Cooperative activation of bovine herpesvirus 1 productive infection and viral regulatory promoters by androgen receptor and Krüppel-like transcription factors 4 and 15.

Bovine herpesvirus 1 (BoHV-1), a significant viral pathogen, establishes latency in sensory neurons. The viral genome contains more than 100 consensus glucocorticoid receptor (GR) regulatory elements (GREs): consequently, stress stimulates viral replication and reactivation from latency. The immediate early transcription unit 1 (IEtu1) and bICP0 early promoters are transactivated by GR and synthetic corticosteroid dexamethasone. The androgen receptor (AR), like GR, is a Type 1 nuclear hormone receptor that binds and stimulates certain promoters containing GREs. Consequently, we hypothesized AR and 5α-Dihydrotestosterone (DHT) stimulate productive infection and key viral promoters. New studies demonstrated AR, DHT, and Krüppel like transcription factor 4 (KLF4) cooperatively stimulated productive infection and bICP0 E promoter activity in mouse neuroblastoma cells (Neuro-2A). KLF15 also cooperated with AR and DHT to stimulate IEtu1 promoter activity. We suggest AR and testosterone increase the prevalence of virus in semen by stimulating viral gene expression and replication.

Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6ß,10ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11ß,17ß-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of ß-glucuronidase enzyme (IC50 = 42.98 ± 1.24 µM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 µM). Its transformed products 3 (IC50 = 79.09 ± 0.06 µM), and 8 (IC50 = 70.09 ± 0.05 µM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 µM), and its metabolite 8 (IC50 = 34.16 ± 5.3 µM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 µM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 µM) and 4 (IC50 = 152.5 ± 2.15 µM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 µM), and its transformed products 2 (IC50 = 43.3 ± 7.7 µM), 3 (IC50 = 65.6 ± 2.5 µM), and 4 (IC50 = 89.4 ± 2.7 µM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.

Sigma1 Targeting to Suppress Aberrant Androgen Receptor Signaling in Prostate Cancer.

Suppression of androgen receptor (AR) activity in prostate cancer by androgen depletion or direct AR antagonist treatment, although initially effective, leads to incurable castration-resistant prostate cancer (CRPC) via compensatory mechanisms including resurgence of AR and AR splice variant (ARV) signaling. Emerging evidence suggests that Sigma1 (also known as sigma-1 receptor) is a unique chaperone or scaffolding protein that contributes to cellular protein homeostasis. We reported previously that some Sigma1-selective small molecules can be used to pharmacologically modulate protein homeostasis pathways. We hypothesized that these Sigma1-mediated responses could be exploited to suppress AR protein levels and activity. Here we demonstrate that treatment with a small-molecule Sigma1 inhibitor prevented 5α- dihydrotestosterone-mediated nuclear translocation of AR and induced proteasomal degradation of AR and ARV, suppressing the transcriptional activity and protein levels of both full-length and splice-variant AR. Consistent with these data, RNAi knockdown of Sigma1 resulted in decreased AR levels and transcriptional activity. Furthermore, Sigma1 physically associated with ARV7 and ARv567es as well as full-length AR. Treatment of mice xenografted with ARV-driven CRPC tumors with a drug-like small-molecule Sigma1 inhibitor significantly inhibited tumor growth associated with elimination of AR and ARV7 in responsive tumors. Together, our data show that Sigma1 modulators can be used to suppress AR/ARV-driven prostate cancer cells via regulation of pharmacologically responsive Sigma1-AR/ARV interactions, both in vitro and in vivoCancer Res; 77(9); 2439-52. ©2017 AACR.

Regio- and stereoselective reduction of 17-oxosteroids to 17ß-hydroxysteroids by a yeast strain Zygowilliopsis sp. WY7905.

The reduction of 17-oxosteroids to 17ß-hydroxysteroids is one of the important transformations for the preparation of many steroidal drugs and intermediates. The strain Zygowilliopsis sp. WY7905 was found to catalyze the reduction of C-17 carbonyl group of androst-4-ene-3,17-dione (AD) to give testosterone (TS) as the sole product by the constitutive 17ß-hydroxysteroid dehydrogenase (17ß-HSD). The optimal conditions for the reduction were pH 8.0 and 30°C with supplementing 10g/l glucose and 1% Tween 80 (w/v). Under the optimized transformation conditions, 0.75g/l AD was reduced to a single product TS with >90% yield and >99% diastereomeric excess (de) within 24h. This strain also reduced other 17-oxosteroids such as estrone, 3ß-hydroxyandrost-5-en-17-one and norandrostenedione, to give the corresponding 17ß-hydroxysteroids, while the C-3 and C-20 carbonyl groups were intact. The absence of by-products in this microbial 17ß-reduction would facilitate the product purification. As such, the strain might serve as a useful biocatalyst for this important transformation.

