Measurement of Diethylstilbestrol in Plasma from Patients with Cancer of the Prostate1

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[CANCER RESEARCH 41, 4693-4697, November 1981] 0008-5472/81 /0041-OOOOS02.00 Measurement of Diethylstilbestrol in Plasma from Patients with Cancer of the Prostate1

Heather A. Kemp, Graham F. Read,2 Diana Riad-Fahmy, Adrian W. Pike, Simon J. Gaskell, Kenneth Queen, Maureen E. Harper, and Keith Griffiths

Tenovus Institute for Cancer Research, Welsh National School of Medicine, Heath, Cardiff. CF4 4XXÂ¡H. A. K.. G. F. P., D. R-F., A. W. P., S. J. G., M. E. H., K. G.Â¡, and Department of Urology, St. Woolos Hospital, Newport. Gwent[K. Q.Â¡.United Kingdom

ABSTRACT on the prostate gland (6), and it is possibly this latter effect which prompted the use of large doses (>200 mg/day) of DES A specific radioimmunoassay has been developed for dieth- phosphate (Honvan) for the treatment of prostatic cancer. ylstilbestrol (DES), using an antiserum raised against DES Endocrine studies of the type undertaken by this Institute (11 ), monocarboxymethyl ether and a tritium-labeled radioligand. however, have clearly indicated that a dose of 1 mg DES Ids Prior to radioimmunoassay, a fraction enriched in DES is ob effectively decreases plasma testosterone levels and does not tained from a dichloroethane extract of plasma using Sephadex result in concomitant increases in the concentrations of prolac- LH-20. The specificity of the assay is good, and the sensitivity tin and growth hormone. Such observations, and the concern (130 pg/ml) is adequate for accurate determination of DES in over side effects (3), have tended to direct clinicians towards plasma from prostatic cancer patients treated with the drug. treatment schedules using low-dosage DES. The precision is satisfactory, with an interassay coefficient of Despite its extensive use, there is no information on the variation of approximately 10% at concentrations of approxi levels of DES in patients being successfully treated or in those mately 1 ng/ml, and the blank values are negligible. Excellent who apparently fail to respond to such therapy. Initially, 70% agreement (r = 0.96) is observed between data obtained by of treated patients respond clinically to DES therapy, but the radioimmunoassay and those obtained by a procedure using majority eventually relapse. It remains to be determined gas chromatography-high-resolution mass spectrometry. DES whether this is related to a change in the hormone sensitivity concentrations in the plasma of six treated (1 mg DES three of the tumor resulting from dedifferentiation or to changes in times daily) patients were in the range 0.15 to 6.0 ng/ml. the pharmacokinetics of DES in certain patients. RIAs with the Increases in plasma concentration were observed within 2 hr potential to measure the plasma concentrations of DES have of administration, with secondary rises occurring 5 to 6 hr later. been described (2), but no data on the levels in patients have Plasma testosterone concentrations were low in four of the been reported. Here we report a sensitive RIA for DES and patients; in a single subject, relatively high levels of testoster illustrate its application to the determination of concentrations one were further elevated following administration of luteinizing in the plasma of patients receiving 1 mg tds. hormone-releasing hormone. MATERIALS AND METHODS INTRODUCTION Materials. [monoef/iy/-3H]DES (50 to 100 Ci/mmol) was purchased from the Radiochemical Centre, Amersham, United Kingdom, and Although there is still controversy as to the most effective stored in either benzene or toluene solution (50/iCi/ml) at -20Â°. For form of therapy for early prostatic cancer and on the relative use in the assay, an aliquot was taken, the solvent was evaporated merits of surgery and radiotherapy (16), it has long been under nitrogen, and the residue was dissolved in assay buffer (see accepted that endocrine therapy, generally in the form of DES3 below) containing antiserum. The working solution contained 35 to 40 treatment, is the most effective for the management of the pg/100 fi\ (about 0.125 /iCi/ml). Nonradioactive DES, hexestrol, and advanced progressive disease. There is still discussion con various steroids were purchased from Sigma London Chemical Com cerning the precise mode of action of DES (10), though it is pany, Poole. Dorset, United Kingdom; the sodium salt of DES diphos generally accepted that its principal effect is indirect, decreas phate (Honvan) was obtained from Ward Blenkinsop & Co., Ltd., London, United Kingdom; u-hydroxydienestrol, stored as the triacetate, ing testicular synthesis and secretion of testosterone by inhibit and /?-dienestrol were a gift from Dr. M. Metzler, Institut fÃ¼rPharmak ing luteinizing hormone secretion from the pituitary. There is, ologie und Toxikologie der UniversitÃ¤t Wurzburg, Wurzburg, West however, evidence for a direct action of DES on the testis and Germany; DES monoglucuronide and DES diglucuronide were kindly provided by Dr. K. Krohn, Institut fÃ¼rOrganische Chemie und Bio ' This work was generously supported by the Tenovus Organization. chemie. Hamburg, West Germany. Solutions of these compounds were 2 To whom requests for reprints should be addressed. stored in ethanol (1 mg/10 ml) at 4Â°.Dichloroethane, purchased from 3 The abbreviations used are: DES. diethylstilbestrol; RIA. radioimmunoassay; Koch-Light Laboratories, Ltd.. Colnbrook, Buckinghampshire, United LH-RH, luteinizing hormone-releasing hormone; GC-MS, gas chromatography- high-resolution mass spectrometry; DMS, dimethylstilbestrol; TBDMS, ferf-bu- Kingdom, and dichloromethane from BDH Chemicals, Poole, Dorset. tyldimethylsilyl; Ids, ter die sumendum. three times daily; Trivial names used are: United Kingdom, were used without further purification. Ethanol and diethylstilbestrol (DES), E-3.4-/j/s(p-hydroxyphenyl)hex-3-ene; dimethylstilbes methanol were obtained from James Burroughs, Ltd., London. A solu trol (DMS), E-3.4-b/s(p-hydroxyphenyl)but-2-ene; dienestrol. Â£,Â£-3,4-bis(p-hydroxyphenyl)hexa-2,4-diene; hexestrol, 3.4-bis(p-hydroxyphenyl)hexane; u-hy- tion of 0.01 M phosphate buffer (pH 7.4) containing 0.9% sodium chloride and 0.1% sodium azide was stored at 4Â°; the assay buffer droxydienestrol. E,E-3,4-b/s(p-hydroxyphenyl)hexa-2.4-dien-l-ol; dihydrodibu- tylstilbestrol. 5,6-b/s(p-hydroxyphenyl)decane; diethylstilbestrol diphosphate. additionally included gelatin (0.1 %). Dextran-coated charcoal was pre 3,4-b/s( p-phosphoxyphenyl)hex-3-ene; estradiol, 1,3,5(10)-estratriene-3.17/3- pared by dissolving dextran T-70 (0.06%, Pharmacia Ltd., London) in diol; estrone. 3-hydroxyestra-1,3.5(10)-tnen-17-one; cholesterol. 5-cholesten- assay buffer and then adding HCI-washed activated charcoal (No. C- 3ÃŸ-ot; progesterone, 4-pregnene-3,20-dione; testosterone, 17/8-hydroxy-4-an- drosten-3-one. 4386, 0.6%; Sigma London Chemical Company). The suspension was Received February 20, 1981 ; accepted July 29, 1981. stirred at 4Â°for 4 hr before use. Sephadex LH-20 from Pharmacia,

