Measurement of thyroid stimulating immunoglobulins using a novel thyroid stimulating hormone receptor-guanine nucleotide-binding protein, (GNAS) fusion bioassay.

Hyperthyroidism, defined by overproduction of thyroid hormones, has a 2-3% prevalence in the population. The most common form of hyperthyroidism is Graves' disease. A diagnostic biomarker for Graves' disease is the presence of immunoglobulins which bind to, and stimulate, the thyroid stimulating hormone receptor (TSHR), a G-protein coupled receptor (GPCR). We hypothesized that the ectopically expressed TSHR gene in a thyroid stimulating immunoglobulin (TSI) assay could be engineered to increase the accumulation of the GPCR pathway second messenger, cyclic AMP (cAMP), the molecule measured in the assay as a marker for pathway activation. An ectopically expressing TSHR-mutant guanine nucleotide-binding protein, (GNAS) Chinese hamster ovary (CHO) cell clone was constructed using standard molecular biology techniques. After incubation of the new clone with sera containing various levels of TSI, GPCR pathway activation was then quantified by measuring cAMP accumulation in the clone. The clone, together with a NaCl-free cell assay buffer containing 5% polyethylene glycol (PEG)6000, was tested against 56 Graves' patients, 27 toxic thyroid nodule patients and 119 normal patients. Using receiver operating characteristic analysis, when comparing normal with Graves' sera, the assay yielded a sensitivity of 93%, a specificity of 99% and an efficiency of 98%. Total complex precision (within-run, across runs and across days), presented as a percentage coefficient of variation, was found to be 7·8, 8·7 and 7·6% for low, medium and high TSI responding serum, respectively. We conclude that the performance of the new TSI assay provides sensitive detection of TSI, allowing for accurate, early detection of Graves' disease.

Effects of testosterone replacement in human immunodeficiency virus-infected women with weight loss.

The objective of this study was to determine whether physiological testosterone replacement increases fat-free mass (FFM) and muscle strength and contributes to weight maintenance in HIV-infected women with relative androgen deficiency and weight loss. Fifty-two HIV-infected, medically stable women, 18-50 yr of age, with more than 5% weight loss over 6 months and testosterone levels below 33 ng/dl were randomized into this double-blind, placebo-controlled trial of 24-wk duration. Subjects in the testosterone group applied testosterone patches twice weekly to achieve a nominal delivery of 300 mug testosterone over 24 h. Data were evaluable for 44 women. Serum average total and peak testosterone levels increased significantly in the testosterone group, but did not change in the placebo group. However, there were no significant changes in FFM (testosterone, 0.7 +/- 0.4 kg; placebo, 0.3 +/- 0.4 kg), fat mass (testosterone, 0.3 +/- 0.7 kg; placebo, 0.6 +/- 0.7 kg), or body weight (testosterone, 1.0 +/- 0.9 kg; placebo, 0.9 +/- 0.8 kg) between the two treatment groups. There were no significant changes in leg press strength, leg power, or muscle fatigability in either group. Changes in quality of life, sexual function, cognitive function, and Karnofsky performance scores did not differ significantly between the two groups. High-density lipoprotein cholesterol levels decreased significantly in the testosterone group. The patches were well tolerated. We conclude that physiological testosterone replacement was safe and effective in raising testosterone levels into the mid to high normal range, but did not significantly increase FFM, body weight, or muscle performance in HIV-infected women with low testosterone levels and mild weight loss. Additional studies are needed to fully explore the role of androgens in the regulation of body composition in women.

Augmented pituitary corticotropin response to a threshold dosage of human corticotropin-releasing hormone in depressives pretreated with metyrapone.

We studied pituitary corticotropin response to exogenous corticotropin-releasing hormone infusion and attempted to control for the confounding effect of variable serum cortisol levels between depressed and control subjects. If metyrapone was given during the time of day when hypothalamic pituitary adrenal activity was otherwise low, the relative increase in the corticotropin concentration was small. Pituitary response to exogenous corticotropin-releasing hormone can be defined under conditions in which the amount of glucocorticoid-mediated negative feedback present at the level of the pituitary gland is equal in all subjects. When the ambient cortisol level was equalized (and suppressed) in all subjects at the time of study with a threshold dosage of corticotropin-releasing hormone, we found an augmented response to corticotropin-releasing hormone in depressives. This raises the possibility that either increased pituitary sensitivity to corticotropin-releasing hormone or an increased intracellular pool of corticotropin is available for release in subjects with major depressive illness.

