Citations
All
Search in:AllTitleAbstractAuthor name
Publications
(331)
Patents
Grants
Pathways
Clinical trials
Publication
Journal: Clinical Endocrinology
October/16/1997
Abstract
OBJECTIVE
Polycystic ovary syndrome (PCOS) is a common endocrinopathy of unknown aetiology. The aims of this study were to identify whether ovarian thecal cell steroidogenesis is abnormally regulated in PCOS by measuring steroid responses to a single dose of hCG before, and during, suppression of endogenous LH levels by GnRH analogue (GnRHa).
METHODS
Serum levels of LH, FSH, 17 alpha hydroxyprogesterone (<em>17OHP</em>), androstenedione, testosterone and dehydroepiandrosterone sulphate were measured before, and 48 hours after, a single intramuscular injection of 10,000 IU hCG. The test was repeated 4 weeks after suppression of endogenous LH levels by GnRHa.
METHODS
The ovarian responses to hCG were compared in three groups of women. Eleven women had normal ovaries and regular cycles, eight had polycystic ovaries but not clinical features of the syndrome (PCO group) and eight had polycystic ovaries, anovulation and either severe hirsutism or alopecia (PCOS group).
RESULTS
Before GnRHa treatment, LH levels were significantly higher in the PCOS group but hCG stimulated a similar rise in <em>17OHP</em> in all three groups. Following analogue, LH levels were suppressed in all three groups but the <em>17OHP</em> responses to hCG were significantly higher in both the PCO and PCOS groups compared with normal controls.
CONCLUSIONS
These findings provide further evidence in favour of an intrinsic abnormality of thecal cell steroidogenesis in the polycystic ovary.
Publication
Journal: Fertility and Sterility
October/11/2001
Abstract
OBJECTIVE
To examine the direct effect of metformin on thecal cell androgen production.
METHODS
Basic science research laboratory, University of Texas Southwestern, Dallas, Texas.
METHODS
Human ovarian theca-like tumor cells were treated with various concentrations of metformin in the presence and absence of forskolin for 48 hours.
METHODS
Media were collected, and radioimmunoassay (RIA) for progesterone, 17 alpha-hydroxyprogesterone (<em>17OHP</em>), androstenedione, and testosterone was performed. The effect of metformin on the expression of various enzymes involved in theca cell steroidogenesis was examined.
RESULTS
Metformin (50 microM and 200 microM) significantly inhibited androstenedione production from both forskolin-stimulated and unstimulated theca cells. Testosterone production was also significantly inhibited in forskolin-treated cells in the presence of 200 microM of metformin-treated compared with forskolin-only-treated cells. Western blot analysis revealed that metformin significantly inhibited the expression of steroidogenic acute regulatory (StAR) protein and 17 alpha-hydroxylase (CYP17) expression in cells stimulated with forskolin compared with forskolin treatment alone. There was no significant change in either 3beta-hydroxysteroid dehydrogenase (3 beta HSD) or cholesterol side-chain cleavage (CYP11A1) protein expression. Northern analysis revealed a significant decrease in the expression of CYP17 mRNA in forskolin-stimulated cells treated with metformin (200 microM) compared with forskolin-only-treated cells, however, there was no significant change in steroidogenic acute regulatory protein mRNA expression.
CONCLUSIONS
Our results suggest that metformin may have a direct effect on thecal cells' androgen production.
Publication
Journal: European Journal of Endocrinology
January/5/2005
Abstract
Congenital adrenal hyperplasia (CAH) is well suited for newborn screening, as it is a common and potentially fatal disease which can be easily diagnosed by a simple hormonal measurement in blood. Moreover, early recognition and treatment can prevent severe salt wasting, dehydration and death and shorten the time of male sex assignment in virilised females.In screening programmes, 17alpha-hydroxyprogesterone (<em>17OHP</em>) is measured in filter paper blood spots obtained by a heel puncture preferably between 2 and 4 days after birth. Three assay techniques are utilised for initial screening: radio-immunoassay (USA), enzyme-linked immunosorbent assay (Japan) and time-resolved fluoro-immunoassay (Europe). Preterm newborns have higher <em>17OHP</em> concentrations in serum than babies born at term. Therefore, cut-off levels are based on gestational age (in Japan and Europe) or on birth weight (in the USA). There is a considerable variation in cut-off levels from one programme to another. This is most likely due to the different antibodies and reagents used, varying thickness and density of filter paper used for sample collection and, most significantly, the characteristics of the reference population (in terms of birth weight and gestational age). More than 30 million newborns have been screened. The prevalence of CAH in the USA and Europe is approximately 1:15 000-16 000, and slightly lower in Japan (1:19 000). In general, severe salt wasting can be prevented, but there is a remarkable variation in the number of false-positives and false-negatives among the various programmes. Ongoing refinement of cut-off levels is needed to improve specificity and sensitivity.