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Ltd. was washed with dichloromethane:methanol (94:6, v/v) and meth- into vials, scintillant (6.5 ml) was added, and the radioactivity was anol before use. Lipidex 5000 (Packard, Downers Grove, III.) was measured for 2 min; the tubes used for determination of total counts converted to the triethylaminohydroxypropyl derivative by the proce contained approximately 10,000 cpm. Plasma concentrations were dure of Axelson and SjÃ¶vall (4). Lipid scintillant was prepared by calculated using a Hewlett-Packard 9810 A programmable desk-top dissolving PRO (5 g) in toluene (1 I) and Triton X-100 (500 ml). Other calculator. The program used a rectangular hyperbola fit to the stand chemicals were Analar grade (BDH Chemicals). All glassware was ard curve. Recoveries were monitored in preliminary assays by adding washed overnight in detergent, rinsed, and soaked in sodium chromate- an internal standard of radioactive DES to the plasma aliquot before sulfuric acid before being rinsed in deionized water and dried. extraction. Recovery of DES (after allowance for the use of aliquots in Patients. Blood was collected from 6 patients with histologically the assay) was calculated to be 87.7% Â±6.8%; this was considered proven carcinoma of the prostate using an indwelling catheter over adequate, and addition of the internal standard was discontinued. periods of approximately 8 hr (5 patients) or 16 hr (one patient). Five Correction for recovery was not included in the calculation. of the patients had received DES, 1 mg tds. for at least 3 months prior Analyses by GC-MS. A solution of DMS in drug-free plasma was to the study. The sixth patient was investigated 6 days, 3 months, and prepared by addition of 50 /il of an ethanolic solution (10 Â¿ig/ml) of 6 months after commencement of therapy. They were hospitalized for DMS to 10 ml of plasma. After mixing and standing overnight, 50 jtl 48 hr and ate, slept, and drank as appropriate. DES was administered were added as internal standard to each aliquot (0.25 to 1.0 ml) of at 6:30 a.m., 12:30 p.m., and 5 p.m. Samples of blood (3 ml) were plasma to be analyzed. The plasma was extracted with diethyl ether (8 collected from 5 patients at 30-min intervals from 7:30 a.m. until 4 p.m. ml), the aqueous layer was frozen, and the extract was removed and except between 9 and 10 a.m. when more frequent sampling occurred. dried under a stream of nitrogen. The residue was dissolved in 3 x Blood was similarly collected on the second day between 7:30 a.m. 100 fil methanol:water:chloroform (80:30:15, v/v; Solvent A) and ap and 12 noon, and LH-RH (100 /ig i.v.) was administered at 9 a.m. The plied to a column (2 x 0.6 cm) of triethylaminohydroxypropyl-Lipidex sixth patient had blood collected at 30-min intervals from 8 p.m. to 12 5000 (4), swollen Â¡nSolvent A. Polar constituents were removed by noon the following day. Samples were centrifuged, and plasma was elution with 2 ml of Solvent A. Nonpolar constituents were subsequently stored at -20Â° until assayed for testosterone (12) and DES. eluted with 2 ml of methanol:chloroform (80:15, v/v; Solvent B). Finally, Synthesis of Antigenic Conjugate. The O-carboxymethyl ether of DES and DMS, together with naturally occurring weakly acidic com DES was prepared according to the general procedure of Rao and ponents, were recovered by elution with 2 ml of Solvent B, acidified by Moore (17). Pure DES monocarboxymethyl ether was recrystallized the addition of a small quantity of solid carbon dioxide. The final column from toluene:hexane (m.p. 157Â°). The product was characterized by eluate was dried under nitrogen, and the DES and DMS were converted field desorption mass spectrometry using a Varian MAT 731 instrument to the TBDMS derivatives by reaction with ferf-butyldimethylchlorosi- (MH+ = m/z 327) and by IR spectroscopy. A conjugate, produced by lane:imidazole:dimethylformamide (1:1:6, v/v; Applied Science Labo coupling the hapten to