Cushing's disease with 'normal suppression' due to decreased dexamethasone clearance.

Suppression of urinary corticosteroids during low-dose dexamethasone testing (0.5 mg every six hours eight times) has commonly been recognized as a response that excludes the diagnosis of Cushing's syndrome. Although "normal suppression" has been reported previously in Cushing's disease, rarely has an explanation been provided for this aberrant response. We report a case of proven Cushing's disease in which normal suppression was observed with low-dose dexamethasone testing. Further study suggested that this phenomenon, which is not widely recognized, was related to an abnormally decreased clearance of dexamethasone. We therefore suggest that whenever responses to testing appear discordant with the clinical index of suspicion, simultaneous plasma dexamethasone and cortisol levels should be obtained to exclude abnormalities in dexamethasone clearance.

Single-dose metyrapone test: review of a four-year experience.

Subnormal plasma 11-deoxycortisol (compound S) responses to metyrapone were found in patients with adrenal insufficiency or with Cushing syndrome caused by adrenal tumors and in those receiving long-term glucocorticoid or diphenylhydantoin sodium therapy. High normal or exaggerated responses were seen in women receiving oral contraceptives, patients with Cushing syndrome caused by adrenal hyperplasia, and those with untreated hypothyroidism. Diabetes mellitus, hypoglycemia, congestive failure, and obesity also were associated with exaggerated responses. Subnormal plasma S responses were observed in 15 patients who responded normally to a repeat test or to the standard metyrapone test. The abnormal response resulted from insufficient metyrapone, administration at the wrong time, or delay in obtaining the blood sample. The single-dose metyrapone test may be the procedure of choice in screening for adrenal insufficiency.

Percutaneous absorption of dexamethasone estimated by a plasma radioimmunoassay.

Precutaneous absorption of dexamethasone and its effect on the pituitary adrenal axis were measured in vivo in normal human subjects after application to skin. Specific plasma dexamethasone and cortisol radioimmunoassays were used. Following application of 1% dexamethasone on 500 cm2 of normal skin, the plasma dexamethasone concentration was maximal at 2 hr, and the average absorption was 0.25% over 8 hr; significant cortisol suppression occurred at 2, 4, and 8 hr. This technique: (1) provides an accurate assessment of the in vivo absorption of dexamethasone applied to human skin, (2) avoids exposure of the subjects to radioactive steroids, (3)permits estimation of the quantity of unmetabolized steroids absorbed, and (4) serves as a possible model for the development of similar assays for other topical steroids.

Familial prostatic cancer risk and low testosterone.

We investigated whether familial factors influence 1) the incidence of prostatic cancer and 2) the plasma content of sex steroids. A 4-fold higher relative risk for the development of prostatic cancer was observed for brothers (n = 257) of prostatic cancer cases (n = 150) compared to their brothers-in-law (n = 202) and males in the general population of the state Utah. The intraclass correlation for plasma testosterone content [intraclass correlation coefficient (r1) = 0.51; P less than 0.01] and the apparent free testosterone concentration (r1 = 0.54; P less than 0.01) were highly significant in nonendocrinologically treated cases and their brothers. Further, sons and their fathers had significant intraclass correlations for both plasma dihydrotesterone (r1 = 0.83; P less than 0.01) and the ratio of testosterone to dihydrotestosterone (r1 = 0.46; P less than 0.05). Probands and their brothers, and sons of the patients with the disease had significantly lower plasma testosterone levels than controls of comparable age. This is the first documentation indicating that familial (possibly genetic) factors are potent risk factors for predisposing men to the development of prostatic cancer and in regulating the plasma content of androgens. Our results indicate that plasma androgen levels in families with prostatic cancer are clustered in the lower range of the normal population. They also suggest that plasma androgen content is more similar within each family with the cancer than among the families without cancer.

Factitious Cushing's syndrome.

A woman with pituitary-dependent Cushing's disease remained hypercortisolemic after bilateral adrenalectomy. A search for an adrenal remnant by venous catheterization study suggested persistent cortisol-secreting tissue in the left adrenal bed. During ACTH stimulation, plasma corticosterone concentrations remained low and cortisol remained high, suggesting the cortisol was of exogenous origin. Cushingoid features resolved after confronting the patient with this evidence. Plasma corticosterone concentrations played a novel role in the diagnosis of factitiously induced Cushing's syndrome in this patient.

