Testolactone-associated high androgen levels, a pharmacologic effect or a laboratory artifact?

Testolactone, an aromatase inhibitor, blocks conversion of androgens to estrogens. In familial male precocious puberty, slowing of pubertal progression and growth velocity occurs with testolactone and spironolactone. Girls with McCune-Albright syndrome, given testolactone, respond similarly. A 2-yr-old female (case 1) on testolactone for non-McCune-Albright gonadotropin independent precocious puberty had marked elevations of androstenedione (18 ng/mL, normal: 0.2-3.1) and testosterone (3.6 ng/mL, normal < 0.2) but no virilization. Investigations were undertaken to determine whether elevations in testosterone and androstenedione were caused by interference in these RIAs. After a single oral dose of testolactone (5 mg/kg in case 1; 4 mg/kg in case 2, a 3-yr-old boy with familial male precocious puberty; 10 mg/kg in a healthy postmenopausal control), serum testosterone and androstenedione were measured serially by RIA for 24 h. Androstenedione went from normal to a mean peak of 45.4 ng/mL at 1-2 h and returned to baseline by 24 h. Testosterone, undetectable at baseline (case 1 and control) or 1.8 ng/mL (case 2) rose to a mean peak of 6.9 ng/mL and returned to baseline by 24 h. Testolactone, in serial dilutions, cross-reacted in the testosterone assay. Testolactone significantly interferes in these serum RIAs, making their use unreliable in follow-up of patients treated with testolactone.

Effect of methimazole, with or without L-thyroxine, on remission rates in Graves' disease.

Medical treatment of Graves' disease involves antithyroid drugs with or without the addition of exogenous T4. There have been conflicting reports as to whether the addition of T4 improves remission rates or delays relapse. To evaluate this issue in a North American population, 199 patients were treated with methimazole until they were euthyroid. They were then randomized to either methimazole alone in a dose sufficient to normalize TSH (group 1), or to 30 mg methimazole daily plus sufficient T4 to maintain TSH in the upper normal range (group 2), or to 30 mg methimazole daily plus sufficient T4 to suppress TSH below 0.6 mIU/L (group 3). After 18 months, methimazole was stopped, and T4 was continued in groups 2 and 3. Because not all patients in groups 2 and 3 achieved their target TSH concentration, they were reassigned to group A (TSH > or = 1.0) or group B (TSH < 1.0), based on the mean TSH achieved during methimazole treatment. One hundred forty-nine patients have been followed for at least 6 months after stopping methimazole (mean 27 months). Fifty-eight percent of patients have relapsed. There were no significant differences in relapse rates after stopping methimazole. Among those patients who did relapse, however, there was a significant difference in the months to relapse after stopping methimazole between groups B and 1 (group 1: 3.3 +/- 0.7, group A: 5.6 +/- 0.8, group B: 7.4 +/- 1.7; P = 0.01 for the comparison between groups B and 1). We conclude that the addition of T4 to methimazole does not improve long-term remission rates in Graves' disease.

Effect of methimazole with or without exogenous L-thyroxine on serum concentrations of thyrotropin (TSH) receptor antibodies in patients with Graves' disease.

