Plasma 17-hydroxyprogesterone Research Articles

Indicaciones e interpretación de los estudios analíticos hormonales de la corteza suprarrenal

In the adrenal cortex, entities ranging from benign diseases such as adrenocortical adenomas to malignant diseases such as adrenocortical carcinoma or adrenal metastases can be found. Regarding functionality, the spectrum of diseases varies widely: from non-functioning adenomas, patients with autonomous cortisol secretion (ACS), primary hyperaldosteronism (PHA), ACTH-independent Cushing's syndrome, excess secretion of sex hormones, congenital adrenal hyperplasia (CAH), and adrenal insufficiency (AI). A hormonal study is essential for the proper characterization of these disorders. Baseline hormonal tests and functional studies are both necessary in some cases to reach the definitive diagnosis. The test of choice for Cushing's syndrome screening is urinary free cortisol, late-night salivary cortisol, or a 1mg dexamethasone suppression test (DST). For ACS, it is a DST. For PAH, it is calculation of the plasma aldosterone concentration and renin activity or concentration ratio. For AI, it is the determination of plasma cortisol between 8:00 and 9:00 a.m. and after cosyntropin stimulation. Lastly, for CAH, it is the measurement of plasma 17-hydroxyprogesterone.

Relationship between plasma concentration of 17-hydroxyprogesterone caproate and gestational age at preterm delivery

The effectiveness of 17-hydroxyprogesterone caproate is unclear as trials have provided conflicting results. With the absence of fundamental pharmacologic studies addressing dosing or the relationship between drug concentration and gestational age at delivery, the effectiveness of the medication cannot be evaluated. This study aimed to evaluate the relationship between plasma concentrations of 17-hydroxyprogesterone caproate and preterm birth rates and gestational age at preterm delivery and to assess the safety of the 500-mg dose. This study recruited 2 cohorts with previous spontaneous preterm birth; 1 cohort (n=143) was randomly assigned to either 250-mg or 500-mg 17-hydroxyprogesterone caproate, and the other cohort (n=16) was receiving the 250-mg dose for routine care. Steady-state trough plasma concentrations of 17-hydroxyprogesterone caproate obtained at 26 to 30 weeks of gestation were correlated to dose, spontaneous preterm birth rates, and measures of gestational length. Furthermore, maternal and neonatal safety outcomes were evaluated according to dose. There was a dose proportional increase in trough plasma concentrations with the 250-mg (median, 8.6 ng/m; n=66) and 500-mg (median, 16.2 ng/mL; n=55) doses. In 116 compliant participants with blood samples, drug concentration was not related to the spontaneous preterm birth rate (odds ratio, 1.00; 95% confidence interval, 0.93-1.08). However, there was a significant relationship between drug concentration and both the interval from the first administration to delivery (interval A: coefficient, 1.11; 95% confidence interval, 0.00-2.23; P=.05) and the interval from the 26- to 30-week blood draw to delivery (interval B: coefficient, 1.56; 95% confidence interval, 0.25-2.87; P=.02). The spontaneous preterm birth rate or measures of gestational length were not related to dose. Postenrollment cerclage adversely affected all pharmacodynamic assessments because it was a powerful predictor of spontaneous preterm birth (odds ratio, 4.03; 95% confidence interval, 1.24-13.19; P=.021) and both measures of gestational length (interval A [coefficient, -14.9; 95% confidence interval, -26.3 to -3.4; P=.011] and interval B [coefficient, -15.9; 95% confidence interval, -25.8 to -5.9; P=.002]). Initial cervical length was significantly related to the risk of postenrollment cerclage (odds ratio, 0.80; 95% confidence interval, 0.70-0.92; P=.001). Maternal and neonatal safety outcomes were similar in both dosing groups. In this pharmacodynamic study, trough plasma 17-hydroxyprogesterone caproate concentrations were significantly associated with gestational age at preterm birth but not with the preterm birth rate. Postenrollment cerclage was a powerful predictor of spontaneous preterm birth rate and gestational length. Initial cervical length predicted the risk of postenrollment cerclage. Adverse events were similar with the 500-mg and 250-mg doses of 17-hydroxyprogesterone caproate.