Publication
Journal: Journal of Biological Chemistry
May/16/2012
Abstract
Steroid 21-hydroxylase (cytochrome P450 21A2, CYP21A2) deficiency accounts for ∼95% of individuals with congenital adrenal hyperplasia, a common autosomal recessive metabolic disorder of adrenal steroidogenesis. The effects of amino acid mutations on CYP21A2 activity lead to impairment of the synthesis of cortisol and aldosterone and the excessive production of androgens. In order to understand the structural and molecular basis of this group of diseases, the bovine CYP21A2 crystal structure complexed with the substrate 17-hydroxyprogesterone (<em>17OHP</em>) was determined to 3.0 Å resolution. An intriguing result from this structure is that there are two molecules of <em>17OHP</em> bound to the enzyme, the distal one being located at the entrance of the substrate access channel and the proximal one bound in the active site. The substrate binding features locate the key substrate recognition residues not only around the heme but also along the substrate access channel. In addition, orientation of the skeleton of the proximal molecule is toward the interior of the enzyme away from the substrate access channel. The <em>17OHP</em> complex of CYP21A2 provides a good relationship between the crystal structure, clinical data, and genetic mutants documented in the literature, thereby enhancing our understanding of congenital adrenal hyperplasia. In addition, the location of certain CYP21A2 mutations provides general understanding of structure/function relationships in P450s.
Publication
Journal: Gynecological Endocrinology
July/15/2008
Abstract
OBJECTIVE
To evaluate the effects the administration of myo-inositol (MYO) on hormonal parameters in a group of PCOS patients.
METHODS
Controlled clinical study.
METHODS
PCOS patients in a clinical research environment.
METHODS
20 overweight PCOS patients were enrolled after informed consent.
METHODS
All patients underwent hormonal evaluations and an oral glucose tollerance test (OGTT) before and after 12 weeks of therapy (Group A (n = 10): myo-inositol 2 gr. plus folic acid 200 mug every day; Group B (n = 10): folic acid 200 mug every day). Ultrasound examinations and Ferriman-Gallwey score were also performed.
METHODS
Plasma LH, FSH, PRL, E2, <em>17OHP</em>, A, T, glucose, insulin, C peptide concentrations, BMI, HOMA index and glucose-to-insulin ratio.
RESULTS
After 12 weeks of MYO administration plasma LH, PRL, T, insulin levels and LH/FSH resulted significantly reduced. Insulin sensitivity, expressed as glucose-to-insulin ratio and HOMA index resulted significantly improved after 12 weeks of treatment. Menstrual cyclicity was restored in all amenorrheic and oligomenorrheic subjects. No changes occurred in the patients treated with folic acid.
CONCLUSIONS
Myo-inositol administration improves reproductive axis functioning in PCOS patients reducing the hyperinsulinemic state that affects LH secretion.
Publication
Journal: Journal of Clinical Endocrinology and Metabolism
November/1/2007
Abstract
BACKGROUND
In male patients with congenital adrenal hyperplasia (CAH), testicular adrenal rest tumors are frequently found that may interfere with gonadal function.
OBJECTIVE
Our objective was to determine steroid-producing features of testicular adrenal rest tumors.
METHODS
The study is descriptive and took place at a university medical center.
METHODS
Eight adult CAH patients with bilateral testicular adrenal rest tumors were treated with testis-sparing surgery.
METHODS
In all but one patient, spermatic veins were cannulated during surgery and blood samples collected to measure the adrenal-specific steroid 21-deoxycortisol (21DF) and 17-hydroxyprogesterone (<em>17OHP</em>) and androstenedione (A). The same parameters were measured in simultaneously taken peripheral blood. mRNA concentrations of adrenal-specific enzymes CYP11B1 and CYP11B2 and ACTH and angiotensin II (AII) receptors were measured in tumor tissue.
