sensitivity Logit B/B0 vs. log dose standard curves were linear between 3.9 and 500 pg of EE2. The smallest amount of EE2 per RIA tube which reduced the number of c.p.m. of labeled EE2 bound at zero mass by 2 standard deviations was 3.9 pg. Based upon an average EE2 recovery of 70% when 1 ml plasma aliquots were extracted, this RIA allows the reliable measurement of 20 pg of EE2 per ml of plasma. Accuracy Accuracy studies were carried out by adding known amounts of EE2 to plasma obtained from women during the follicular phase (blank plasma). Additions of EE2 were made in the range of 16 to 500 pg/ml of plasma and then analyzed by RIA. The results of these measurements are shown in Table I. A plot of measured vs.added EE2 gave a regression line of y = 1.08~ - 2.6, r = A further test of accuracy was made by the measurement of BE2 in the presence of known amounts of NET. To three separate 1 ml blank plasma samples were added 100 pg of EE2 and then respectively 1, 5 and 10 ng of NET. RIA analysis of these samples for EE2 after separation of the NET gave values of 87, 112 and 97. Similarly, in a second experiment 200 pg of EE2 was added in the presence of 1, 5 and 10 ng NET. The EE2 measurements obtained were 237, 195 and 206. These measurements are within experimental error and indicate that there is no substantial interference after removal of the NET. The 460 NOVEMBER 1980VOL.22N0.5

5 corresponding EE2 measurements obtained by RIA without preceding chromatography were 116, 140 and 145 pg as well as 355, 318 and 366 pg, respectively. Blank plasmas gave an RIA value of 12 pg/ml with a standard deviation of +6 pg/ml for thirty-six determinations. TABLE I Radioimmunoassay measurement of ethinylestradiol (EE2) added to blank plasma EE2 EE2* Ratio of Coeff. of Added Measured Measured No. of Std. Variation pg/ml pg/ml Added - Determn. Dev. (%) *Mean of all determinations. Precision Measurement of EE2 at a concentration of 112 pg/ml gave an intra-assay coefficient of variation of 4.8% for six determinations. Consecutive analysis of three sera samples containing 50, 100 and 200 pg/ml of EE2 gave inter-assay coefficients of variation of 16.1, 11.1 and 8.4%, respectively, for thirty-six determinations. Specificity A variety of endogenous and synthetic steroids were tested for their cross-reaction with the EE2 antiserum. The degree of cross-reaction was determined both at the 20% and 50% inhibition points and the results are listed in Table II. The results clearly show that marked interference is indicated at the 20% inhibition point for mestranol, 17S-hydroxy-17a-ethinyl- SB-estran-3-one and estradiol. NOVEMBER 1980 VOL. 22 NO

6 TABLE II Cross-reactions of various steroids in the anti-ethinylestradiol-7-(3-thiopropionic acid)-bovine serum albumin serum/3h-ethinylestradiol radioimmunoassay Steroid Percent Cross- Reaction A* B** ethinylestradiol mestranol norethindrone co hydroxy-17a-ethinyl-58-estran-3-one co a-ethinyl-5a-estrane-3a,l7B_diol co a-ethinyl-5a-estrane-38,17/3-diol <O.Ol a-ethinyl-58-estrane-3a,l7S-diol <O.Ol a-ethinyl-58-estrane-38,178-diol <O.Ol nortestosterone <O.Ol 0.06 estrone estradiol androstenedione co.01 co.01 testosterone co.01 X hydroxy-5a-androst-3-one X0.01 co.01 *Defined as (x/y) x 100% where x is the mass of ethinylestradiol and y the mass of steroid tested at which the binding of H ethinylestradlol is reduced by 50%. **Defined as (x/y) x 100% where x is the mass of ethinylestradiol and y the mass of steroid tested at which the binding of H ethinylestradiol is reduced by 20%. The results listed in Table III show that some if not all interfering factors of the assay were removed by the celite column chromatography because the results after chromatography were 49% lower. The results shown in Table III were obtained by analyzing plasma samples obtained from three women at different time points after ingestion of a single tablet containing EE2 (0.06 mg) and NET (0.5 mg). 462 NOVEMBER 1980VOL.22NO.5

7 TABLE III Plasma ethinylestradiol (EE2) concentrations measured by radioimmunoassay without and subsequent to micro-celite column chromatography in 3 women at different times following ingestion of 0.06 mg of EE2 and 0.5 mg of norethindrone Plasma EE2 Concentration (pg/ml) Without With Chroma- Chroma- Percent Sample tography tography Difference Mean Plasma Levels of EE2 and NET Following Oral Administration of a Tablet Containing EE2 (0.12 mg) and NET (1.0 mg) Plasma clearance profiles for EE2 and NET in three female subjects are shown in Figures 1 and 2, respectively. In the three subjects studied, the EE2 peak plasma levels occurred at 1.0, 1.5 and 1.0 hours (488, 532 and 718 pg/ml, respectively). Thereafter, the EE2 levels decreased rapidly to 30, 35 and 59 pg/ml within 24 hours. The NET peak plasma levels, in the same three subjects, occurred at 1.5, 0.5 and 0.5 hours (11.6, 15.0 and 30.3 ng/ml, respectively). After 24 hours, the levels declined to 0.93, 0.58 and 1.82 ng/ml, respectively. The plasma clearance profiles shown in Figures 1 and 2 indicate a biphasic elimination process during the time period that measurements were made. The half- NOVEMBER 1980VOL.22NO.S 463