Urinary steroidomic profiles by LC-MS/MS to monitor classic 21-Hydroxylase deficiency

21-hydroxylase deficiency, the most common enzyme defect associated with congenital adrenal hyperplasia (CAH) is characterized by an impairment of both aldosterone and cortisol biosynthesis. Close clinical and biological monitoring of Hydrocortisone (HC) and 9α-Fludrocortisone (FDR) replacement therapies is required to achieve an optimal treatment. As frequent and repeated reassessments of plasma steroids, 17-hydroxyprogesterone (17-OHP), androstenedione (Δ4-A) and testosterone (TESTO) is needed in childhood, urine steroid profiling could represent an interesting non-invasive alternative.We developed and validated a LC-MS/MS method for the measurement of 23-urinary mineralocorticoids, glucocorticoids and adrenal androgens. The usefulness of steroid profiling was investigated on single 08h00 am-collected spot urine for discriminating between 61 CAH patients and their age- and sex-matched controls. CAH patients were split into two groups according to their 08h00 am-plasma concentrations of 17-OHP: below (controlled patients, n = 26) and above 20 ng/mL (uncontrolled patients, n = 35).The lower limit of quantification and the wide analytical range allows to assay both free and total concentrations of the main urinary adreno-corticoids and their tetra-hydrometabolites. Extraction recoveries higher than 75% and intra-assay precision below 20% were found for most steroids.Urinary steroids upstream of the 21-hydroxylase defect were higher in uncontrolled CAH patients. Among CAH patients, plasma and urinary 17-OHP were closely correlated. As compared to controls, steroids downstream of the enzyme defect collapsed in CAH patients. This fall was more pronounced in controlled than in uncontrolled patients. Androgens (Δ4-A, TESTO and the sum etiocholanolone + androsterone) accumulated in uncontrolled CAH patients. A strong relationship was observed between plasma and urinary levels of androstenedione. Daily doses and urinary excretion of both FDR and HC were similar in both CAH groups. Urinary FDR was inversely related to the sodium-to-potassium ratio in urine. A partial least squares discriminant analysis model allowed to classify the patient’s classes unaffected, controlled and un-controlled CAH patients based on urinary steroidomic profiles.Our LC-MS/MS method successfully established steroid profiling in urine and represents a useful and non-invasive tool for discriminating CAH patients according to treatment efficiency.

The High Prevalence of Testicular Adrenal Rest Tumors in Adult Men With Congenital Adrenal Hyperplasia Is Correlated With ACTH Levels.

Introduction: The aims of this study were to determine the prevalence of testicular-adrenal rest tumors (T-ARTs) in patients with congenital adrenal hyperplasia (CAH) and to evaluate the related ultrasound (US) features, hormonal profiles, and semen parameters. Therefore, we attempted to understand the potential impact of adrenocorticotropic hormone (ACTH) on the persistence or disappearance of T-ART.Methods: We conducted a longitudinal cohort study including patients with CAH who were undergoing treatment with cortisone and, when indicated, fludrocortisone replacement therapy. We performed andrological examinations, US of the testis, hormone profiling, and semen analysis.Results: Of the 25 patients (mean ± SD age, 32.2 ± 7.5 years), T-ARTs were detected by US in 14 (56.0%) patients. The mean ± SD diameter of the lesions was 13.2 ± 6.8 mm. Among 3 (21.4%) patients, the lesions were observed in one testis, whereas both testes were affected in the remaining 11 (78.6%) patients. The lesions were hypoechoic in 12 (85.7%) patients and hyperechoic in 2 (14.3%). Plasma ACTH and 17-hydroxyprogesterone (17-OHP) levels were significantly higher in patients with T-ART than in patients without lesions (319.4 ± 307.0 pg/ml and 12.4 ± 2.7 ng/ml vs. 33.5 ± 10.7 pg/ml and 8.2 ± 1.8 ng/ml, respectively; p < 0.01). The mean values of sperm concentration and motility were significantly lower in patients with T-ART than in patients without lesions (12.1 ± 12.4 × 106 cells/ml and 18.4 ± 11.1% vs. 41.5 ± 23.2 × 106 cells/ml and 30.8 ± 15.4%, respectively; p < 0.05). Logistic regression analysis showed ACTH level as a significant predictor of T-ART (p < 0.05). In patients with T-ART, the dose of hydrocortisone was increased by ~25–30%, while the fludrocortisone treatment remained unchanged. After 6 months of steroid treatment, patients underwent US and hormonal evaluation. Disappearance and a reduction in T-ART were observed in 6 (42.9%) and 5 (35.7%) patients, respectively; a reduction in ACTH levels (from 319.4 ± 307.0 to 48.1 ± 5.1 pg/ml; p < 0.01) was reported. A significant correlation between ACTH level reduction and T-ART diameter reduction was observed (p < 0.5; r = 0.55).Conclusions: T-ARTs were detected in 56% of patients with CAH and were associated with impaired semen parameters. However, these lesions are potentially reversible, as demonstrated by the disappearance/reduction after adjustment of cortisone therapy and by the reduction in plasma ACTH level. Our study supports the importance of periodic US evaluation and maintenance of plasma ACTH levels within the normal range in men with CAH.