Androgens regulate proliferation of human prostate cancer cells in culture by increasing transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF)/TGF-alpha receptor.

Androgens affect growth of the prostate gland and many prostate cancers. Androgens could mediate their mitogenic effects on prostate cells by an autocrine loop involving epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha that bind to the EGF/TGF-alpha receptor. We examined the effects of 5 alpha-dihydrotestosterone (DHT) and testosterone (T), EGF, and EGF-alpha on cell proliferation and 3H-thymidine incorporation in an androgen-dependent human prostate cancer cell line, ALVA101, in serum-free medium. The regulation of TGF-alpha and EGF/TGF-alpha receptor messenger RNA (mRNA) levels were determined by Northern blot analysis and EGF/TGF-alpha receptor protein by immunoblot. After 24 h of treatment of ALVA101 cells with DHT (10(-8) M) or T (10(-8) M), TGF-alpha mRNA levels increased 3- and 2.5-fold, respectively, and EGF/TGF-alpha receptor mRNA levels 2- and 1.5-fold, respectively. Cell numbers increased at day 5 in response to 10(-8) M DHT (18%, P < 0.01), 10(-8) M T (15%, P < 0.01), 20 ng/ml EGF (16%, P < 0.01), and 50 ng/mL TGF-alpha (34%, P < 0.01). DHT combined with TGF-alpha or T combined with EGF increased cell number 43% and 40% above control, respectively (P < 0.01 vs. DHT, P < 0.05 vs. TGF-alpha, T, EGF alone). The anti-EGF/TGF-alpha receptor antibody (528) blocked the cell proliferation induced by either DHT or TGF-alpha. We conclude that DHT and T stimulate synthesis of TGF-alpha and EGF/TGF-alpha receptor mRNAs and EGF/TGF-alpha receptor content in ALVA101 cells. This mitogenic effect of androgen on ALVA101 cells may involve TGF-alpha and the EGF/TGF-alpha receptor autocrine loop.

Subnormal tissue 3 alpha-androstanediol and androsterone in prostatic hyperplasia.

We investigated the role of 3 alpha-androstanediol (3 alpha-diol) in the development of benign prostatic hyperplasia (BPH) and the apparent equilibrium of enzymes which metabolize it in normal and hyperplastic prostatic tissue of humans. We determined the endogenous concentrations of 3 alpha-diol, androsterone, its 3 alpha-17-keto metabolite or precursor, and 5 alpha-dihydrotestosterone (DHT), its 3-keto,17 beta-hydroxy product of precursor, by RIA after extraction and paper chromatography of the androgens from normal and hyperplastic prostate glands. The mean concentrations of 3 alpha-diol and androsterone were about one-third of normal in BPH. The mean ratio of the concentration of DHT to 3 alpha-diol was significantly higher (P less than 0.005) than normal in BPH, whereas no statistical difference was observed for the mean ratio of the tissue levels of 3 alpha-diol to androsterone in the two groups. Our data do not support the postulate that 3 alpha-diol is causally related to the development of BPH. However, they indicate that the apparent equilibrium of the 3 alpha-hydroxysteroid oxidoreductase favors the formation of the 3-keto-oxidized product, DHT, which may have relevance to the occurrence of the renewed growth of the prostate of aging men.

Kinetics and interconversion of prednisolone and prednisone studied with new radioimmunogassays.

To determine the kinetics of metabolism and interconversion of prednisolone and prednisone, the plasma levels of these synthetic glucocorticoids were measured by new radioimmunoassays (RIA) after intravenous (iv) or oral administration to normal humans. Following the iv injection of prednisolone phosphate in 8 normal subjects, the mean half-time of prednisolone was 240+/-20 min (mean+/-SE) and the metabolic clearance rate was 82+/-7.2 1/24 h/m2. Of 10 mg of prednisone given orally to 5 normal subjects, 69+/-5% was absorbed and converted to peripheral plasma prednisolone within 8 h. The data indicate that prednisone is extensively metabolized to prednisolone before it clears the splanchnic circulation and the formation prednisone by oxidation of the 11-hydorxyl group of prednisolone appears to occur mainly in peripheral tissues.