Detection of 5α-androst-2-en-17-one and variants: Identification of main urinary metabolites in human urine samples by GC-MS and NMR.

Two steroids were identified in a supplement named D-2 following the detection of unknown compounds during the routine testing of an athlete's sample. The main glucuroconjugated metabolites were isolated from this urine by high performance liquid chromatography (HPLC) following enzymatic hydrolysis and identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses as being 2α-hydroxy-5α-androst-3-en-17-one (M1) and 2ß,3α-dihydroxy-5α-androstan-17-one (M2). A third metabolite, 3α,4ß-dihydroxy-5α-androstan-17-one (M3) was also detected, however in lower amounts. The precursor steroids, 5α-androst-2-en-17-one (1) and 5α-androst-3-en-17-one (2) were present in the first D-2 products offered on the Internet. Later, the corresponding 17-hydroxyl compounds were offered as such or as esters (acetate, cypionate) in different relative ratios. Both M2 and M3 were synthesized from the trans-diaxial hydrolysis of the corresponding 2α,3α- and 3α,4α-epoxides (3). These were excreted in the hours following the controlled administration of the commercial product called D-2 R to a male volunteer and were also produced from the incubation of 1 and 2 with S9 liver fractions. Some preparations contain predominantly the alkene in C-2 and, therefore, an efficient detection method must include both primary metabolites M1 and M2. The latter was found equally in the fractions extracted following the enzymatic hydrolysis with ß-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright © 2016 John Wiley & Sons, Ltd.

Steroid Profiling in Male Wobbler Mouse, a Model of Amyotrophic Lateral Sclerosis.

The Wobbler mouse is an animal model for human motoneuron diseases, especially amyotrophic lateral sclerosis (ALS), used in the investigation of both pathology and therapeutic treatment. ALS is a fatal neurodegenerative disease, characterized by the selective and progressive death of motoneurons, leading to progressive paralysis. Previous limited studies have reported steroidal hormone dysregulation in Wobbler mouse and in ALS patients, suggesting endocrine dysfunctions which may be involved in the pathogenesis of the disease. In this study, we established a steroid profiling in brain, spinal cord, plasma, adrenal glands, and testes in 2-month-old male Wobbler mice and their littermates by gas chromatography coupled to mass spectrometry. Our results show in Wobbler mice the following: 1) a marked up-regulation of corticosterone levels in adrenal glands, plasma, spinal cord regions (cervical, thoracic, lumbar) and brain; 2) a strong decrease in T levels in the testis, plasma, spinal cord, and brain; and 3) increased levels of progesterone and especially of its reduced metabolites 5α-dihydroprogesterone, allopregnanolone, and 20α-dihydroprogesterone in the brain, spinal cord, and adrenal glands. Furthermore, Wobbler mice showed a hypothalamic-pituitary-gonadal hypoactivity. Interestingly, plasma concentrations of corticosterone and T correlate well with their respective levels in cervical spinal cord in both control and Wobbler mice. T down-regulation is probably the consequence of adrenal hyperactivity, and the up-regulation of progesterone and its reduced metabolites may correspond to an endogenous protective mechanism in response to motoneuron degeneration. Our findings suggest that increased levels of corticosterone and decreased levels of T in plasma could be a signature of motoneuron degeneration.

Equine 5α-reductase activity and expression in epididymis.

The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies.

Phthalates Exert Multiple Effects on Leydig Cell Steroidogenesis.

Humans are significantly exposed to phthalates via food packaging, cosmetics and medical devices such as tubings and catheters. Testicular Leydig cells (LCs) are suggested to be among the main targets of phthalate toxicity in the body. However, their sensitivity to phthalates is species-dependent. This paper describes the response of the LCs from different species (mouse, rat and human) to phthalate exposure in different experimental paradigms (in vivo, ex vivo and in vitro), with particular focus on mechanisms of phthalate action on LC steroidogenesis. A comprehensive analysis of the impact of phthalate diesters and phthalate monoesters on LCs in different stages of their development is presented and possible mechanisms of phthalates action are discussed. Finally novel, not yet fully elucidated sites of action of phthalate monoesters on the backdoor pathway of 5α-dihydrotestosterone biosynthesis in immature mouse LCs and their effects on steroidogenesis and redox state in adult mouse LCs are reported.