SAT-011 Quantification of Testosterone, Androstenedione and 17-Hydroxyprogesterone Collected Using Mitra®Micro Sampling Devices

Introduction Measurement of total plasma testosterone (T), androstenedione (A4) and 17-hydroxyprogesterone (17OHP) typically requires a venous sample. A highly sensitive and specific assay was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after extraction from a Mitra® volumetric absorptive microsampling (VAMs) device. VAMs devices are small absorptive polymer tips which absorb a fixed amount of blood (1); they can be dried after collection of finger prick blood and stored prior to analysis. Differences in binding in whole blood compared to serum require the quantification of haematocrit using a published protocol (2), thus allowing an estimated serum result to be generated. Method Pre-mixed calibrator/sample (10 µL) was combined with deionised water and mixed internal standard, after vortexing MTBE was added and mixed by inversion for 1 minute. The supernatant was transferred to a clean 2 mL deep-well plate, dried down and re-constituted with 100 µL 50:50 methanol/water. Mitra® tips were prepared from anonymised whole blood samples in the laboratory and compared to the corresponding plasma using the same method. Results Mean recovery was 86% for A4, 102% for T and 97% for 17OHP, the lower limit of quantification was 1 nmol/L for A4, 1 nmol/L for testosterone and 4 nmol/L for 17OHP. Ion suppression was within acceptable criteria for all the analytes measured. For the samples prepared in the laboratory, comparisons for T, A4 and 17OHP the R-squared values were 0.95, 0.93 and 0.95 respectively with a minimal bias. Summary We have developed a robust assay for LC-MS/MS measurement of VAMs for T, A4, and 17OHP in a routine clinical laboratory. It is simple, reproducible and easy to perform and may have implications for monitoring of T therapy in hypogonadal males or screening/monitoring of patients with congenital adrenal hyperplasia. In-house comparisons of T, A4 and 17OHP compared well when haematocrit was used to adjust for the space occupying effect of red cells.

Performance enhancement in the measurement of 5 endogenous steroids by LC–MS/MS combined with differential ion mobility spectrometry

BackgroundChallenges for steroid analysis by LC–MS/MS include low ionization efficiency, endogenous isobars with similar fragmentation patterns and chromatographic retention. Differential ion mobility spectrometry (DMS) provides an additional degree of separation prior to MS/MS detection, and shows promise in improving specificity of analysis. We developed a sensitive and specific method for measurement of corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone and progesterone in human serum and plasma using an ABSciex 5500 mass spectrometer equipped with a differential ion mobility interface. Methods250μL aliquots of serum were spiked with deuterated internal standards and extracted with MTBE. The samples were analyzed using positive mode electrospray LC–DMS–MS/MS. The method was validated and compared with immunoassays and LC–MS/MS methods of reference laboratories. ResultsInter and intra assay imprecision was <10%. Limits of quantification and detection in nmol/L were 0.18, 0.09 for corticosterone and 17-hydroxyprogesterone, 0.30, 0.16 for 11-deoxycortisol, 0.12, 0.06 for progesterone and 0.06, 0.03 for 11-deoxycorticosterone. Comparison for progesterone and 17-hydroxyprogesterone with immunoassay showed slopes of 0.97 and 1.0, intercepts of 0.16 and 0.10 and coefficients of determination (r2) of 0.92 and 0.97, respectively. Progesterone by immunoassay showed positive bias in samples measuring <3.18nmol/L. Reference intervals for progesterone and 11-deoxycorticosterone in post-menopausal women were found to be <2.88 and <0.28nmol/L respectively. ConclusionsWe developed and validated an LC–DMS–MS/MS method for analysis of five endogenous steroids suitable for routine measurements in clinical diagnostic laboratories. Specificity gained with DMS allows reducing the complexity of sample preparation, decreasing LC run times and increasing speed of the analysis.