bovine serum albumin by the mixed anhydride ratories, State College, Pa.) Excess reagent was removed by filtration reaction (7), had a molar incorporation ratio of 12:1 (DES:bovine serum through a short column of Sephadex LH-20 (9). albumin). Standard mixtures of DES and DMS for the preparation of the Antiserum. Rabbits were immunized with the conjugate (15), and standard curve were obtained in 2 ways. First, aliquots of stock one antiserum with a titer of 1/8000 was selected for use in the assay. solutions of DES and DMS in ethanol were mixed in appropriate The antiserum was stored at -20Â° and diluted to 1/100 with assay proportions. Second, solutions of DES in plasma were prepared, and buffer. appropriate aliquots were diluted to 1 ml with drug-free plasma to give Extraction from Plasma. Dichloroethane (3 ml) was added to plasma total amounts of DES equal to those in standard mixtures prepared (0.5 ml). The tubes were shaken for 12 min and centrifuged (2.5 x 103 from stock ethanolic solutions. The plasma samples were then treated rpm) for 2 min, and 2 ml of the lower dichloroethane layer were as for samples of unknown DES concentration. removed to a clean tube. The solvent was evaporated under nitrogen. GC-MS was performed using a Varian MAT 731 double-focusing Sephadex LH-20 Chromatography. Each extract was dissolved in instrument. Separations were achieved on a glass column (length 3 m, the chromatography solvent (dichloromethane:methanol, 94:6, v/v; 2 inside diameter 3.5 mm) packed with 1% OV-1 on Gas-Chrom Q (100 x 0.2 ml) and applied to a column (4 x 0.6 cm) of Sephadex LH-20, to 120 mesh) at 280Â°. The mass spectrometric resolution was 8000. swollen in the same solvent. The first 3.0-ml eluate was discarded, and The ion source temperature was 200Â°, and the electron energy was 70 the DES fraction was collected in the next 3.0 ml. The fraction was eV. Analyses were carried out in the single-ion monitoring mode with dried and dissolved in methanol (500 fil). The reproducibility of the sequential detection of ions of m/z 468.2880 for cis- and trans-DMS Chromatographie separation was improved by the addition of a layer b/s-TBDMS ether and m/z 496.3193 for cis- and frans-DES bis- (1 to 2 mm) of sand to the top of each Sephadex LH-20 column. TBDMS ether. (Each massxharge ratio corresponded to the respective Replicate experiments using [3H]DES indicated excellent reproducibil ity: mean recovery was 96.3 Â± 2.9% (n = 6). Eluate (30 ml) was Table 1 collected from a large column (30 x 1.3 cm) containing swollen Seph Cross-reaction of compounds with anti-DES antiserum at a final dilution of adex LH-20. This eluate was used in the preparation of the standard 1:8000Compound curve to correct for column blank effects. Aliquots (3 ml) were mea aDES/3-Dienestrolu>-Hydroxydienestrolmeso-DihydrodibutylstilbestrolHexestrolDES% of cross-reaction sured into tubes, one for each point on the standard curve, and dried, and methanol (500 ,Â«l)was added. RIA. Triplicate aliquots (100 /il) of the methanolic solution were dried under Nj. Antiserum:radioligand solution (200 jul) was added, and the mixture was incubated (37Â°; 2 hr). For the standards, solutions of 20 monoglucuronideDES to 200 pg DES per 10 fil were prepared in ethanol. Triplicate aliquots diglucuronideDES (10 /il) were mixed with aliquots (100 n\) of column solvent residue in (Honvan)EstroneEstradiol-1diphosphate methanol, dried, and treated as the samples. After equilibration, the 7ÃŸ5a-Androstane-3o,1 free and bound fractions were separated by addition of a suspension 7/8-diolCholesterolProgesteroneTestosterone1000.250.100.0610.967.47.80.44<0.04<0.04<0.04<0.04<0.04<0.04a of dextran-coated charcoal (1 ml) to all tubes except those used for determination of total counts, to which assay buffer (1 ml) only was added. The solutions were mixed, allowed to stand for 10 min, and centrifuged (12 min; 2.5 x 103 rpm). The supernatant was decanted Determined according to the criteria of Abraham (1).