Medical treatment of Graves' disease involves use of antithyroid drugs with or without the addition of exogenous L-T4. There have been conflicting reports as to whether the addition of T4 reduces TSH receptor antibodies and improves remission rates more than antithyroid drugs alone. To further examine the effect of drug therapy on serum concentrations of TSH receptor antibodies. 70 patients with Graves' disease were treated with methimazole (Tapazole) alone until they were euthyroid. Then they were randomized to receive either: 1) methimazole alone in a dose sufficient to normalize TSH (0.3-5.4 mIU/L; Group 1); 2) 30 mg methimazole daily plus sufficient T4 (Synthroid) to maintain TSH in the high-normal range (2.0-5.4 mIU/L; Group 2); or 3) 30 mg methimazole daily plus sufficient T4 to suppress TSH to below 0.6 mIU/L (Group 3). The duration of treatment in all groups was 18 months. At baseline and after 6 and 18 months, TSH receptor antibodies were measured both by the ability of patients' sera to stimulate cAMP production by FRTL-5 cells (thyroid-stimulating Ig) and by the ability of patients' sera to inhibit binding of radiolabeled TSH to solubilized porcine thyroid membranes (TSH-binding, inhibiting Ig). Thyroid-stimulating Ig(TSI) and TSH-binding, inhibiting Ig(TBII) concentrations were similar among the three groups at baseline. Mean baseline TSI (expressed as the percent of normal control) for all patients combined was 306 +/- 21%. Mean baseline TBII (expressed as percent inhibition of TSH binding) was 38 +/- 2%. TSI was elevated in 85% and TBII was elevated in 75% of patients at baseline. After 18 months, TSI was elevated in 64% of patients, and TBII was elevated in 28%. Serum TSI decreased by 36 +/- 5% during the study, and there was no significant difference in the degree of reduction among the three groups (P = 0.99). Serum TBII decreased by 59 +/- 3%, and there also was no significant difference among the groups (P = 0.83). At baseline, serum TBII correlated with free T4 (r = 0.33, P < 0.01), total T3 (r = 0.55, P < 0.01), and thyroid size (r = 0.35, P < 0.01). There was no correlation between TSI and any of the baseline parameters or between TSI and TBII at any timepoint. In conclusion, we found that the addition of T4 to methimazole does not result in a greater decrease in TSH receptor antibody concentrations than treatment with methimazole alone. From these results, we would predict no difference in remission rates among these patients, but confirmation of this prediction will need to await long-term follow-up of these subjects.

Effect of daily and alternate day low dose prednisone on serum cortisol and adrenal androgens in hirsute women.

To test the hypothesis that alternate day prednisone treatment more effectively suppresses adrenal androgen secretion (compared to cortisol secretion) than daily prednisone treatment, we measured serum dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), and cortisol concentrations during these two prednisone treatment schedules in eight hirsute women. The women were assigned randomly to receive either a daily nighttime dose of prednisone (100 micrograms/kg) or an alternate nighttime prednisone dose (200 micrograms/kg) for 4 months. During the following 4 months the women received the other schedule. Serum hormone levels were measured 0, 4, and 8 months before and after iv administration of 25 U synthetic ACTH. To optimally compare the daily and alternate day prednisone regimens, hormonal determinations were made on 2 successive days (days 1 and 2) after the last dose of prednisone. We found no evidence for greater suppression of adrenal androgens or lesser suppression of cortisol with alternate day prednisone treatment. Basal serum DHEA and DHEAS concentrations were suppressed to a greater degree than was cortisol during both daily and alternate day prednisone treatments. ACTH-stimulated DHEA and cortisol concentrations were equally suppressed. Only two of the eight women noted improvement in hirsutism during the study, and four women gained weight. Thus, adrenal androgen secretion was more easily suppressed than was cortisol secretion by this low dose of glucocorticoid, but there was no advantage to alternate day therapy.

Cyclosporine absorption in two patients with short-bowel syndrome.

We present the cases of two patients with short-bowel syndrome who failed to achieve therapeutic cyclosporine serum concentrations on oral drug but were successful on intravenous administration. One patient received cyclosporine after renal transplantation for renal failure secondary to enteric oxalosis; the second received cyclosporine for active Crohn's disease. The rapid bowel transit time was the critical factor in limiting cyclosporine absorption in both cases. In studying oral and intravenous pharmacokinetic profiles, we support a zero-order kinetic model for oral cyclosporine absorption.

Polycystic kidney and liver disease and corticosterone changes in the cpk mouse.