Innovative Approach to the Accumulation of Rubrosterone by Fermentation of Asparagus filicinus with Fusarium oxysporum.

Rubrosterone, possessing various remarkable bioactivities, is an insect-molting C19-steroid. However, only very small amounts are available for biological tests due to its limited content from plant sources. Fungi of genus Fusarium have been reported to have the ability to convert C27-steroids into C19-steroids. In this study, Asparagus filicinus, containing a high content of 20-hydroxyecdysone, was utilized to accumulate rubrosterone through solid fermentation by Fusarium oxysporum. The results showed that F. oxysporum had the ability to facilitate the complete biotransformation of 20-hydroxyecdysone to rubrosterone by solid-state fermentation. The present method could be an innovative and efficient approach to accumulate rubrosterone with an outstanding conversion ratio.

Comparing older and younger Japanese primiparae: fatigue, depression and biomarkers of stress.

This cohort study of primiparae was conducted to answer the following questions: Do older (≧ 35 years) and younger (20-29 years) Japanese primiparous mothers differ when comparing biomarkers of stress and measures of fatigue and depression? Are there changes in fatigue, depression and stress biomarkers when comparing older and younger mothers during the postpartum period? The Postnatal Accumulated Fatigue Scale and the Edinburgh Postnatal Depression Scale were administered in a time-series method four times: shortly after birth and monthly afterwards. Assays to measure biomarkers of stress, urinary 17-ketosteroids, urinary 17-hydroxycorticosteroids and salivary chromogranin-A, were collected shortly after delivery and at 1 month postpartum in both groups and a third time in older mothers at the 4th month. Statistical testing showed very little difference in fatigue, depression or stress biomarkers between older and younger mothers shortly after birth or 1 month later. Accumulated fatigue and depression scores of older mothers were highest 1 month after delivery. Additional cohort studies are required to characterize physical/psychological well-being of older Japanese primiparae.

Computational determination of binding structures and free energies of glucose 6-phosphate dehydrogenase with novel steroid inhibitors.

Glucose 6-phosphate dehydrogenase (G6PD), the first and the rate-limiting enzyme in the pentose phosphate pathway (PPP), catalyzes the oxidation of G6P to 6-phosphogluconolactone and the reduction of NADP(+) to NADPH. Its key role in cancer promotes the development of a potent and selective inhibitor that might increase cancer cell death when combined with radiotherapy. In the present study, we investigated the detailed binding modes and binding free energies for G6PD interacting with a promising series of recently developed inhibitors, i.e., the steroid derivatives, by performing molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations. The docking indicates that the inhibitors occupy the binding sites of both G6P and NADP(+). The calculated binding free energies on the basis of the MD-simulated enzyme-inhibitor complexes are in good agreement with the experimental activity data for all of the examined inhibitors. The valuable insights into the detailed enzyme-inhibitor binding including the important intermolecular interactions, e.g., the hydrogen bond interaction and the hydrophobic interaction, have been provided. The computational results provide new insights into future rational design of more potent inhibitors of G6PD as a treatment for cancer.

Adrenal Function in females with low plasma HDL-C due to mutations in ABCA1 and LCAT.

INTRODUCTION: Adrenal steroidogenesis is essential for human survival and depends on the availability of the precursor cholesterol. Male subjects with low plasma levels of high density lipoprotein (HDL) cholesterol are characterized by decreased adrenal function. Whether this is also the case in female subjects with low plasma HDL-C levels is unresolved to date. FINDINGS: 15 female ATP binding cassette transporter AI (ABCAI) and 14 female lecithin-cholesterol acyltransferase (LCAT) were included in the study. HDL-C levels were 38% and 41% lower in ABCA1 and LCAT mutation carriers compared to controls, respectively. Urinary steroid excretion of 17-ketogenic steroids or 17-hydroxy corticosteroids did not differ between 15 female ABCA1 mutation carriers (p = 0.27 vs 0.30 respectively) and 30 matched normolipidemic controls or between 14 female LCAT mutation carriers and 28 matched normolipidemic controls (p = 0.10 and 0.14, respectively). Cosyntropin testing in an unselected subgroup of 8 ABCA1 mutation carriers and 3 LCAT mutation carriers did not reveal differences between carriers and controls. CONCLUSION: Adrenal function in females with molecularly defined low HDL-C levels is not different from controls. The discrepancy with the finding of impaired steroidogenesis in males with molecularly defined low HDL-C levels underscores the importance of gender specific analyses in cholesterol-related research.