Determinants of Adrenal Androgen Hypofunction in Premenopausal Females With Rheumatoid Arthritis

The aim of our study was to investigate adrenocortical function in the context of disease activity and inflammatory status in premenopausal RA females. Adrenal glucocorticoid and androgen responses to the 1 microg ACTH 1-24 test were investigated in 23 premenopausal RA and in 15 age- and BMI-matched healthy females. Twelve RA patients were on low-dose prednisone (<8.5 mg/day). Patients with DAS28>3.2 had lower (p<0.05) total plasma cortisol, 17-hydroxyprogesterone, dehydroepiandrosterone and androstenedione responses in the ACTH test compared to healthy controls. Patients with DAS28>3.2 had lower (p<0.05) dehydroepiandrosterone response in the ACTH test compared to patients with DAS28</=3.2. C-reactive protein (CRP), DAS28, and interleukin (IL)-6 negatively correlated with androstenedione response to ACTH 1-24. Responses of all measured adrenal steroids were lower (p<0.05) in patients on low-dose glucocorticoids compared to healthy controls. RA patients not treated with glucocorticoids had lower total cortisol response (p=0.038) but did not differ in free plasma cortisol in the ACTH test. The results indicate an association of increased disease activity with a decrease in adrenal androgen production in RA and normal cortisol bioavailability in patients not treated with glucocorticoids.

Adrenal Enzyme Impairment in Neonates and Adolescents Treated with Ritonavir and Protease Inhibitors for HIV Exposure or Infection

Background: Human deficiency virus (HIV) protease inhibitors (PIs) are widely used drugs whose effects are pharmacologically enhanced by ritonavir, a potent cytochrome P450 inhibitor. We reported previously that prophylactic postnatal ritonavir-PI therapy in HIV-exposed neonates was associated with increases in plasma 17-hydroxyprogesterone (17-OHP) and dehydroepiandrosterone sulfate (DHEA-S). Aims: To further investigate adrenal function in neonates and adolescents given ritonavir-PI. Methods: Adrenal function was assessed prospectively in 3 HIV-exposed neonates given short-term prophylactic treatment and 3 HIV-infected adolescents given long-term treatment. Plasma cortisol, 17-OHP, 17-OH-pregnenolone, DHEA-S, and androstenedione were measured before and after ACTH administration. Results: None of the patients had clinical signs of adrenal dysfunction. The only neonate exposed to ritonavir-PI in utero had up to 3-fold increases in plasma 17-OHP. Increases in 17-OH-pregnenolone of up to 3.1-fold were noted in 4 of the 6 patients, and all 6 patients had elevations in DHEA-S (up to 20.4-fold increase) and/or DHEA (up to 4.7-fold) and/or androstenedione (up to 5.2-fold). All these parameters improved after treatment completion. Conclusion: Neonates and adolescents given ritonavir-PI exhibit a similar adrenal dysfunction profile consistent with an impact on multiple adrenal enzymes. These abnormalities require evaluation, given the potentially long exposure times.

Excessive Ovarian Production of Nerve Growth Factor Facilitates Development of Cystic Ovarian Morphology in Mice and Is a Feature of Polycystic Ovarian Syndrome in Humans

Although ovarian nerve growth factor (NGF) facilitates follicular development and ovulation, an excess of the neurotrophin in the rodent ovary reduces ovulatory capacity and causes development of precystic follicles. Here we show that ovarian NGF production is enhanced in patients with polycystic ovarian syndrome (PCOS) and that transgenically driven overproduction of NGF targeted to the ovary results in cystic morphology, when accompanied by elevated LH levels. NGF levels are increased in the follicular fluid from PCOS ovaries and in the culture medium of granulosa cells from PCOS patients, as compared with non-PCOS patients. Ovaries from transgenic mice carrying the NGF gene targeted to thecal-interstitial cells by the 17alpha-hydroxylase gene promoter produce more NGF than wild-type (WT) ovaries and are hyperinnervated by sympathetic nerves. Antral follicle growth is arrested resulting in accumulation of intermediate size follicles, many of which are apoptotic. Peripubertal transgenic mice respond to a gonadotropin challenge with a greater increase in plasma 17-hydroxyprogesterone, estradiol, and testosterone levels than WT controls. Transgenic mice also exhibit a reduced ovulatory response, delayed puberty, and reduced fertility, as assessed by a prolonged interval between litters, and a reduced number of pups per litter. Sustained, but mild, elevation of plasma LH levels results in a heightened incidence of ovarian follicular cysts in transgenic mice as compared with WT controls. These results suggest that overproduction of ovarian NGF is a component of polycystic ovarian morphology in both humans and rodents and that a persistent elevation in plasma LH levels is required for the morphological abnormalities to appear.