METHODS
Adrenal-specific steroids/enzymes were assessed.
RESULTS
21DF, <em>17OHP</em>, and A levels were measurable in all spermatic vein samples. The ratio (mean +/- SD) between spermatic vein and simultaneously taken peripheral blood samples was 37.8 +/- 56.3 (21DF), 132.0 +/- 249 (<em>17OHP</em>), and 57.0 +/- 68.2 (A). CYP11B1, CYP11B2, and ACTH and AII receptor mRNAs were detected in all tumors with a strong correlation between ACTH receptor mRNA in tumors and 21DF (r = 0.85; P = 0.015), <em>17OHP</em> (r = 1; P = 0.01) and A (r = 0.89; P = 0.007) concentrations in peripheral blood.
CONCLUSIONS
Testicular adrenal rest tumors produce adrenal-specific steroids and express adrenal-specific enzymes and ACTH and AII receptors, confirming the strong resemblance with adrenal tissue. Because AII receptors are present in tumor tissue, it can be hypothesized that AII may be an additional factor responsible for testicular adrenal rest tumor growth.
Publication
Journal: Steroids
June/23/2011
Abstract
BACKGROUND
The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects.
METHODS
0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis.
RESULTS
Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone>1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone<1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and <em>17OHP</em> Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established.
CONCLUSIONS
Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.
Publication
Journal: Journal of Clinical Endocrinology and Metabolism
July/2/1999
Abstract
Recent reports indicate that girls with premature adrenarche are at risk of developing functional ovarian hyperandrogenism and polycystic ovarian syndrome (PCOS). As insulin and insulin-like growth factors (IGFs) have been implicated in the pathogenesis of PCOS, we hypothesize that they may also have a role in the hyperandrogenism of premature adrenarche. Thirty-five prepubertal girls (23 Caribbean Hispanics and 12 Black African-Americans) underwent a 60-min ACTH and LH-releasing hormone test. Insulin sensitivity (S(I)) was assessed using the frequently sampled i.v. glucose tolerance test with tolbutamide. Fasting levels of IGF-I, IGF-binding protein-1 (IGFBP-1), IGFBP-3, sex hormone-binding globulin, and free testosterone (T) were also obtained. The mean age of the patients was 6.8 yr, and bone age was 8.0 yr. Twenty-five patients had a family history of noninsulin-dependent diabetes mellitus and 19 patients had acanthosis nigricans. The mean S(I) for the entire group was 6.78 +/- 5.21 x 10(-4) min/microU x mL (normal prepubertal S(I), 6.5 +/- 0.54 x 10(-4) min(-1) x microU(-1) x mL(-1)). However, 15 of the 35 girls had an S(I) that was more than 2 SD below the mean reported for normal prepubertal children. Of these 15 patients, 13 were obese, and 14 had acanthosis nigricans. For the entire group of girls, the mean ACTH-stimulated levels of 17-hydroxypregnenolone (<em>17OHP</em>reg), dehydroepiandrosterone (DHEA), androstenedione (AS), 17-hydroxyprogesterone (<em>17OHP</em>), and T and the ACTH-stimulated ratios of <em>17OHP</em>reg/<em>17OHP</em>, <em>17OHP</em>reg/DHEA, <em>17OHP</em>/AS, and DHEA/AS did not differ from the levels reported for Tanner stage II-III pubertal girls. The girls were divided into two groups based on their S(I) (group I, S(I) >2 SD below the mean for age; group II, normal S(I)). The group I girls with a reduced S(I) had significantly higher ACTH-stimulated levels of <em>17OHP</em>reg (group I, 760 +/- 87.84 ng/dL; group II, 428.9 +/- 46.28 ng/dL; P = 0.002), <em>17OHP</em>reg/<em>17OHP</em> ratio (group