Mice homozygous for the mutation cpk develop a lethal infantile polycystic kidney disease (PKD) and have abnormal elevations of corticosterone in the postnatal period. We examined the development of these elevated glucocorticoid levels in early life. The histological progression of a cystic lesion of the biliary ducts in older heterozygotes was also studied. Normal mice had low (less than 85 nmol/liter) or undetectable (less than 36 nmol/liter) levels from the sixth to twelfth day of life. Corticosterone levels were significantly higher in all homozygote (cpk/cpk) and some of the normal sibs, who were presumed to be heterozygote (cpk/+) mice compared to age-matched controls from day seven to 12. Corticosterone levels were similar from the fifteenth day. A significant proportion of adult obligate heterozygotes from breeding pairs were found to have focal areas of cystic dilatation of the biliary ducts, in the absence of any renal abnormality. The incidence of this lesion increased with age (10% at 150 days, 23% at 300 days, 29% at 450 days, 65% at greater than 450 days of age). The livers of homozygote mice dying in infancy, and control adult mice of the C57BL/6J background strain were free of cysts. The finding of hepatic cysts is reminiscent of adult type PKD. The cpk model thus supports the concept that the different forms of PKD may represent varying expression of a similar gene complex.

Gonadal regulation in men with flaccid paraplegia.

Previous clinical studies have shown that some men with spinal cord injury (SCI) have aspermatogenesis and germinal epithelial atrophy. Additionally, an altered response to a luteinizing hormone releasing hormone (LHRH) challenge has been observed in men having SCI with abnormal testicular biopsy. In the present study, an LHRH challenge was given to 13 SCI and able-bodied men, and serum levels of follicular stimulating hormone (FSH), luteinizing hormone (LH), prolactin, and testosterone was determined by RIA. Based on physical examinations, the 13 subjects were divided into four groups: (A) men with SCI and a lower motor neuron lesion but no evidence of lumbosacral spinal cord function, (B) men with SCI and a lower motor neuron lesion but evidence of sacral cord function, (C) men with SCI and a spastic upper motor neuron lesion, and (D) able-bodied men as the control group. The only significant differences were associated with group A which showed increased levels of FSH before and after LHRH challenge and increased LH levels 30 min after injection relative to all other groups. The data suggest that SCI which results in a persistent bladder atonia and flaccid paraplegia, also produces an alteration in gonadal regulation. Thus, assessment of serum FSH and LH levels may provide information concerning somatic, visceral, and gonadal function after SCI.

Medrogestone and an LHRH analogue as potential combination therapy for hormone-dependent cancers.

Treatment (5 mg/kg s.c. for 1 to 4 weeks) of adult male rats with medrogestone (Colprone), a compound with progestational and antiandrogenic properties, induced significant atrophy of the ventral prostate without affecting testicular weight, testicular LH/hCG receptor levels or plasma testosterone. The potent LHRH agonist (D-Ala6, des-Gly-NH210) LHRH ethylamide at 500 ng s.c. for 2-4 weeks suppressed the testicular weight, testicular LH/hCG receptor levels, plasma testosterone levels, and caused atrophy of the androgen-dependent seminal vesicles and ventral prostate. The combination 4 week-treatment of medrogestone and LHRH agonist led to the most significant decrease of prostatic weight. The potential usefulness of this combination therapy in hormone-dependent cancers is discussed.

LHRH and analogues as potential therapy for benign prostatic hyperplasia and hormone-dependent cancers.

In adult male rats, daily s.c. injections of 0.008-125 microgram luteinizing hormone releasing hormone (LHRH) or its analogue (D-Ala, des-Gly-NH2)LHRH ethylamide, led to significant differences in inhibitory effects on testicular function and accessory sex organ weights. The analogue was at least 35 times more potent than LHRH in reducing testicular LH/hCG receptors and 350 times more effective in decreasing plasma testosterone concentrations. In a second study, adult male rats treated with 25-2500 microgram LHRH daily for a 3-week period showed 85%-90% decrease in plasma testosterone concentrations. Treatment with 25 or 250 microgram LHRH effected a maximal 25% and 40% decrease of the ventral prostate and seminal vesicles, respectively, without affecting the testicular weight, the latter being reduced by 15% with the higher dose of 2500 microgram. The LHRH analogue is proposed for the treatment of prostatic and breast cancers while native LHRH could be an effective therapy for benign prostatic hyperplasia.

Effect of medrogestone on conversions of pregnenolone and 17 alpha-hydroxypregnenolone in rat testis.

