Free testosterone levels during danazol therapy*

FERTILITY AND STERILITY Copyright 1983 The American Fertility Society Printed in U.8A. Free testosterone levels during danazol therapy* Bo Nilsson, M.D. t:j: Ragnar Stidergard, Ph.D. Mats-GQran Damber, M.D.t Jan-Erik Damber, M.D. Bo von Schoultz, M.D.t University of Umea, Umea, Sweden Danazol is a testosterone (T) derivative widely used in the clinical treatment of endometriosis. Its mechanism of action is poorly understood, but is side effects are mainly androgenic. Previously it was demonstrated that danazol can displace T from sex-hormone-binding globulin (SHBG). The binding properties of danazol to SHBG and albumin were studied with the use of labeled danazol in an aqueous two-phase equilibrium partition system. Levels of total T, SHBG, and albumin were measured in 16 women undergoing danazol treatment for endometriosis. Thereafter, free and protein-bound T levels were calculated. A marked rise in free T was found during danazol therapy as compared with pretreatment levels. The data suggest that many of the effects of danazol could be explained by increased levels of free T during treatment. Fertil Steril 39:505, 1983 Danazol is an ethinyl testosterone (T) derivative that is frequently used in the clinical treatment of endometriosis. Its mechanism of action is unclear, but side effects during therapy are mainly androgenic. 1-5 In a previous study6 it was found that danazol binds to sex-hormone-binding globulin (SHBG) and that it competes with T for the binding sites, thereby displacing T. Increased levels of free, metabolically active T might explain several of the endocrine effects following danazol administration.2, 4, 5, 7-9 However, T displacement from SHBG does not necessarily Received October 18, 1982; revised and accepted December 22,1982. *Supported by the Swedish Medical Research Council (5982), the Medical Faculty University of Umea, Umea, Sweden, and Fertility Research Institute, Malmo, Sweden. tdepartment of Obstetrics and Gynecology. *Reprint requests: Bo Nilsson, M.D., Department ofobstetrics and Gynecology, University of Umea, S-901 87 Umea, Sweden. Department of Physiology. cause increased circulating levels of the free steroid, because albumin binding and feedback mechanisms may have a modulating influence. Therefore, in the present study, we used labeled danazol to determine its binding properties to SHBG and albumin. Thereafter, levels of free and protein-bound T were calculated in women undergoing danazol treatment for endometriosis. MATERIALS AND METHODS THE TWO-PHASE SYSTEM An aqueous two-phase system composed of two water-soluble polymers, polyethylene glycol (PEG) (upper phase 7% wt/wt) and dextran (lower phase 12% wt/wt), was used for equilibrium partition.io, 11 A 0.002 M K-phosphate buffer containing 0.1 M K-thiocyanate with a ph of 7.4 was used. In this system, serum proteins are almost exclusively confined to the lower dextran-contain- -ingphase, while steroids distribute more equally Nilsson et al. Free T levels during danazol therapy 505