I, 3.95 +/- 0.36; group II, 2.96 +/- 0.35; P = 0.05), <em>17OHP</em>reg/DHEA (group I, 2.06 +/- 0.21; group II, 1.4 +/- 0.13; P = 0.01), and free T (group I, 1 +/- 0.23 ng/dL; group II, 0.49 +/- 0.19 ng/dL; P = 0.014). Levels of sex hormone-binding globulin were lower in the group I girls. Furthermore, for the entire group of girls, the S(I) correlated inversely with ACTH-stimulated levels of <em>17OHP</em>reg, DHEA, and AS and the ACTH-stimulated ratio of <em>17OHP</em>reg/<em>17OHP</em>. IGF-I correlated inversely with S(I) (r = -0.94; P < 0.001) and correlated directly with the ACTH-stimulated levels of <em>17OHP</em>reg (r = 0.8; P < 0.001) and AS (r = 0.63; P < 0.05). IGF-I also correlated with the ACTH-stimulated ratios of <em>17OHP</em>reg/<em>17OHP</em> (r = 0.61; P < 0.05), <em>17OHP</em>reg/DHEA (r = 0.9; P < 0.001), <em>17OHP</em>/AS (r = 0.79; P < 0.001), and DHEA/AS (r = 0.96; P < 0.001). IGFBP-1 correlated inversely with the ACTH-stimulated levels of <em>17OHP</em>reg (r = -0.38; P < 0.05) and DHEA (r = -0.36; P < 0.05). To summarize, the ACTH-stimulated delta5-steroid levels were higher in prepubertal girls with premature adrenarche and reduced S(I). There was a significant inverse correlation among ACTH-stimulated hormone levels, S(I), and IGFBP-1, whereas IGF-I correlated directly with ACTH-stimulated androgens. These findings support the hypothesis that insulin and IGFs may have a role in the hyperandrogenism of premature adrenarche just as they do in PCOS. Hence, in certain girls with premature adrenarche, hyperandrogenism may be the first presentation of PCOS and/or insulin resistance.
Publication
Journal: Journal of Clinical Endocrinology and Metabolism
May/28/2009
Abstract
OBJECTIVE
Our objective was to determine the ovarian function of asymptomatic volunteers with a polycystic ovary (V-PCO).
METHODS
Non-hirsute eumenorrheic V-PCO (n = 32) and volunteers with ultrasonographically normal ovaries (V-NO) (n = 21) were compared with one another and with polycystic ovary syndrome (PCOS) patients who met National Institute of Health criteria (n = 90). DESIGN/SETTING/INTERVENTIONS: GnRH agonist (GnRHag), ACTH, and oral glucose tolerance tests were prospectively performed in a General Clinical Research Center.
RESULTS
The distribution of 17-hydroxyprogesterone (<em>17OHP</em>) responses to GnRHag of V-PCO formed a distinct population intermediate between that of V-NO, the reference population, and PCOS. Nevertheless, the V-PCO population was heterogeneous. There were 53% (seventeen of 32) that were functionally normal, with <em>17OHP</em> responses and free testosterone levels like V-NO. A total of 25% (eight of 32) had an elevated free testosterone, thus meeting Rotterdam criteria for PCOS; one third of these had <em>17OHP</em> hyperresponsiveness to GnRHag testing. The remaining 22% (seven of 32) had <em>17OHP</em> hyperresponsiveness to GnRHag, but normal free testosterone. Of PCOS, 69% had elevated <em>17OHP</em> hyperresponsiveness to GnRHag. Ovarian volume correlated significantly with <em>17OHP</em> responses only in PCOS, accounting for just 10% of the variance.
CONCLUSIONS
Many asymptomatic volunteers have a PCO. They are a distinct, but heterogeneous, population with respect to ovarian function, ranging from normal (53%) to occult PCOS by Rotterdam criteria (25%). Nearly one quarter (22%) had the typical PCOS type of ovarian dysfunction without hyperandrogenemia, termed a "dysregulated PCO"; they or their offspring may be at risk for PCOS. Ovarian ultrasonographic characteristics must be considered when establishing norms for ovarian function.