Medrogestone, viz., 6,17-dimethylpregn-4,6-diene,3,20-dione (Colprone), a synthetic compound with progestational and antiandrogenic properties, was studied for its effects on the metabolism of pregnenolone and 17 alpha-hydroxypregnenolone by a 10,000 g supernatant fraction of testicular homogenates. This compound inhibited the conversion of pregnenolone to progesterone (91%), 17 alpha-hydroxyprogesterone (83%), delta 4-androstenedione (43%), and testosterone (30%) and the conversion of 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone (84%), delta 4-androstenedione (56%), and testosterone (38%).

Inhibition of testosterone metabolism by medrogestone in rat ventral prostate in vivo and in vitro.

Medrogestone (MDG), viz., 6,17-dimethyl-4,6-pregnadiene-3,20-dione (Colprone), a synthetic compound with progestational and antiandrogenic properties was studied for its effect on the conversion of testosterone (T) to 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 alpha(beta)-17 beta-diol (Adiol) by ventral prostatic preparations of the rat. MDG, 20 mg/kg b.w./day s.c. for 14 days, inhibited the conversion of T to DHT by ventral prostatic homogenates and cytoplasm and at the same dose and route, but for 28 days, it reduced the conversion of T to DHT by ventral prostatic nuclei and T to Adiol by ventral prostatic cytoplasm and nuclei. MDG, from 1 X 10(-4) to 1 X 0(-5) M final concentration in vitro, induced dose-dependent decreases in the conversions of T to DHT and Adiol by ventral prostatic nuclei.

A simple and sensitive nonradioactive method for the detection of urinary human chorionic gonadotropin and diagnosis of early human pregnancy. I. Multiple-unit test.

A simple, sensitive, and reproducible method for the detection of urinary human chorionic gonadotropin (hCG) and diagnosis of early human pregnancy is reported. A 5-ml aliquot of filtered early-morning urine sample was concentrated in a microconcentrator (M) to 0.1 ml of retentate which was diluted with 0.4 ml of distilled water and tested in a hemagglutination inhibition test (M-HIT). Also, a 0.1-ml aliquot of filtered unconcentrated urine sample was diluted with 0.4 ml of distilled water and tested in the same hemagglutination inhibition test (HIT). Urine samples from women of reproductive age; from perimenopausal, menopausal, and proteinuric women; and from adult males were tested in the HIT and M-HIT. Some of these urine samples were also tested in the mouse ovulation bioassay (MOB). The M-HIT was significantly more reliable than the HIT for diagnosis of early pregnancy 25 to 55 days after menses. Correct negative results with the M-HIT were obtained in urine samples of most of the nonpregnant cycling, perimenopausal, and menopausal women, and adult males. Urine samples from subjects with severe proteinuria gave false-positive types of reactions in the M-HIT. Positive results were obtained in the MOB with a number of urine samples from pregnant, perimenopausal, and menopausal women. A properly conducted M-HIT should be very valuable in diagnosing pregnancy as early as the 26th day of the cycle in regularly menstruating women.

PIP: This article discusses a simple, sensitive, reproducible method for detecting HCG (human chorionic gonadotropin) in the urine and the subsequent early diagnosis of pregnancy. 5 ml of filtered urine sample (early morning) was concentrated in an M (microconcentrator) to 0.1 ml of retentate diluted with 0.4 ml of distilled water. It was then tested in a M-HIT (hemagglutination test). Another 0.1 ml aliquot of urine sample (filtered and unconcentrated) was diluted with the same amount of distilled water and tested in the same HIT (hemagglutination test). Urine samples from women of reproductive age, from perimenopausal, menopausal, and proteinuric women, and from adult males were tested in both the HIT and M-HIT, as well as in the MOB (mouse ovulation bioassay). The M-HIT Proved to be significantly more reliable than the HIT for diagnosis of early pregnancy, 25-55 days following menses. Appropriate negative results were obtained with the M-HIT in those urine samples from most of the nonpregnant, cycling, perimenopausal and postmenopausal women, and the adult males. False-positive reactions in the M-HIT resulted from the urine specimens of those with severe proteinuria. The MOB yielded positive results in a number of urine samples from pregnant, perimenopausal and menopausal women. The M-HIT, if properly done, indicates high reliability in diagnosing pregnancy as early as the 26th day in the cycles of menstruating women.