Familial Glucocorticoid Deficiency with a Point Mutation in the ACTH Receptor: A Case Report

Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder characterized by severe glucocorticoid deficiency associated with failure of adrenal responsiveness to ACTH but no mineralocorticoid deficiency. We report a 2 month-old boy of nonconsanguineous parents, presented with hyperpigmentation. Physical examination showed diffuse dark skin of body including, oral mucosa, gum, hands, nails and scrotum. Laboratory evaluation revealed low serum cortisol (0.3 µg/dL), with very high plasma ACTH level (18,000 pg/mL), and serum cortisol level did not increase after ACTH stimulation test. Serum sodium, potassium, plasma renin activity, aldosterone and 17-hydroxyprogesterone were normal. Sequence analysis of the ACTH receptor (MC2R) gene showed a homozygous mutation of D103N. Diagnosis of FGD was made and treatment started with oral hydrocortisone.

Lack of ACTH and Androgen Receptor Expression in a Giant Adrenal Myelolipoma Associated with 21-hydroxylase Deficiency

Myelolipomas of the adrenal gland are benign, nonfunctioning tumors. Patients with congenital adrenal hyperplasia sometimes develop large and bilateral myelolipomas. Although the precise pathogenesis of myelolipomas remains unclear, prolonged stimulation with high levels of adrenocorticotropic hormone (ACTH) or adrenal androgens are assumed to have a causative role. To clarify the role of ACTH and androgen in the pathogenesis of myelolipoma, we report a case of giant adrenal myelolipoma in a patient with poorly controlled congenital adrenal hyperplasia. A 43-year-old female was diagnosed with congenital adrenal hyperplasia at 6 years of age because of ambiguous genitalia. She had high plasma ACTH and 17-hydroxyprogesterone levels. Abdominal computed tomography showed a huge mass on the left adrenal gland, and an enlarged right adrenal mass. Genetic testing for CYP21A2 was performed and revealed that her genotype was IVS2-13A/C>G/I172N. Adrenalectomy for the left-side tumor was performed. Histological study revealed that the tumor consisted of fat cells and myeloid components, findings compatible with adrenal myelolipoma. Neither ACTH receptors nor androgen receptor was over-expressed in the tumor. Our finding that the tumor did not over-express ACTH or androgen receptor suggests a limited direct role for these hormones in the development of the myelolipoma.

Congenital adrenal hyperplasia due to 21 hydroxylase deficiency: Case report

A girl with congenital adrenal hyperplasia due to 21 hydroxylase (CYP 21, p450c21) deficiency is the reviewed case. The clinical features (virilisation, clitoromegaly, rapid somatic growth, accelerated skeletal maturation) and laboratory findings (high levels of plasma 17hydroxy-progesterone, corticotrophin--ACTH, testosterone and dehydroepiandrostenedione--DHEA, low level of plasma cortisol, high level of urine 17-ketosteroids, synacthen and luteinising hormone releasing hormone--LHRH test) and the response to hydrocortisone therapy pointed at heterosexual gonadotrophin independent puberty due to irregular production of cortisol caused by 21 hydroxylase deficiency that leads to elevated ACTH and 17-hydroxy progesterone secretion and makes congenital adrenal hyperplasia as entity. The six-month therapy resulted in the clinical and laboratory findings improvement, such as the decreased annual growth of body height and the stagnation in the development of the secondary sexual features.

Increased plasma 17-hydroxyprogesterone and milt production in response to gonadotropin-releasing hormone agonist in captive male starry flounder, Platichthys stellatus

In captivity, reproduction of male fish is often dysfunctional, resulting in abnormally viscous milt. Treatment with exogenous gonadotropin or gonadotropin-releasing hormone agonist (GnRHa) has been found useful in overcoming this reproductive failure in some fish species. In this study, captive male starry flounder ( Platichthys stellatus) were treated with GnRHa at three different dosages (50, 100 or 200 μg kg −1 fish body weight) by implantation with cholesterol pellets during their natural spawning season. The GnRHa treatment increased the levels of plasma testosterone (T) at the early spawning season and plasma 17-hydroxyprogesterone (17P4) throughout the spawning season. Both milt volume and sperm count were increased in a dose-dependent manner by GnRHa treatment, but the increase of milt volume was twice as rapid as the increase of sperm count. The treatment decreased sperm concentration. The increased milt volume was strongly correlated with the level of plasma 17P4 ( r=0.690), but not with T ( r=0.250). The level of 17P4 was also associated with sperm motility. The sperm obtained both from control and GnRHa-treated groups was used to fertilize starry flounder eggs, and the resultant embryos were grown until metamorphosis. No negative effects of the GnRHa treatment on fertilization, hatching and larval growth were found. The results suggest that GnRHa treatment can render captive male flounders to recover their ability to hydrate milt.