8 Figure 1. Plasma ethinylestradiol (EE2) concentrations in three women prior to and 0.5, 1.0, 1.5, 3.0, 6.0, 12.0 and 24.0 hours following the ingestion of 0.12 mg of EE2 and 1.0 mg of norethindrone. 464 NOVEMBER 1980VOL.22N0.5

9 ; HOURS Figure 2. Plasma norethindrone (NET) concentrations in three women prior to and 0.5, , 3.0, 6.0 and 24.0 hours following the ingestion fo 1.0 mg of NET and 0.12 mg of ethinylestradiol. NOVEMBER 1980 VOL. 22 NO

10 life values for EE2 and NET for both the a-and S-elimination phases are listed in Table IV. TABLE IV Half-lives of ethinylestradiol (EE2) and norethindrone (NET) for the a-and S-elimination phases in 3 women following the ingestion of 0.12 mg of EE2 and 1.0 mg of NET Phase Subject 1 Subject 2 Subject 3 EE2 NET EE2 NET EE2 NET a S DISCUSSION Separation of EE2 from NET by celite column partition chromatography is a distinctive feature of the present EE2 RIA. Measurement of EE2 by RIA without prior separation of NET from EE2 gave values which were overestimated by an average of 50 pg/ml (Table III). The chromatographic step undoubtedly renders the assay more specific since lower levels are observed after chromatography. Dose-response curves for a variety of synthetic and endogenous steroids were not parallel with EE2. For this reason,cross-reactivity calculations were made both at the 20% inhibition point as well as the customary 50% point. The effect of this non-parallelism is clearly indicated by 17fGhydroxy-17a-ethinyl-5S-estran-3-one where negligible interference is shown at 50% inhibition but extensive cross-reactivity at 20% inhibition (Table II). Accuracy of the method was determined by the measurement of EE2 in the presence of NET. At the selected NET concentrations, namely 1, 5 and 10 ng/ml,there was no interference with the EE2 measurements. An EE2 RIA method described by Ahluwalia et al. (6) used an LH-20 column to separate NET from EE2 but the accuracy of the method was not determined in the presence of NET. The plasma clearance profiles in Figure 1 show EE2 to be rapidly absorbed after oral administration. Peak concentrations were reached within 1.0 to 1.5 hours. de la Pena et al.(2) showed that peak values of plasma EE2 were usually attained within 1 hour after oral administration of 0.05 or 0.08 mg of marketed EE2 tablets. Speck et al. 466 NOVEMBER 1980VOL.22NO.5

11 (13) administered 0.05 mg of tritiated EE2 in tablet form to eight women and found the tl/2 of absorption to be 12 minutes and maximum plasma levels were reached at 55 minutes. In our study, with a 0.12 mg dose of EE2, peak plasma levels ranged from 488 to 718 pg/ml. These results are similar to levels reported by several investigators (2,5,14,15) following EE2 oral doses of mg or 0.05 mg with a gestagen (NET or levonorgestrel), or EE2 doses of 0.05 mg or 0.08 mg alone. The peak plasma levels cited in the above instances are however at considerable variance with those reported by Lahteenmaki and Nilsson (7) and by Nilsson and Nygren (8). For roughly equivalent doses, that is at EE2 doses of 0.03 mg and 0.05 mg, the peak plasma levels attained were only about one fourth of those reported above. The reason for these lower levels in the latter studies is not known. The plasma clearance curves exhibited a biphasic elimination process and were accordingly divided into an a phase (1.5 to 6.0 hours) and a B phase (6.0 to 24.0 hours). The EE2 a phase and B phase half-life values (Table IV) are similar to those reported by Humpel et al. (16) and Goldzieher -- et al. (17). The latter investigators studied a total of 98 women in 5 widely different localities of the world. They found substantial differences in the pharmacokinetics of EE2, following the administration of single oral doses of this steroid ranging from 35 to 100 ug. The a and B phase half-life values ranged from 1 to 3 hours and from 6 to 14 hours, respectively. The NET peak plasma levels were reached within 0.5 to 1.5 hours and in this respect were similar to that obtained for EE2. The half-life values obtained for the.t3 phase for NET were similar to those obtained for EE2 (Table IV). The range for the NET a half-life values was 0.8 to 1.9 hours and for the 6~ half-life the range was 9.2 to 10.9 hours. These values are in agreement with those reported in the literature (11,18-20). In these studies the a half-life values ranged from 0.3 to 4.4 hours and the $ half-life values ranged from 5.0 to 16.5 hours. Although there appears to be a similarity between B halflife values for NET and EE2, it may be premature to assess this relationship with the limited data available. To make a valid comparison between any pharmacokinetic parameters of EE2 and NET, data from a much larger number of subjects will be required. NOVEMBER 1980VOL.22NO.5 467