Publication
Journal: Steroids
January/15/2002
Abstract
We studied influences of dental care, food and storage on the reproducibility of salivary steroid levels. Cortisol (F), 17OH-progesterone (<em>17OHP</em>) and Progesterone (P) were measured using adapted commercial radioimmunoassays. Saliva samples of healthy adults (n = 15; m:8; f:7) were collected directly before and after dental care, and directly before and after breakfast with various foodstuffs. A second experiment investigated stability of steroids under different storage conditions. Four series of identical saliva portions (I: Native saliva; II: Centrifuged saliva; III: Saliva with trifluor acetate (TFA); IV: Saliva with 0.5% NaN(3)) were stored at room temperature and at 4 degrees C for up to three weeks. To demonstrate influences of repeated thawing and re-freezing of saliva on steroid values, saliva samples (n = 15) were divided into identical portions. These portions were frozen and re-thawed up to 5 times before measurement. Neither dental care nor intake of bread or milk effected the reproducibility of F, 170HP, and P. Steroid levels decreased significantly in the course of three weeks under different storage conditions (P < 0.001). This decrease was clinically relevant from the second week onward, with exception of NaN(3) treated samples. After repeated freezing and re-thawing <em>17OHP</em> and P decreased slightly (about 5%). Only F decreased significantly after the third thawing (P < 0.001). The results show the usefulness of standardized handling of saliva samples for improving reproducibility and reliability of salivary steroid measurements.
Publication
Journal: Journal of Clinical Endocrinology and Metabolism
August/9/2005
Abstract
OBJECTIVE
In newborn screening programs for congenital adrenal hyperplasia, 17-alpha-hydroxyprogesterone (<em>17OHP</em>) cutoff levels are based on birth weight (BW) or on gestational age (GA). We investigated which approach would result in the greatest specificity and sensitivity.
METHODS
For the determination of <em>17OHP</em>, a neonatal <em>17OHP</em> assay was used in filter paper blood of 9492 newborns. The relationships between <em>17OHP</em> and BW and between <em>17OHP</em> and GA were studied by regression analysis. Reference curves with a specificity of 99.95% were constructed with the method that summarizes the distribution by three smoothed curves representing the skewness (L curve), the median (M curve), and the coefficient of variation (S curve). Median cutoff levels for BW and for GA according to the 99.95% reference curves were calculated.
RESULTS
Regression analysis showed that GA is a better predictor of <em>17OHP</em> than BW (R(2) was 50.6 vs. 35.8%, respectively). At a specificity of 99.95%, the calculated median <em>17OHP</em> cutoff level was lower for GA [12.6 microg/liter (38 nmol/liter)] than for BW [17.6 microg/liter (54 nmol/liter)], thus leading to a greater sensitivity.
CONCLUSIONS
This study demonstrates that GA is a better predictor of <em>17OHP</em> in newborns and will result in greater specificity than BW despite the fact that the determination of GA might be less reliable than BW.
Publication
Journal: Steroids
March/16/2007
Abstract
Saliva analysis is an accepted non-invasive alternative to plasma in pediatric endocrinology. Although commercial saliva collectors are available, the reliability of these devices for the analysis of salivary hormones has not been proved. We investigated the recovery and linearity of salivary steroids (cortisol, cortisone, 17-hyroxyprogesterone, testosterone, androstenedione) being relevant in endocrine research and therapy control. Pooled saliva was spiked with ascending concentrations of the steroids and applied onto a variety of absorbents, such as the cotton and the polyester (PE) Salivette (Sarstedt), the foam-tip applicator (Whatman) and strips of blood-spot collection paper (Whatman). Analysis was performed by LC-MS/MS. Best results were achieved using the PE Salivette, yielding recoveries (%) of 99.8 (cortisol), 98.7 (cortisone), 91.8 (<em>17OHP</em>), 96.3 (testosterone), 98.9 (androstendione) with a volume recovery of 98+/-1%. Using the blood-spot paper, recoveries (%) were 92.0 (cortisol), 89.1 (cortisone), 72.0 (<em>17OHP</em>), 70.3 (testosterone) and 77.1 (androstendione). The recovery of glucocorticoids was significantly higher compared to androgens (p<0.001). The recovery of liquid volume was 95+/-2%. The cotton Salivette yielded weak recoveries of 88.7 (cortisol), 86.2 (cortisone), 60.9 (<em>17OHP</em>), 62.0 (testosterone) and 72.4 (androstendione). The recovery of the glucocorticoids differed significantly from the androgens (p<0.001). Liquid recovery was most variable with 89+/-8%. The weakest recoveries were found in the foam-tips being 76.2 for cortisol, only 41.8 for cortisone, 31.1 for <em>17OHP</em>, 38.5 for testosterone and 36.1 for androstendione. The volume recovery here was 97+/-1%. We assume only the PE version of the Salivette suitable for salivary steroid analysis. The weak recovery from the cotton version is a severe problem due to lacking comparability with values obtained with the polyester wads and the weak homogeneity as observed over a physiological concentration range.