Hormonal evaluation and mutation screening for steroid 21-hydroxylase deficiency in patients with unilateral and bilateral adrenal incidentalomas.

The aims of the present study were (a) to examine the occurrence of 21-hydroxylase gene (CYP21) mutations in patients with unilateral and bilateral adrenal incidentalomas and (b) to correlate the results of mutation screening with hormonal parameters of 21-hydroxylase deficiency. The frequency of the eight commonly occurring CYP21 mutations in blood DNA samples of 19 patients with bilateral, as well as in blood and tumoral tissue DNA samples of 31 patients with unilateral adrenal incidentalomas, was determined. In all patients, hormonal evaluation for 21-hydroxylase deficiency was performed using measurements of basal and ACTH-stimulated plasma 17-hydroxyprogesterone (17-OHP) concentrations. Blood and tumoral DNA samples were analyzed by allele-specific PCR for the detection of the eight commonly occurring CYP21 mutations (deletion/large gene conversion, intron 2 splicing, Ile172Asn, exon 6 cluster, Val281Leu, Leu307insT, Gln318Stop and Arg356Trp mutations). Plasma 17-OHP concentrations were measured by radioimmunoassay. Of the 19 patients with bilateral adrenal incidentalomas, one patient had homozygous (Val281Leu) and three patients had heterozygous germline CYP21 mutations (Val281Leu in two cases and Arg356Trp in one case). Heterozygous germline CYP21 mutations were also detected in five of the 31 patients with unilateral adrenal incidentalomas (Ile172Asn in three cases and Val281Leu in two cases). Mutation screening of tumoral DNA in unilateral incidentalomas showed the presence of corresponding germline mutations but no additional somatic mutations were found. ACTH-stimulated plasma 17-OHP concentrations were above 1500 ng/dl in all patients with bilateral incidentalomas who had homozygous and heterozygous CYP21 mutations, but heterozygous carriers with unilateral incidentalomas had highly variable ACTH-stimulated plasma 17-OHP levels (between 111 and 1705 ng/dl). These results suggest a similar frequency of germline CYP21 mutations in patients with bilateral and unilateral adrenal incidentalomas (21.1% and 16.1% respectively). Therefore, it cannot be ruled out that, in at least some patients, CYP21 mutations may play a role in the pathomechanism of bilateral and unilateral adrenal incidentalomas. However, the lack of clear association of CYP21 mutations with increased ACTH-stimulated plasma l7-OHP response, especially in patients with unilateral incidentalomas, suggests that the effect of CYP21 mutations on adrenocortical tumor formation may also involve mechanism(s) independent of ACTH-induced changes in 17-OHP secretion.

Comparative analysis of plasma 17-hydroxyprogesterone and cortisol responses to ACTH in patients with various adrenal tumors before and after unilateral adrenalectomy

Patients with non-hyperfunctioning adrenal adenomas often have an increased plasma 17-hydroxyprogesterone response to ACTH stimulation. The effects of adrenal surgery on this abnormality have rarely been investigated. One hundred and sixty-one patients with unilateral adrenal tumors (non-hyperfunctioning adenomas, 78; cortisol-producing adenomas, 8; aldosterone-producing adenomas, 37; adrenal cysts, 12; pheochromocytomas, 26) were studied. Patients before and after adrenal surgery as well as 60 healthy subjects underwent an ACTH stimulation test using 2 mg synthetic ACTH(1-24) (Cortrosyn Depot, Organon). Basal and ACTH-stimulated plasma 17-hydroxyprogesterone and cortisol concentrations are reported. Before adrenal surgery, the basal plasma 17-hydroxyprogesterone concentrations were normal in patients with all types of tumors. However, the ACTH-stimulated plasma 17-hydroxyprogesterone levels were abnormally increased in 53% and 31% of patients with non-hyperfunctioning adenomas and aldosterone-producing adenomas, respectively. In addition, a few patients with adrenal cysts and pheochromocytomas also showed an increased ACTH-stimulated 17-hydroxyprogesterone response. After unilateral adrenalectomy, this hormonal abnormality disappeared in most, although not all patients with adrenal tumors. In patients with non-hyperfunctioning adrenal tumors, ACTH-stimulated plasma 17-hydroxyprogesterone and cortisol concentrations significantly correlated with the size of the tumors. These results firmly indicate that the tumoral mass itself may be responsible for the increased plasma 17-hydroxyprogesterone and cortisol responses after ACTH stimulation in patients with non-hyperfunctioning and hyperfunctioning adrenal adenomas.