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1981 American Association for Cancer Research. DES in Plasma molecular ion.) The ratio (fl) of peak areas corresponding to the DES Table 2 and DMS derivatives were determined using the expression R = Recovery of DES from spiked plasma samples (HTcoEs + HTTDEs)/(HTCDMs+ HÃ•TDMS),where HÃŽCDESisthe product of (ng/ml)Added2.00DES the height of the peak attributed to c/s-DES TBDMS and the gas Chromatographie retention time (which is proportional to peak width) of of recovery85 that component. Values for peak area ratios were converted to DES concentrations by reference to the standard curves derived as de 1.17 0.97 1116% 63 0.60Measured1.69 0.53n"15 88 scribed. Linear regression of ratio values derived from analyses of "spiked" plasma samples (y) on values derived from analyses of Number of replicate analyses. standard mixtures (x) gave the equation y = 1.067x - 0.038 ng/ml Table 3 (correlation coefficient, r = 0.997), indicating excellent agreement Intra- and interassay variance in high-, medium-, and low-concentration quality between the 2 methods and confirming the ability of the assay proce control samples dure to determine accurately the concentrations of DES in plasma IntraassayPlasma supplemented with the drug. The standard curve derived from mixtures of aliquots of stock ethanolic solutions of DES and DMS was used for the determination of unknown concentrations. poolHigh (ng/ml)2.00 (ng/ml)1 (%)11.7 Â±0.20C .69 Â±0.20 Medium 0.78 Â±0.09 11.4 0.97 Â±0.10 1116CV 10.0 RESULTS LowDES 0.52 Â±0.10n"121212cv"(%)10.320.0InterassayDES0.53 Â±0.10n15 19.3 Number of replicate analyses. Characteristics of the RIA. The cross-reactions of the anti- 6 CV. coefficient of variation. serum (used at a final dilution of 1:8000) are listed in Table 1. c Mean Â±S.D. There is a negligible degree of cross-reaction with the naturally occurring steroids and only a small degree of cross-reaction y.0 878x +0051 with the major metabolites (13) /8-dienestrol and u)-hydroxydÂ¡- r-096 enestrol, indicating acceptable specificity for the hex-3-ene substructure of DES. The degree of cross-reaction with the glucuronides of DES is high, but, since an extraction step is â€”¿20 E s included in the analytical procedure, no glucuronides are ex XÂ» pected in the RIA. The cross-reaction with hexestrol (11 %), 1 although relatively high, is clinically irrelevant. A typical standard curve for the DES assay, over the range of 0 to 200 pg, with a corresponding precision profile, is shown