Publication
Journal: Journal of Steroid Biochemistry and Molecular Biology
May/4/2012
Abstract
In order to overcome many limitations of immunoassays, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has the potential to find its place in the clinical laboratory medicine for quantification of steroid hormones. A prerequisite for the application of a new analytical procedure in clinical diagnostics is standardization to minimize analytical intra- and interlaboratory variability and inaccuracy. We evaluate a newly standardized HPLC-MS/MS assay in kit-format, developed for routine determination of 16 steroid hormones in human serum samples. Fifteen metabolites can be measured quantitatively, which include aldosterone, androstenedione, androsterone, corticosterone, cortisol, cortisone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), 17β-estradiol (E2), estrone (E1), etiocholanolone, 17α-hydroxyprogesterone (<em>17OHP</em>), progesterone, and testosterone. 11-Deoxycorticosterone is the only compound rated as semi-quantitative in this kit. The sample preparation is performed by solid phase extraction (SPE) on a 96-well plate. The standardized assay has been validated for human serum in terms of lower and upper limit of quantification (LLOQ 0.01-32 ng/mL, ULOQ 5-8000 ng/mL), linear correlation coefficient of calibration (R(2)>0.9966), intra- and inter-day precision (intra-day 1.1-8.8%, inter-day 5.2-14.8% and 8.2-18.6% for 11-deoxycorticosterone), accuracy (intra-day 88.3-115.5% and 109.3-128.2% for 11-deoxycorticosterone, inter-day 91.4-117.2% and 102.3-137.1% for 11-deoxycorticosterone), analytical total error (3.6-17.8%), proficiency test accuracy (85.4-113.4%), recovery (68-99%), and metabolite stability (freeze/thaw stability 95.5-108.1%, short term stability 86.9-107.2%). Inter-assay comparison with a routine reference HPLC-MS/MS assay and seven immunoassays demonstrates the outstanding high performance of this HPLC-MS/MS based kit by improvements in accuracy for progesterone, androstenedione, and <em>17OHP</em>. Finally, results of two metyrapone tests demonstrate the potential of the standardized HPLC-MS/MS assay for the analysis of a comprehensive steroid hormone profile in clinical diagnostics.
Publication
Journal: Journal of Clinical Endocrinology and Metabolism
February/12/2009
Abstract
BACKGROUND
21-Hydroxylase deficiency (21OHD) is caused by CYP21A2 gene mutations disrupting the adrenal 21-hydroxylase, P450c21. CYP21A2 mutations generally correlate well with the 21OHD phenotype, but some children with severe CYP21A2 mutations have residual 21-hydroxylase activity. Some hepatic P450 enzymes can 21-hydroxylate progesterone, but their physiological relevance in modifying 21OHD is not known.
OBJECTIVE
We determined the ability of CYP2C19 and CYP3A4 to 21-hydroxylate progesterone and 17-hydroxyprogesterone (<em>17OHP</em>), determined the impact of the common P450 oxidoreductase (POR) variant A503V on these activities, and examined correlations between CYP2C19 variants and phenotype in patients with 21OHD.
METHODS
Bacterially expressed, N-terminally modified, C-His-tagged human P450c21, CYP2C19, and CYP3A4 were combined with bacterially expressed wild-type and A503V POR. The 21-hydroxylation of radiolabeled progesterone and <em>17OHP</em> was assessed, and the Michaelis constant (Km) and maximum velocity (Vmax) of the reactions were measured. CYP2C19 was genotyped in 21OHD patients with genotypes predicting severe congenital adrenal hyperplasia.