12 ACKNOWLEDGEMZNTS The authors wish to thank the Research Triangle Institute, Research Triangle Park, N.C., for kindly donating the anti-ethinylestradiol-7-(3-thiopropionic acid)-bovine serum albumin serum. This antiserum was prepared at the Research Triangle Institute under a contract with the National Institute of General Medical Sciences. REFERENCES Inman, W.H.W., Vessey, M.P., Westerholm, B. and Engelund, A. Thromboembolic disease and the steroidal content of oral contraceptives. A report to the committee on safety of drugs. Br. Med. J. 2: , de la Pena, A., Chenault, C.B. and Goldzieher, J.W. A radioimmunoassay of unconjugated plasma ethynylestradiol in women given a single oral dose of ethynylestradiol or mestranol. Steroids 25: , Kundu, N., Keeman, S.P. and Slaunwhite, W.R., Jr. Radioimmunoassay (RIA) of ethynylestradiol (EE). The Endocrine Society, 56th Annual Meeting, Atlanta, Georgia, 1974, Program book, p. A-263 (416). Warren, R.J. and Fotherby, K. Radioimmunoassay of ethynylestradiol. J. Endocrinol. 63:30-31, Pasqualini, J.R., Castellet, R., Portois, M.C., Hill, J.L. and Kincl, F.A. Plasma concentrations of ethynyloestradiol and norethindrone after oral administration to women. J. Reprod. Fertil. 49: , Ahluwalia, B.S., Curry, C.L., Cracker, C.L. and Verma, P.S. Evidence of higher ethynylestradiol blood levels in human hypertensive oral contraceptive users. Fertil. Steril. 28: , Lahteenmaki, P. and Nilsson, C.G. Plasma concentrations of ethinylestradiol and d-norgestrel during two immediate postabortal oral contraceptive cycles. Contraception 17:9-17, Nilsson, S. and Nygren, K.-G. Ethinyl estradiol in peripheral plasma after oral administration of 30 ug and 50 ug to women. Contraception 18: , NOVEMBER 1980VOL.22N0.5

13 Cook, C.E., Tallent, C.R. and Christensen, H.D. Antigens from synthetic and natural estrogens: Formation by conjugation with protein through a thiopropionic acid link. Life Sciences 14: , Rodbard, D. and Lewald, J.W. Computer analysis of radioligand assay and radioimmunoassay data. Acta Endocrinol, Copenh. Suppl. 147:78-103, Stanczyk, F.Z., Brenner, PFD., Mishell, D.R., Jr., Ortiz, A., Gentzschein, E.K.E. and Goebelsmann, U.: A radioimmunoassay for norethindrone (NET): Measurement of serum NET. concentrations following ingestion of NET-containing oral contraceptive steroids. Contraception 18: , Dvorchik, B.H. and Vesell, E.S. Pharmacokinetic interpretation of data gathered during therapeutic drug monitoring. Clin. Chim. 22: , Speck, U., Wendt., H., Schylze, P.E. and Jentsch, D. Bio-availability and pharmacokinetics of cyproterone acetate-14c and ethinylestradiol-3h after oral administration as a coated tablet (SH B 209 AB). Contraception 14: , Fotherby, K. and Warren, R.J. Bioavailability of contraceptive steroids from capsules. Contraception 14: , Morris, S.E., Scarisbrick, J.J., Cameron, E.H.D., Groom, G.V., Buckingham, M.S., Everitt, J. and Elstein, M. Comparison of plasma hormone changes using a "conventional" and a "paper" pill formulation of a low-dose oral contraceptive. Fertil. Steril. 29: , Humpel, M., Nieuweboer, B., Wendt, H. and Speck, U. Investigations of pharmacokinetics of ethinyloestradiol to specific considerations of a possible firstpass effect in women. Contraception 19: , Goldzieher, J.W., Dozier, T-S., de la Pena, A., Ojo, O.A., Lean, T.S., Chinnatamby, S., Basnayake, S., and Koetsawang, S., Plasma levels and pharmacokinetics of ethinyl estrogens in various populations. Contraception 21:1-16, Warren, R.J. and Fotherby, K. Radioimmunoassay of synthetic progestogens, norethisterone and norgestrel. J. Endocrinol. 62: , NOVEMBER 1980VOL.22NO.5 469

14 19. Nygren, K.-G., Lindberg, P., Martisson, K., BOSU, W.T.K. and Johansson, E.D.B. Radioimmunoassay of norethindrone: peripheral plasma levels after oral administration to humans and Rhesus monkeys. Contraception 9: , Mills, T.M., Lin, T.J., Hernandez-Ayup, S., Greenblatt, R.B., Ellegood, J-0. and Mahesh, V.B. The metabolic clearance rate and urinary excretion of oral contraceptive drugs. I. Norethindrone. Am. J. Obstet. Gynecol. 120: , NOVEMBER 1980 VOL. 22 NO. 5

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