A simple and sensitive nonradioactive method for the detection of urinary human chorionic gonadotropin and diagnosis of early human pregnancy. II. Single-unit test.

A simple, sensitive, and reliable single-unit nonradioactive method for the detection of human chorionic gonadotropin (hCG) in concentrated urine and the diagnosis of early pregnancy is reported. This unit, presently termed the Ayerst pregnancy test kit (APTK), consists of four components: a sampler-filter paper cone, an ultrafilter-concentrator to which a vial holder is attached, a support stand with a mirror, and an immunologic reagent vial. In the APTK, 5 to 6 ml of urine were sampled, filtered, and concentrated, and the hCG in the retentate was detected by Ayerst immunologic reagents [APTK(AY)] and by the Pregnosticon "All In" [APTK(P)]. Some of the unconcentrated urine samples (0.1 ml) were also tested in hemagglutination inhibition tests (HIT) using Ayerst [HIG(AY)] and Pregnosticon "All In" [HIT(P)] reagents. Urine samples from pregnant, nonpregnant (ovulating and nonovulating), perimenopausal, and menopausal women were tested. It was found that the APTK(AY) and APTK(P) were significantly more sensitive and reliable than the HIT(AY) and HIT(P) in detecting low levels of urinary hCG for early diagnosis of pregnancy. The sensitivity and specificity of the APTK(AY) were better than those of the APTK(P). The APTK(AY) give significantly more correct positive and negative results than the other tests performed simultaneously. The APTK(AY) is simpler and safer than the serum radioimmunoassays and radioreceptor assay presently used to detect low levels of hCG for the early diagnosis of pregnancy and other hCG-producing states.

Effects of a luteinizing hormone-releasing hormone analog on mating and fertility in rats.

A rhythmic antifertility effect of a luteinizing hormone-releasing hormone (LH-RH) analog, [D-Ala6-des-Gly-NH210]-LH-RH-ethylamide (AY-25,205), administered intramuscularly every 3rd day staring in the afternoon of diestrus, was demonstrated in 4-day cyclic rats. The antifertility effect was achieved for a period of approximately four cycles when the females were allowed constant cohabitation with fertile males except for 24 hours following treatment. Unrestricted cohabitation resulted in some matings and pregnancies in the group treated every 3rd day and also in some of the groups treated every 4th day with restricted cohabitation. The antifertility effect of AY-25,205, with unrestricted cohabitation, disappeared when the second treatment was given 4 days after the first. It is presumed that the antifertility effect of AY-25,205 was achieved through its capacity to induce ovulation at a physiologically "wrong time" (i.e., 1 day before the expected day of proestrus) and through its effect on mating behavior. The present experimental models suggest that AY-25,205 or similar analogs could be potentially useful for a more reliable rhythm method of birth control in humans, by timing ovulation and narrowing the fertile period of the cycle.

PIP: The rhythmic antifertility effect of an LH-RH analog, AY-25,205, was investigated in 4-day cyclic rats. All groups (10 animals in each) received 20-120 ng/rat im between 3:00 and 3:30 P.M. the day of diestrus. Treatment continued for either 2 or 4 consecutive cycles, and subsequent injections were given either on the 3rd or 4th consecutive day; cohabitation was either unrestricted or prevented for 24 hours posttreatment. Only 1 rat of 40 in the 3-day. restricted cohabitation groups became pregnant during the 1st cycle, and the antifertility effect continued. However, for those groups treated every 4th day and allowed unrestricted cohabitation, 25 of 30 animals became pregnant the night of the 2nd injection. It is suggested that the antifertility effect of AY-25,205 is due to an ability to induce ovulation 1 day before the appearance of gonadotropin-induced lordosis. AY-25,205 or similar analogs may have application in humans by making more precise the time of ovulation, which would permit more effective use of the "rhythm method" of birth control.