Rabbit 3-hydroxyhexobarbital dehydrogenase is a NADPH-preferring reductase with broad substrate specificity for ketosteroids, prostaglandin D2, and other endogenous and xenobiotic carbonyl compounds.

3-Hydroxyhexobarbital dehydrogenase (3HBD) catalyzes NAD(P)⁺-linked oxidation of 3-hydroxyhexobarbital into 3-oxohexobarbital. The enzyme has been thought to act as a dehydrogenase for xenobiotic alcohols and some hydroxysteroids, but its physiological function remains unknown. We have purified rabbit 3HBD, isolated its cDNA, and examined its specificity for coenzymes and substrates, reaction directionality and tissue distribution. 3HBD is a member (AKR1C29) of the aldo-keto reductase (AKR) superfamily, and exhibited high preference for NADP(H) over NAD(H) at a physiological pH of 7.4. In the NADPH-linked reduction, 3HBD showed broad substrate specificity for a variety of quinones, ketones and aldehydes, including 3-, 17- and 20-ketosteroids and prostaglandin D2, which were converted to 3α-, 17ß- and 20α-hydroxysteroids and 9α,11ß-prostaglandin F2, respectively. Especially, α-diketones (such as isatin and diacetyl) and lipid peroxidation-derived aldehydes (such as 4-oxo- and 4-hydroxy-2-nonenals) were excellent substrates showing low K(m) values (0.1-5.9 µM). In 3HBD-overexpressed cells, 3-oxohexobarbital and 5ß-androstan-3α-ol-17-one were metabolized into 3-hydroxyhexobarbital and 5ß-androstane-3α,17ß-diol, respectively, but the reverse reactions did not proceed. The overexpression of the enzyme in the cells decreased the cytotoxicity of 4-oxo-2-nonenal. The mRNA for 3HBD was ubiquitously expressed in rabbit tissues. The results suggest that 3HBD is an NADPH-preferring reductase, and plays roles in the metabolisms of steroids, prostaglandin D2, carbohydrates and xenobiotics, as well as a defense system, protecting against reactive carbonyl compounds.

[Effect of chronic sensorineural hearing loss on several indicators of immune and endocrine systems of 7-11 year-old children].

We investigated the effects of chronic bilateral sensorineural hearing loss of III-IV degree on the performance of interleukins, immunoglobulins serum and saliva, the functional activity of granulocyte-monocyte cell immunity, evaluated the activity of the hypothalamic-pituitary-adrenal system in children aged 7-11 years. It was found that due to stress activation of the sympathetic-adrenal system the function of granulocytes and monocytes is suppressed, with a predominance of production of anti-inflammatory interleukins. This leads to the dominance of T-helper type 2. Products granulocytes and T-helper type-2 anti-inflammatory interleukins IL-4, IL-5, IL-10, IL-13 leads to the activation of B-cells. Thus, in children 7-11 years of age with congenital bilateral sensorineural hearing loss is a decrease of non-specific humoral immunity dominated type of immune response to increased levels of IgG.

Rete testis-associated nodular steroid cell nests: description of putative pluripotential testicular hilus steroid cells.

A putative hilus interstitial cell has been proposed as the cell of origin for testicular tumors of adrenogenital syndrome, but its normal histology is not documented. We present hitherto undescribed nodular steroid cell nests associated with the rete testis that are distinctive in their morphology and immunohistochemical profile from Leydig cells and do not have the morphology of typical extra-adrenal cortical rests. These nodules measured 1, 1, 1.8, 2, and 2.5 mm in size with a distinct sinusoidal vasculature. Individual cells were rounded to polygonal with evenly distributed moderate-to-abundant eosinophilic cytoplasm. The nuclei were homogenous and round, with fine chromatin and ocasionally with prominent nucleoli. The differential diagnosis included adrenocortical rests, testicular adnexal Leydig cells, carcinoid tumorlets, paraganglionic rests, and adenomatoid mesothelial proliferation. Immunohistochemistry showed positivity for melan A (5/5), inhibin (3/5), and calretinin (2/4), although the immunoreactivity was distinctively different from the concurrent intratesticular Leydig cells and testicular adnexal Leydig cells in all cases. The unique morphology, immunophenotype, and distinctive location of these cells in the testicular mediastinum raises the possibility that these cells represent testicular hilus steroid cells, the putative histogenetic cell implicated for testicular tumors of adrenogenital syndrome. We propose to name these proliferations rete testis-associated nodular steroid cell nests.