Determination of 17-Hydroxyprogesterone in Plasma by Stable Isotope Dilution/Benchtop Liquid Chromatography-Tandem Mass Spectrometry

An assay based on stable isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS-MS) was developed for the quantification of 17-hydroxyprogesterone, the most important indicator of 21-hydroxylase deficiency in human plasma. Plasma was extracted using ethyl acetate and Extrelut^® columns. LC was performed on a reversed-phase C18 column using a water/methanol gradient. A benchtop triple quadrupole mass spectrometer, operating in selected reaction monitoring mode, served as mass detector. The analytical run time was 9 min per sample. The sensitivity was high: 0.06 pmol of 17-hydroxyprogesterone yielded a signal-to-noise ratio of 13. Precision (CV) and accuracy (relative error) derived from the analyses of unspiked and spiked validation samples were 7.4–12.0% and 6.4%, respectively. When analyzing the same samples – median (range), in nanomoles per liter – from neonates and adults independently by ID/LC-MS-MS as well as by ID/gas chromatography (GC)-MS, corresponding results were obtained: neonates (n = 10), ID/LC-MS-MS 3.99 (0.48–16.05), ID/GC-MS 5.39 (1.57–13.02); adults (n = 10), ID/LC-MS-MS 2.66 (1.39–6.15), ID/GC-MS 2.54 (0.51–5.12). The technique permitted reliable detection of classical and nonclassical forms of 21- hydroxylase deficiency. The much simpler sample preparation, the faster analytical run time and the operational ease possible with ID/LC-MS-MS permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity. We expect that benchtop tandem mass spectrometry will open new avenues in clinical steroid analysis.

Effect of carbenoxolone on the plasma renin activity and hypothalamic-pituitary-adrenal axis in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

To test the hypothesis that carbenoxolone, an inhibitor of 11beta-hydroxysteroid dehydrogenase, might augment the ACTH-suppressing and mineralocorticoid activities of hydrocortisone without a corresponding increase in peripheral hydrocortisone effects, we assessed the effects of carbenoxolone in patients with congenital adrenal hyperplasia. Six patients with classic 21-hydroxylase deficiency (5 salt-losing, 1 nonsalt-losing) were enrolled in this study. The study protocol involved 3 treatment periods (except for patient 3): phase 1, hydrocortisone and fludrocortisone; phase 2, hydrocortisone, fludrocortisone and carbenoxolone; phase 3, hydrocortisone and carbenoxolone. Patient 3 was not treated with fludrocortisone at baseline, so she participated only in phase 1 (hydrocortisone only) and phase 2 (hydrocortisone and carbenoxolone). Hydrocortisone and fludrocortisone dosages were kept the same during the study except for the discontinuation of fludrocortisone during phase 3. Plasma adrenal androgens or their precursors (androstenedione, 17-hydroxyprogesterone, and testosterone, and urine pregnanetriol); plasma cortisol, cortisol-binding globulin, ACTH, apparent cortisol metabolic clearance, 24-h urine 17-hydroxysteroids, and urine free cortisol; mineralocorticoid activity, as measured by plasma renin activity, body weight, plasma potassium, and mean blood pressure; fasting insulin/glucose ratio, protein balance, % eosinophils in peripheral blood, and total urine pyridinoline and deoxypyridinoline; TRH stimulation of TSH and pyridostigmine/GHRH stimulation of growth hormone. Compared to phase 1, the addition of carbenoxolone (with or without concurrent fludrocortisone administration) produced statistically significant decreases of 20-50% in mean plasma 17-hydroxyprogesterone, androstenedione, and renin activity. Since carbenoxolone also decreased the apparent metabolic clearance rate of cortisol by 20%, other measures of systemic glucocorticoid activity were examined. Carbenoxolone did not produce a cushingoid appearance or increase body weight, blood pressure, blood glucose or plasma insulin levels. Carbenoxolone also did not suppress stimulated GH levels, but did decrease TRH-stimulated TSH levels by approximately 20% (P < 0.05). Carbenoxolone can augment the adrenal androgen-suppressing activity of hydrocortisone in patients with 21-hydroxylase deficiency. These observations support the hypothesis that selective inhibition of enzymes that metabolize cortisol may lead to new approaches to improve the treatment of congenital adrenal hyperplasia.