Assessment of 11beta-hydroxylase activity with plasma corticosterone, deoxycorticosterone, cortisol, and deoxycortisol: role of ACTH and angiotensin.

UNLABELLED: In this study we evaluated the role of ACTH and angiotensin on regulation of activities of 11beta-hydroxylases of the adrenal cortex. The ratio of the plasma concentrations of 11 deoxycorticosterone (DOC) to plasma corticosterone (B) reflected the activity of the enzyme of the B and/or aldosterone pathways, and the ratio of plasma 11-deoxycortisol (S) to plasma cortisol (F) as the activity of the enzyme in the F pathway. In normal subjects, both ratios were significantly lower at 0800-0900 h (Doc to B, .01+/-.004, mean+/-SE, n=10; and S to F, .01+/-.003) than at 2000 h (DOC to B, .028+/-.024 and S to F, .015+/-.005). The plasma levels of DOC, B, S and F were all significantly lower at 2000-2100 h than at 0800-0900 h. In contrast 9 patients with Cushing's syndrome exhibited no diurnal change in the ratios. The ratios increased substantially following dexamethasone or metyrapone administration. A high or low salt diet and an angiotensin infusion produced no significant effect on the ratios. The plasma concentration of all four steroids was increased by more than 50% by an infusion of angiotensin. Four hours after administration of 80 mg of Lasix at 0800 h to 10 normal subjects, the ratios of DOC to B and S to F increased significantly (P less than .02), an effect possibly related to a decreased secretion of ACTH. CONCLUSIONS: 1) 11beta-hydroxylase activity of the B and/or aldosterone and F pathways appears to change in parallel with ACTH secretion, and 2) although angiotensin stimulates steroidogenesis of the pathways, it has no apparent effect on 11beta-hydroxylase activity.

Hereditary and environmental influences on the variation of thyroid hormones in normal male twins.

Heritability of the variation of the plasma total and unbound T4 (free T4), T3, T4-binding globulin (TBG), and TSH concentrations was investigated in 15 monozygotic and 15 dizygotic male twin pairs. The variability in plasma of total (58%) and free T4 (72%) concentrations was significantly less (P less than 0.01) in the twin pairs than in unrelated men. Half of the variability of T3 (P less than 0.05), TBG (P less than 0.05), and TSH (P less than 0.05) was affected by influences shared by the twin pairs in both monozygotic and dizygotic twin pairs. The heritability index for variability of the plasma total T4 and free T4 was greater than 28% (P less than 0.05), and it was 25% or less for T3 and TBG. Variation in TBG accounted for less than 20% (P less than 0.001) of the variation in thyroid hormone concentrations. The results indicate that familial factors, which are affected by genetic and/or environmental factors, influence the variation of plasma TBG, TSH, T4, free T4, and T3 concentrations among normal men. Genetic factors influenced the variation in plasma T4 and free T4 levels.

Quantitating genetic and nongenetic factors influencing androgen production and clearance rates in men.

Both hereditary and nonhereditary factors have a decided influence on plasma sex steroid concentrations in men. We studied the relative contributions of genetic and nongenetic factors on the production rate (PR) and MCR of testosterone and dihydrotestosterone (DHT) and their conversion ratios to other metabolites in monozygotic (MZ; n = 22) and dizygotic (DZ; n = 24) male twins. Zygosity was determined by measurement of 10 blood proteins and enzymes. The kinetic studies were conducted with isotope dilution techniques. The genetic effect was determined from the equation: 2[rMZ - rDZ], where r is intraclass correlation. A heritability of over 40% was found for the PRs of DHT/body surface area and of testosterone/body surface area. Nongenetic factors accounted for 50% or more of the variation of the conversion ratios for testosterone/3 alpha-androstanediol and DHT/3 alpha-androstanediol. The results suggest that genetic factors markedly influence the PRs of testosterone and DHT, suggesting that the PR of these potent androgens is under genetic control despite the decided influence of environmental factors on their clearance.

Pharmacokinetics and metabolism of a permeation-enhanced testosterone transdermal system in hypogonadal men: influence of application site- -a clinical research center study.