17beta-Hydroxysteroid dehydrogenase inhibitors: a patent review.

IMPORTANCE OF THE FIELD: 17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) mainly catalyze the reduction of C17-ketosteroids to their corresponding hydroxylated forms as well as the reverse reaction (oxidation). Able to convert inactive or less active steroid hormones into more potent ones and vice versa, certain 17beta-HSDs play a key role, especially in the regulation of estrogen and androgen levels. The therapeutic potential of this enzyme family, especially for the treatment of breast cancer, prostate cancer, acne and osteoporosis, then stimulated the development of inhibitors of 17beta-HSDs and important progress was achieved over the last years. AREAS COVERED IN THIS REVIEW: This review article reports all patent applications related to the inhibitors of 17beta-HSDs, including some articles needed to complement the information presented. WHAT THE READER WILL GAIN: Readers will be informed about the role and function of 17beta-HSDs in the first section and about the history of inhibitor development in the second section. Furthermore, in the third and main section, the readers will learn about the structures of patented inhibitors originating from different companies and academic groups. TAKE HOME MESSAGE: The increase in the number of 17beta-HSD inhibitors reported in the last years augurs well for the future. The challenge is now to translate these results into clinical studies to allow determination of the therapeutic usefulness of 17beta-HSD inhibitors.

Identification of early response genes and pathway activated by androgens in the initial segment and caput regions of the regressed rat epididymis.

To identify the initial response to androgens and estrogens in the orchidectomized, regressed epididymis, we determined the gene expression changes triggered by the administration of either of two metabolites of testosterone, 5alpha-dihydrotestosterone (DHT) or 17beta-estradiol (E2), in the regressed rat epididymis. Adult rats were orchidectomized and 8 d later implanted with either empty implants (control), DHT-filled-, or E2-filled-polydioxanone implants. Rats were euthanized 12 h, 1 d, and 7 d later, and RNA was extracted and probed on Rat230-2.0 Affymetrix arrays. Probe sets that respond to DHT or E2 were identified at early time points; although the expression of some was repressed, the expression of many others was either transiently or chronically elevated. Nerve growth factor receptor (Ngfr) and S100 calcium binding protein G (S100g) were two E2 up-regulated genes detected at 12 h. Among the genes that showed a dramatic early response to DHT were endothelin 1 (Edn1), bone morphogenetic protein 4 (Bmp4), and IGF binding protein 3 (Igfbp3), which were suppressed, and IGF-I (Igf1), which was induced. Genes that were up- or down-regulated by DHT were classified based on biological function. Using PathwayStudio 4.0, we identified genes that were linked and directly influenced either the expression or regulation of one another. Epidermal growth factor and IGF-I play an important role in the pathway due to their function in regulation and expression of many other genes. These results provide novel insights into the impact of androgen action on the expression of genes that are important for epididymal function.

Monitoring of therapy in congenital adrenal hyperplasia.

BACKGROUND: Congenital adrenal hyperplasia is a group of disorders caused by defects in the adrenal steroidogenic pathways. In its most common form, 21-hydroxylase deficiency, patients develop varying degrees of glucocorticoid and mineralocorticoid deficiency as well as androgen excess. Therapy is guided by monitoring clinical parameters as well as adrenal hormone and metabolite concentrations. CONTENT: We review the evidence for clinical and biochemical parameters used in monitoring therapy for congenital adrenal hyperplasia. We discuss the utility of 24-h urine collections for pregnanetriol and 17-ketosteroids as well as serum measurements of 17-hydroxyprogesterone, androstenedione, and testosterone. In addition, we examine the added value of daily hormonal profiles obtained from salivary or blood-spot samples and discuss the limitations of the various assays. SUMMARY: Clinical parameters such as growth velocity and bone age remain the gold standard for monitoring the adequacy of therapy in congenital adrenal hyperplasia. The use of 24-h urine collections for pregnanetriol and 17-ketosteroid may offer an integrated view of adrenal hormone production but target concentrations must be better defined. Random serum hormone measurements are of little value and fluctuate with time of day and timing relative to glucocorticoid administration. Assays of daily hormonal profiles from saliva or blood spots offer a more detailed assessment of therapeutic control, although salivary assays have variable quality.