Increased Plasma 17-Hydroxyprogesterone Response to ACTH in Patients with Nonhyperfunctioning Adrenal Adenomas Is Not Due to a Deficiency in 21-Hydroxylase Activityd

Increased Plasma 17-Hydroxyprogesterone Response to ACTH in Patients with Nonhyperfunctioning Adrenal Adenomas Is Not Due to a Deficiency in 21-Hydroxylase Activityd

Carrier status for steroid 21-hydroxylase deficiency is only one factor in the variable phenotype of acne.

Previous endocrine studies of women with acne have produced diverse results. This study was designed to seek evidence, from endocrine and genetic studies, for impaired steroid biosynthesis in patients with acne. Adrenal stimulation tests with synthetic adrenocorticotrophic hormone (ACTH) were performed. Steroid hormones were measured basally and 30 minutes after ACTH. The results were correlated with analysis of the steroid 21-hydroxylase gene (CYP21). Fifty-one consecutive female patients (mean age 27.1 years) referred with acne. The median plasma 17-hydroxyprogesterone (17-OHP) before and 30 minutes after ACTH were 2.5 nmol/l (range 1.1-8.2) and 7.3 (2.1-17.8) nmol/l which were significantly above normal female controls (n = 11, mean age 25.6 years) at 1.5 (0.9-4.2) and 4.6 (2.6-8.4) nmol/l. Eighteen of 51 acne patients showed an abnormal 17-OHP response. The 21-hydroxylase gene (CYP21) was examined for major deletions and for three common point mutations in 31 of the patients (14 with exaggerated 17-OHP response). One patient had a deletion of CYP21 on one allele consistent with carrier status for the classical congenital adrenal hyperplasia (CAH). Five patients, one of whom had a normal 17-OHP response to Synacthen, were heterozygous for the val 281 leu mutation in exon 7 of the CYP21 and were therefore carriers for a mutation associated with late-onset CAH. One patient with a raised 17-OHP response was homozygous for the splice site mutation in intron 2 and one patient with a normal 17-OHP response was heterozygous for the mutation. None of the patients had the ile 172 asn mutation. Eight of the 31 acne patients who had CYP21 gene analysis were carriers for mutations in the 21-hydroxylase gene but only six would have been detected by an abnormal response of 17-OHP on stimulation. Although alterations of the CYP21 gene were more common in acne than in controls there is a poor correlation between these events and raised steroids and acne. Factors other than mild impairment of CYP21 contribute to the variability of the clinical phenotype in hyperandrogenic states including acne.

Gonad histology and plasma steroid profiles in wild New Zealand freshwater eels ( Anguilla dieffenbachii and A. australis ) before and at the onset of the natural spawning migration. I. Females

Immature and maturing male New Zealand freshwater eels, the shortfinned Anguilla australis and the longfinned A. dieffenbachii, were caught from the wild to obtain data on the natural reproductive physiology of these fish. Plasma samples were analysed for steroid hormones by radioimmunoassay and values related to the developmental stage of the testes. Our histological observations on testes largely confirmed those reported previously. Thus, the gonad of non-migrating eels often appeared undifferentiated or poorly developed, containing only type A or early type B spermatogonia. In contrast, the testes of migrating shortfins were in early spermatogenesis as evidenced by the presence of late type B spermatogonia. Similarly, early spermatogenic stages were common in migratory longfins, but eels in midspermatogenesis (all germ cell stages present) were also encountered. Unlike a previous study, patches of testicular regression were commonly seen in migrants of both species. Levels of several androgens, androstenedione (AD), testosterone and 11-ketotestosterone (KT), were elevated in migrants compared to non-migrants. AD was higher in early to midspermatogenic A. dieffenbachii (0.63 ng ml−1) than in A. australis (0.25 ng ml−1) in the spermatogonial proliferation stage, while the inverse was observed for KT (27.78 ng ml−1 and 50.52 ng ml−1, respectively). Levels of 17,20β-dihydroxy-4-pregnen-3-one were nearly undetectable (less than 0.12 ng ml−1) in all animals. Plasma 17-hydroxyprogesterone concentrations in fyke-caught eels were elevated to a greater extent in non-migrants (up to 1.92 ng ml−1) than in migrants (around 0.5 ng ml−1), and correlated well with levels of cortisol in all groups. Histological results are compared to previous studies and the presence of regression in the testes is discussed. In addition, the role of steroid hormones, in particular AD and KT, in reproduction and stress is considered.