As part of a phase III multicenter study, the pharmacokinetics and metabolism of a permeation-enhanced testosterone (T) transdermal (TTD) system and the influence of application site were investigated in 34 hypogonadal men (21-65 yr of age). After an 8-week androgen washout period, two TTD systems were applied to the back for 24 h. Serum concentrations of total T, bioavailable testosterone (BT), dihydrotestosterone (DHT), and estradiol (E2) increased from hypogonadal levels into the respective normal physiological ranges and declined to baseline levels within 24 h after system removal. Peak concentrations occurred approximately 8 h after application for T and BT and at 13 h for DHT and E2. The baseline-subtracted time-average steady state concentrations (C'ss) for T and BT were 18.1 +/- 7.49 (+/- SD) and 9.08 +/- 3.99 nmol/L, respectively. DHT/T and E2/T ratios, derived from the C'ss values, were 0.063 +/- 0.018 and 0.0033 +/- 0.0018, comparable to the precursor-product conversion ratios reported in healthy men. The estimated half-lives of each hormone were: T, 1.29 +/- 0.71 h; BT, 1.21 +/- 0.75 h; DHT, 2.83 +/- 0.97 h; and E2, 3.53 +/- 1.93 h. The influence of application site was then evaluated by applying two TTD systems for 24 h to the abdomen, back, chest, shin, thigh, or upper arm, according to a sequential cross-over design. Hormone profiles were qualitatively similar at each site, but C'ss values showed significant differences (by ANOVA, P < 0.0001). Based on the BT levels, the rank ordering of the sites were: back > thigh > upper arm > abdomen > chest > shin. DHT/T and E2/T ratios showed negligible site to site variation and were comparable to the results from the initial study. Estimates of T input, based on hormone levels and analysis of the systems used, averaged 4-5 mg/day for the abdomen, back, thigh, and upper arm and were lower and more variable for the chest and shin. Individual C'ss values for T and BT increased linearly with the T input rates (derived from used system analysis) across all studies (n = 235; r = 0.564 for T and r = 0.754 for BT). From these data, T and BT clearance rates were estimated for each patient, averaging 1248 +/- 518 and 2435 +/- 778 L/day, respectively. T clearance rates were proportional to the BT/T ratio (nonsex hormone-binding globulin-bound fraction). On the basis of these studies, the optimal sites of TTD system application were identified as the back, thigh, upper arm, and abdomen

Effects of age and sex hormones on transition and peripheral zone volumes of prostate and benign prostatic hyperplasia in twins.

Benign prostatic hyperplasia has been shown to increase with age and be influenced by sex hormones. The relationship between aging and hormonal influences on growth of zones of the prostate is unresolved. We studied the relationship of age and sex hormones on volume of prostate zones in 214 male twins between 25 and 75 yr old. Volumes of the total prostate (TV), transition zone (TZ), and peripheral zones (PZ) were measured using transrectal ultrasound, and sex steroid concentrations were measured using RIA. Using transformed data corrected for age, TV (r = 0.54, P < 0.00001), TZ (r = 0.58, P < 0.00001), and PZ (r = 0.39, P < 0.00001) volumes increased with age. However, the PZ volume rose more rapidly than the TZ before age 50, and TZ showed a steeper increase after age 50 yr than the PZ volume. The TZ, PZ, and ratio TZ/PZ correlated significantly (r = 0.87, 0.90, and 0.52, respectively; P < 0.00001). After a TV exceeded 30 g, the rise of the PZ became attenuated, and the slope of the TZ became steeper. Age-adjusted sex hormone concentration was not evaluated in men with larger prostate volumes. Men with American Urological Association symptom scores above 10 had significantly (P < 0.001) larger total prostate volume (TV) and TZ volume, but not PZ volumes, than men with scores below 10. Prostate volumes correlated inversely with age-adjusted serum testosterone (T), dihydrotestosterone, sex hormone binding globulin, and sex hormone binding globulin-bound T concentrations. These results demonstrate that before age 50 yr or before a prostate weight exceeds 30 g, prostate growth may be mainly from enlargement of the PZ and after age 50, the TZ. In addition, elevated T and dihydrotestosterone concentrations do not predispose men to prostate enlargement or symptoms of benign prostatic hyperplasia.

"Normal suppression" to dexamethasone in Cushing's disease: an expression of decreased metabolic clearance for dexamethasone.