between the two phases in a well-defined manner, different for each steroid. The partition coefficient is determined with the use of labeled steroids in serum-free separation experiments. Then, with serum in the system, it is possible from the specific radioactivity in the upper phase for us to calculate the amounts of free and protein-bound steroid in the lower phase. REAGENTS The labeled steroids [1,2-3Hl-5a-dihydrotestosterone, [1,2-3Hl-testosterone (New England Nuclear Corporation, Boston, MA) and [G-3Hldanazol (Sterling-Winthrop Research Institute, New York, NY) were purified by chromatography with the Bush B3 system.12 Unlabeled dihydrotestosterone (DHT), T, cortisol (F), bovine serum albumin, activated charcoal Norit A, dextran (mol wt 17,500), and PEG (mol wt 6000) were purchased from Sigma Chemical Company, St. Louis, MO. Unlabeled danazol was obtained from Sterling-Winthrop AB, Stockholm, Sweden, Celite from Analytical Filter Aid, Johns-Manville, Denver, CO, and T antiserum from Dr. L. Edqvist at the Royal Veterinary College, Stockholm, Sweden. All salts were of analytical grade, and the water was twice distilled in quartz. SERUM Venous blood samples were drawn from healthy women in the third trimester ofpregnancy taking no drugs; from men treated with high doses of estrogen for carcinoma of the prostate; from 16 women with endometriosis before and after 2 to 3 months of treatment with danazol, 600 mg daily (200 mg x 3); and from 1 apparently healthy nonpregnant woman taking no drugs. After coagulation, all sera were decanted and stored at - 20 C until used. DANAZOL BINDING TO SHBG AND ALBUMIN Pooled sera from pregnant women and from men treated with high doses of estrogen were used because of their high SHBG concentrations. We incubated 100 ILl of serum for 15 minutes in glass tubes with 5000 dpm [G-3Hl-danazol, 0.2 ng F (to avoid any possible disturbance due to the presence of corticosteroid-binding globulin [CBG1), and various amounts of unlabeled danazoi. Thereafter, 2.4 gm of the phase system was added. After equilibration for 30 minutes and centrifugation at 1000 x g for 10 minutes, 800 ILl of the upper phase was analyzed in a liquid scintillation counter. All incubations were performed at 37 C. All experiments were performed on triplicate samples. Scatchard analyses were performed for the ratio of bound and free danazol plotted as a function of bound danazoi. 13 SHBG binding of danazol was calculated by subtraction of albumin binding according to RosenthalY Albumin binding was calculated from experiments using heat-treated (60 C for 30 minutes) serum.lo,l1 SHBG BINDING CAPACITY AND ALBUMIN LEVELS DURING DANAZOL THERAPY Individual serum samples from 16 women were analyzed before and after 2 to 3 months of daily treatment with danazol, 200 mg x 3. We pretreated the serum with activated charcoal in order to remove endogenous steroids and danazo16 and, using the two-phase system, calculated SHBG binding capacity by the Scatchard plot method as previously described.6* Albumin concentrations in all serum samples were determined by the method described by Rodkey.15 DETERMINATION OF T LEVELS Serum T levels were measured by radioimmunoassay (RIA)16 in 16 women before and during danazol treatment. Danazol is known to cross-react in T assays.2, 7 Using chromatography on Celite microcolumns as described by Brenner et ai.,16 we found that labeled T and danazol could easily be separated. In order to study whether danazol cross-reaction could be avoided, we used a control serum sample from a healthy, nonpregnant woman. Danazol was added to part of the serum to a final concentration of 3000 nm, which is above reported therapeutic levels.17 T concentrations were measured with RIA 16 with and without purification of the steroid extract on Celite microcolumns. Danazol was found to have considerable cross-reaction with the antibody, giving falsely high values of serum T. Values were increased about 50% when danazol was added. After chromatography, the interference of danazol was abol- *Reference values for serum total T in women range from 0.4 to 3.5 nm (0.12 to 1.0 ng/mi). Levels of free T range from 1% to 3% of total T. Reference values for SHBG binding capacity range from 40 to 120 nm, corresponding to 11.5 to 35 ng DHT/ml. 506 Nilsson et al. Free T levels during danazol therapy Fertility and Sterility

Oanazol Bound/free 8 7 6 5 4 3 2 b c 100 200 300 400 500 600 700 Bound Danazol ng/l00}j1 Figure 1 Scatchard analysis of danazol binding in serum. (a), Total protein binding. (b), Nonspecific albumin binding. (c), Specific SHBG binding of danazol. ished. Mean values ± standard deviation (SD) of T for five separate determinations in the same sample with and without the addition of danazol were 0.512 ± 0.071 and 0.497 ± 0.079 nm, respectively. All samples from the clinical material were purified by chromatography before T levels were measured. STATISTICAL ANALYSES The significance of differences was tested by the Wilcoxon signed rank test. RESULTS DANAZOL BINDING TO SHBG AND ALBUMIN As shown in Figure 1, danazol binds to SHBG with high affinity. The mean value ± SD of the association constant (K A ) for this binding as determined in five experiments was 0.404 ± 0.076 x 10 8 M- 1 The corresponding value for danazol binding to albumin was 11.2 ± 1.54 x 10 4 M- 1. SHBG BINDING CAPACITY, TOTAL T LEVELS, AND ALBUMIN LEVELS During treatment with danazol, 600 mg daily for 2 to 3 months, a marked reduction in SHBG binding capacity was found in all women. The pretreatment mean value was reduced by approximately 80% (P < 0.001). Levels of albumin and total T were unaffected throughout therapy (Table 1). CALCULATION OF FREE T LEVELS The binding of steroids to proteins follows the law of mass action. The distribution of T between protein-bound and free fractions can be calculated if all factors that influence the state of equilibrium are known. The serum concentrations of SHBG, albumin, and total T and the KA values for danazol binding to SHBG and albumin were determined in the present study. The corresponding KA values for T have previously been determined.ii Using these data, the levels of free, albumin-bound and SHBG-bound T for each individual woman were calculated by a computer.ll During treatment, a serum concentration for danazol of 800 nm was assumed. (Serum concentrations of danazol between 800 and 2100 nm have been reported at a daily dose of 400 mgp) Estradiol, DHT, other androgen metabolites, F, and CBG, when present in physiologic concentrations, have minimal effects on the distribution of T,l1 18 and therefore they were omitted from the calculations. Before danazol treatment, an average of 60.15% of total T was bound to SHBG, 38.57% was albumin-bound, and the fraction offree Twas 1.28%. During treatment, the corresponding figures were 17.91%, 79.44%, and 2.64%, respectively. In all women, values of free T (nm) were found to increase during danazol therapy. The pretreatment mean value was increased by 80% (P < 0.001). DISCUSSION Previous reports on total T levels following danazol therapy show considerable disagree- Table 1. Mean Values ± SD for the Serum Concentrations of SHBG (nm), Albumin (nm), Total and Free Testosterone (nm) in 16 Women Before and After 2 to 3 Months of Treatment with Danazol, 600 mg Daily Before danazol During danazol Significance SHBG 81.9 ± 28.92 15.6 ± 5.08 P < 0.01 Albumin 73.6 ± 6.85 73.8 ± 3.03 Nsa Total tes- 1.93 ± 0.54 1.69 ± 0.48 NS tosterone Free tes- 0.025 ± 0.0076 0.045 ± 0.0142 P < 0.01 tosterone ans, not significant. Nilsson et al. Free T levels during danazol therapy 507