lOO-i

K> 20 Â»0 40 GC-MS (ng/ml) Chart 2. Correlation of plasma concentrations determined by RIA and by GC- MS. A single outlier (circled point) was not included in the linear regression.

75- in Chart 1. Addition to the standards of column solvent eluate, in methanol, decreases the binding by approximately 8% and is a necessary part of the procedure. The sensitivity of the assay, calculated using the expression ts/>/N (5), gave a curve value of 8.8 pg corresponding to 0.132 ng/ml. The accuracy of the method, determined by repeatedly measuring 3 pools of plasma, spiked with known amounts of DES, was similar for 100 200 high, medium, and low values (Table 2). Assay precision, DES pg determined by calculating within-assay and between-assay 30-1 coefficients of variation for 3 quality control plasma pools, is shown in Table 3. Comparison of Analytical Data from RIA and GC-MS. Twelve plasma samples from patients treated with DES, rep resenting a spread of concentrations across the range ob served in this study, were analyzed by both RIA and GC-MS procedures (Chart 2). Linear regression of RIA values (y) on GC-MS results (x) gave the equation y = 0.878x + 0.051 (correlation coefficient, r = 0.96; if a single outlier is omitted). 100 200 MS pg Agreement between the methods was thus excellent, particu Chart 1. A typical standard curve for diethylstilbestrol covering the range of larly since no allowance for recovery was made in the RIA. The 20 to 200 pg, with a corresponding precision profile. The coefficients of variation were calculated from the DES valuus of 8 replicates for each point on the single discrepancy when the DES concentration was high may standard curve. suggest incomplete extraction in the RIA in this example. With

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few samples of relatively high concentration, it has not, how Patient J.W. day 1 30-1 1mg DES ever, been possible to evaluate this further. l mg DES DES Levels in Treated Patients with Prostatic Cancer. Plasma samples from 6 patients were assayed for levels of DES and testosterone. Patient F. C. showed an expected marked rise in the concentration of DES approximately 1.5 to "Ãˆ 2-0- -10 2.0 hr after DES administration (1-mg tablet; 6:30 a.m.) and a "2i less significant rise 6 hr later (Chart 3). The first peak was also observed on Day 2. Similar patterns of DES concentration were j observed with Patient J. W. (Chart 4) and 3 others not illus trated. Plasma testosterone concentrations in Patient F. C. (Chart 3) were higher than expected after DES therapy for several months. The circadian rhythm (18) suggests a substantial adrenal contribution to the plasma androgen level, although in 0J 07.00 09.00 11OO 13.00 15.00 this patient an LH-RH stimulation test also resulted in a signifi Clock time cant elevation in the concentration of plasma testosterone (Chart 3), indicating a responsive pituitary-testicular axis. Low levels of testosterone were found in Patient J. W. (Chart 4) and Patient J.W. doy 2 in the 3 other patients studied; there was no response in any 3-0-| l mg DES IHRH of these to the administration of LH-RH. Plasma DES concentrations in a single patient (A. F.) I throughout the night, 6 days, 3 months, and 6 months after B