The adrenal cortical function of a patient with pituitary-dependent Cushing's syndrome exhibited normal responsiveness to conventional doses of dexamethasone (Dex) over several years of evaluation. "Periodic hormonogenesis" did not seem to explain the phenomenon. Plasma concentrations of Dex were measured to ascertain whether an abnormality in Dex metabolism might explain the apparent discrepancy in Dex responsiveness. Plasma levels of Dex after oral administration of the steroid were higher than normal, suggesting that decreased clearance of Dex accounts for the phenomenon of "normal suppression" in this patient with Cushing's syndrome.

Reduction of testosterone synthesis after high dose interleukin-2 therapy of metastatic cancer.

We have investigated the effect of high dose iv bolus interleukin-2 (IL-2) therapy on sex hormone and adrenal steroid concentrations in six men treated for metastatic renal cell carcinoma or malignant melanoma. Blood concentrations of testosterone, 17 beta-estradiol, LH, FSH, cortisol, dehydroepiandrosterone (DHEA), and DHEA sulfate (DHEA-S) were measured before and after a 5-day course of IL-2 therapy. Cortisol levels rose and DHEA-S decreased insignificantly. DHEA declined, reaching a nadir (P less than 0.001) on day 6, and testosterone decreased significantly on day 2 and reached a nadir on day 6 (P less than 0.0001). Concentrations of both steroids then gradually rose. Estradiol rose on day 4 (P less than 0.001) and then declined. Neither LH nor FSH was affected significantly, although there was a rise in the mean level of LH after IL-2 therapy. Our results suggest that high dose IL-2 therapy in men affects both adrenal and testicular androgen production without inhibiting pituitary trophic hormone secretion. These effects of IL-2 on plasma sex steroids may be the result of cytokines stimulated by IL-2 therapy, rather than direct responses to IL-2.

Supranormal nuclear content of 5 alpha-dihydrotestosterone in benign hyperplastic prostate of humans.

We investigated the concentration of nuclear and cytosolic 5 alpha-dihydrotestosterone and 3 alpha-androstanediol in benign prostatic hyperplasia (BPH) in men. The endogenous content of the androgens was quantitated by radioimmunoassay after isolation of the nuclear and cytosolic fractions by classical differential centrifugation techniques. The mean dihydrotestosterone content was about four-fold higher (P < .01) than normal in the nuclear fraction and two-fold higher (P = .05) in the cytosolic fraction in periurethral tissue from hyperplastic prostates. The mean 3 alpha-androstanediol values were approximately one-third of normal in both the nuclear (P < .01) and cytosolic (P < .01) fractions in glands with BPH. In BPH tissue the ratios of dihydrotestosterone to androstanediol content in both the nuclear and cytosolic fractions was significantly higher (P < .01) than the ratio in normal prostate. Our data suggest that elevated intranuclear dihydrotestosterone but not 3 alpha-androstanediol may be causally related to the development of BPH in aging men.

Single dose metyrapone test: 11 beta-hydroxylase inhibition by metyrapone and reduced metyrapone assayed by radioimmunoassay.

To assess the effects of metyrapone and reduced metyrapone on 11 beta-hydroxylase inhibition, the plasma levels of cortisol, 11-deoxycortisol, and the inhibitors were measured by radioimmunoassays in 34 normal subjects 8 h after they received a single oral dose of metyrapone at midnight. The ratio of 11-deoxycortisol to cortisol, as an index of 11 beta-hydroxylase inhibition, was compared to plasma levels of metyrapone and reduced metyrapone. One subject received an infusion of metyrapone ditartrate in order to study the sequential conversion of metyrapone to reduced metyrapone. A new radioimmunoassay was developed for measurement of plasma concentrations of metyrapone and reduced metyrapone. Following intravenous administration of metyrapone, it is rapidly converted to an active metabolite, reduced metyrapone. At 8 h after a dose was given, the average reduced metyrapone level was 1.5 times higher than the average metyrapone level. Following oral administration of the drug, we found a high correlation when plasma levels of metyrapone were compared to reduced metyrapone and when the ratio of 11-deoxycortisol to cortisol was related to metyrapone or to total metyrapone levels. In conclusion, the conversion of metyrapone to reduced metyrapone is such that by 8 hours after a single oral dose, more than one-half of the inhibitory effect on 11 beta-hydroxylase appears to be produced by reduced metyrapone. The inhibitory action of metyrapone and reduced metyrapone on the enzyme system is reflected by their concentration in plasma.