Distribution of tes tos terone (per cent) a therapeutic interval b therapeutic Interval Albumin bound o 1000 2000 3000 4000 0 1000 2000 3000 4000 Serum danazol, nm Figure 2 Percentage of distribution oft (SHBG-bound, albumin-bound, and free fractions) as a function of danazol serum concentration. (a), At the beginning of danazol therapy (total T = 1.9 nm, SHBG = 80 nm, albumin = 74 nm). (b), After 2 to 3 months ofdanazol therapy (total T = 1.7 nm, SHBG = 16 nm, albumin = 74 nm). men t " 2 7-9. 19, 20 one reason b' elng th e cross-reactivity of danazol and its metabolites in T assays. In the present study, using chromatographic purification, we were able to avoid this problem. The addition of danazol in amounts exceeding reported therapeutic serum levels17 did not affect the T determinations. The KA value for danazol binding to SHBG was found to be 0.404 x 108 M-1. This figure could be compared with the KA for T binding of 5.97 x 108 M-1 as determined previously be the same method. 11 The relation between these values (1:15) allows significant T displacement during therapy; and, in fact, an even higher ratio (1:7) for the KA values has been suggested. 18 However, unspecific albumin binding "buffers" the release of T following danazol administration, thereby diminishing the potential. increase in free T. During treatment with danazol, 600 mg daily, levels of total T were unchanged, while a marked increase of the free fraction was recorded. There are two principal mechanisms behind this effect: displacement and SHBG suppression. Figure 2 is an attempt to illustrate these two mechanisms in a theoretical model. At the beginning of therapy, when SHBG levels are still unchanged (Fig. 2a), displacement is dominating and the release of T follows the serum concentration of danazol. Within therapeutic levels, the increase offree T ranges from 25% to 50%, while the SHBG-bound fraction is reduced from 60% to 45% of total values. A marked reduction in SHBG binding capacity is well known to occur during danazol therapy6, 21 and was further confirmed in the present investigation. Figure 2b illustrates the situation after 2 to 3 months of therapy when SHBG levels are suppressed and probably stable at around 20% of pretreatment values. There is now an 80% to 90% increase in free T, compared with the concentrations before danazol treatment. However, only a small part (12% to 15%) of this increase is due to displacement, whereas the fall in SHBG concentration accounts for most of the effect. Within therapeutic danazollevels, the SHBG-bound fraction of total T is now only about 15%, while the main part is bound to albumin. Thus, the interaction of danazol with T distribution is characterized by an initial displacement, followed by a gradual decrease in SHBG binding capacity. The present data suggest that the endocrine effects of danazol could be explained by its T-displacing ability and as a consequence of increased levels of free T during treatment. Acne, anabolic weight gain, oily skin, hirsutism, hoarseness, and changes of serum lipoproteins into a "male" pattern are well-known side effects of danazol in women. I - 5 Certainly, they all might be caused by raised levels of circulating free T. Also, the decline of SHBG might be an androgenic response following T displacement, because T is known to have this effect.22 On the other hand, danazol competition for binding sites may also be protective against excess amounts of free T. In animal experiments, binding to androgen receptors in target cells has been reported.23 Thus, at the cellular level, danazol could reduce the availability of receptors and the androgenic effect. To what extent such blocking actually occurs in women during treatment needs further elucidation. Acknowledgments. Skillful technical assistance was provided by Mrs. Monica Isaksson and Mrs. Mette Wallen, and excellent typing was provided by Mrs. Margareta Holmberg. 508 Nilsson et ai. Free T levels during danazol therapy Fertility and Sterility

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