commencing therapy are shown in Chart 5. The rise in DES -2-OHI -10 levels after the 6:30 a.m. DES tablet was the same at each period. Rises in DES concentration were in each case observed 4 to 6 hr after administration of the 5 p.m. tablet, presumably

corresponding to the secondary rises noted above. 0 K Patient F.C.dayl 1mg DES

2O- -10 0J L0 0700 09.00 11.00 1100 15.00 Clock time Chart 4. Plasma concentrations of diethylstilbestrol and testosterone for Pa I tient J. W., Day 1 (A) and Day 2 (B). Times of administration of DES and LH-RH I are indicated by arrows 5 _

DISCUSSION

The development of this routine RIA for DES will permit i 1 studies of the relationships between DES concentration in 0700 0900 IIJOO 1100 15.00 Clock time plasma, hormone secretion, and clinical response of treated prostatic cancer patients. The assay described has satisfied accepted performance criteria and has been validated by ref erence to a highly specific GC-MS procedure. The antiserum displayed high specificity, although inclusion of a Sephadex B LH-20 chromatography step in the procedure was essential to remove interfering plasma constituents. The range of DES concentrations during the day in the 6 patients studied was 0.15 to 5.9 ng/ml. The observed second peak of DES concentration in plasma after the p.o. dose could arise when DES glucuronides are hydrolyzed in, or reabsorbed from, the large intestine approximately 5 hr after the tablet has been taken (14). Administration p.o. of [14C]DES glucuronide results in a rise in plasma radioactivity 3 to 6 hr later (8). The data from Patient F. C. clearly indicate that, although 0700 09.00 1U>0 13.00 1500 treatment at the level of 1 mg DES tds is sufficient to suppress Clock time testicular activity in most patients (11 ), higher doses may be Chart 3. Plasma concentrations of diethylstilbestrol and testosterone for Pa tient F. C.. Day 1 W) and Day 2 (B). Times of administration of DES and LH-RH required for others. A responsive pituitary-testicular axis was are indicated by arrows. demonstrated in Patient F. C., and furthermore, rhythmic se-

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REFERENCES

2-0-1 6 Do/s 1. Abraham, G. E. Solid-phase radioimmunoassay of oestradiol-17/9. J. Clin. ImgDES' 1700 ImgDES Endocrinol. Metab.. 29: 866-870, 1969. 10630 2. Abraham, G. E., Reifman, E. M., Buster, J. E., Di Stephano, J., and Marshall, J. R. Production of specific antibodies against diethylstilboestrol. Anal. Lett., 5: 479-486, 1972. 3. Arduino, L. J., Ballar, J. C , Becker, L. E., era/. (The Veterans Administration Cooperative Urological Research Group). Carcinoma of the prostate: treat ment comparisons. J. Urol., 98. 516-522, 1967. 4. Axelson, M., and SjÃ¶vall,J. Analysis of unconjugated steroids in plasma by liquid-gel chromatography and glass capillary GC-MS. J. Steroid Biochem., 8:682-692, 1977. 5. Brown, J. B . Bulbrook, R. D . and Greenwood, F. C. An evaluation of a chemical method for the estimation of oestriol, oestrone and oestradiol-17/5 2-0-, 3 Months in human urine. J. Endocrinol.. 76. 41-48, 1957. 6. Danutra, V., Harper, M. E., and Griffiths, K. The effect of stilboestrol analogues on the metabolism of steroids by the testis and prostate of the rat in vitro. J. Endocrinol., 59. 539-544, 1973. 7. Erlanger, B. F.. Borek, F., Beiser. S. M., and Lieberman, S. Steroid-protein conjugates. 1. Preparation and characterisation of conjugates of bovine serum albumin with testosterone and cortisone. J. Biol. Chem., 228: 713- 727, 1957. 8. Fischer, L. J., Weissinger, J. L., Richet, D. E., and Hintze, K. L. Studies on the biological disposition of diethylstilboestrol in rats and humans. J. Toxicol. Environ. Health, I: 587-605. 1976. 9. Gaskell, S. J., and Brooks, C. J. W. f-Butyldimethylsilyl derivatives in the 6 Months gas chromatography-mass spectrometry of steroids. Biochem. Soc. Trans.. 4: 111-113, 1976. l mg DES Imo. DES I 1700 I 06 30 10. Griffiths, K., Davies, P., Harper, M. E., Peeling, W. B., and Pierrepoint, C. G. The etiology and endocrinology of prostatic cancer. In: D. P. Rose (ed.), Endocrinology of Cancer. Vol. II. pp. 1-55. Boca Raton. Florida: CRC Press. 1979. 11. Harper. M. E.. Peeling, W. B., Cowley. T.. Brownsey, B. G.. Phillips. M. E. A.. Groom. G., Fahmy, D. R., and Griffiths. K. Plasma steroid and protein hormone concentrations in patients with prostatic carcinoma, before and 24.00 04.00 during oestrogen therapy. Acta Endocrinol.. 8f: 409-426, 1976. TimÂ« 12. Joyce, B. G.. Fahmy, D.. and Millier. S. G. Specific determination of testos Chart 5. Plasma diethylstilbestrol concentrations measured Â¡nPatient A. F. terone in female plasma by radioimmunoassay : a rapid and reliable proce dure for the routine clinical laboratory. Clin. Chim. Acta, 62. 231-238, 6 days. 3 months, and 6 months after the start of DES treatment. Times of administration of DES are indicated by arrows. 1975. 13. Metzler, M. Metabolic activation of carcinogenic diethylstilbestrol in rodents and humans. J. Toxicol. Environ. Health. ! (Suppl.): 21-35, 1976. cretion of testosterone was observed. This testosterone profile 14. Mroszczak, E. J., and Riegelman. S. Disposition of diethylstilboestrol in the rhesus monkey. J. Pharmacokinet. Biopharm., 3. 303-327, 1975. may be related to the secretion of adrenal androstenedione, 15. Nieschlag, E., Kley, H. K., and Usadel. K.-H. Production of steroid antisera the precursor of plasma testosterone. These studies again in rabbits. In: E. H. D. Cameron. S. G. Hillier, and K. Griffiths (eds.), Steroid illustrate the difficulties of analyzing single daily samples for immunoassay: Proceedings of the Fifth Tenovus Workshop, pp. 87-96. the assessment of the endocrine status of patients with pros- Cardiff, United Kingdom: Alpha Omega Publishing, 1975. 16. Peeling, W. B.. and Griffiths. K. Prostatic cancer. In: J. Lumley and J. Craven tatic cancer (19). The development of a specific RIA procedure (eds.). Surgical Review I, pp. 303-327. Tunbridge Wells, United Kingdom: for the measurement of DES allows the investigation of plasma Pitman Medical, 1979. 17. Rao. P. R.. and Moore. P. H. Synthesis of new steroid haptens for radioim levels of the drug in relation to changes in the rhythmic secre munoassay. Part IV. 3-O-Carboxymethyl ether derivatives of estrogens. tion of hormones during treatment and will facilitate a study of Specific antisera for the assay of esterone. estradiol-1 f/< and estriol. Ste roids. 29. 461-469. 1977. patient variability in relation to response. 18. Resko, J. A., and Eik-Nes, K. B. Diurnal testosterone levels in peripheral plasma of human male subjects. J. Clin. Endocrinol. Metab., 26: 573-576, ACKNOWLEDGMENTS 1966. 19. Walker, R. F., Wilson, D. W., Read, G. F., and Riad-Fahmy, D. Assessment We thank Dr. M. Metzler and Dr. K. Krohn for gifts of various DES metabolites of testicular function by the radioimmunoassay of testosterone in saliva. Int. and Dr. B. Q. Joyce for the determinations of plasma testosterone. J. Androl., 3: 105-120, 1980.

NOVEMBER 1981 4697

Heather A. Kemp, Graham F. Read, Diana Riad-Fahmy, et al.

Cancer Res 1981;41:4693-4697.

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