RESULTS
Compared to P450c21, the Vmax/Km for 21-hydroxylation of progesterone by CYP2C19 and CYP3A4 were 17 and 10%, respectively. With both forms of POR, the Km for P450c21 was approximately 2.6 microm, the Km for CYP2C19 was approximately 11 microm, and the Km for CYP3A4 was approximately 110 microm. Neither CYP2C19 nor CYP3A4 could 21-hydroxylate <em>17OHP</em>. The CYP2C19 ultrametabolizer allele CYP2C19 17 was homozygous in one of five patients with a 21OHD phenotype that was milder than predicted by the CYP21A2 genotype.
CONCLUSIONS
CYP2C19 and CYP3A4 can 21-hydroxylate progesterone but not <em>17OHP</em>, possibly ameliorating mineralocorticoid deficiency, but not glucocorticoid deficiency. Multiple enzymes probably contribute to extraadrenal 21-hydroxylation.
Publication
Journal: Pediatric Research
December/11/2000
Abstract
Some very preterm neonates admitted to the neonatal intensive care unit show circulatory and respiratory problems that improve after administration of steroids. It is unclear whether these symptoms could be caused by adrenal insufficiency. The objective of our study was to investigate the cortisol levels and the cortisol release from the adrenals after ACTH in very preterm infants with and without severe illness and to find whether a relation exists between adrenal function and outcome. An ACTH test (0.5 microg) was performed on d 4 in 21 very preterm infants (gestational age, 25.6-29.6 wk; birth weight, 485-1265 g). Baseline cortisol and 17-hydroxyprogesterone (<em>17OHP</em>) levels and the cortisol levels 30, 60, and 120 min after ACTH administration were measured. The Score for Neonatal Acute Physiology was used to measure illness severity. All infants showed an increase in cortisol levels after ACTH, but the cortisol levels were significantly lower in the ventilated more severely ill infants. After adjusting for birth weight and gestational age, the mean baseline cortisol levels and cortisol/<em>17OHP</em> ratios were significantly lower and the <em>17OHP</em> levels significantly higher in the ventilated infants compared with the nonventilated infants. Patients with an adverse outcome had significantly lower baseline cortisol/<em>17OHP</em> ratios and 60-min cortisol levels during ACTH testing (p = 0.002 and p = 0.03, respectively). These data suggest an insufficient adrenal response to stress in sick ventilated very preterm infants with gestational ages younger than 30 wk compared with nonventilated less sick preterm infants. Further studies are required to investigate whether supplementation with physiologic doses of hydrocortisone may benefit the outcome.
Publication
Journal: JAMA - Journal of the American Medical Association
July/7/2011
Abstract
BACKGROUND
Lopinavir-ritonavir is a human immunodeficiency virus 1 (HIV-1) protease inhibitor boosted by ritonavir, a cytochrome p450 inhibitor. A warning about its tolerance in premature newborns was recently released, and transient elevation of 17-hydroxyprogesterone (<em>17OHP</em>) was noted in 2 newborns treated with lopinavir-ritonavir in France.
OBJECTIVE
To evaluate adrenal function in newborns postnatally treated with lopinavir-ritonavir.
METHODS
Retrospective cross-sectional analysis of the database from the national screening for congenital adrenal hyperplasia (CAH) and the French Perinatal Cohort. Comparison of HIV-1-uninfected newborns postnatally treated with lopinavir-ritonavir and controls treated with standard zidovudine.
METHODS
Plasma <em>17OHP</em> and dehydroepiandrosterone-sulfate (DHEA-S) concentrations during the first week of treatment. Clinical and biological symptoms compatible with adrenal deficiency.
RESULTS
Of 50 HIV-1-uninfected newborns who received lopinavir-ritonavir at birth for a median of 30 days (interquartile range [IQR], 25-33), 7 (14%) had elevated <em>17OHP</em> levels greater than 16.5 ng/mL for term infants (>23.1 ng/mL for preterm) on days 1 to 6 vs 0 of 108 controls having elevated levels. The median <em>17OHP</em> concentration for 42 term newborns treated with lopinavir-ritonavir was 9.9 ng/mL (IQR, 3.9-14.1 ng/mL) vs 3.7 ng/mL (IQR, 2.6-5.3 ng/mL) for 93 term controls (P < .001). The difference observed in median <em>17OHP</em> values between treated newborns and controls was higher in children also exposed in utero (11.5 ng/mL vs 3.7 ng/mL; P < .001) than not exposed in utero (6.9 ng/mL vs 3.3 ng/mL; P = .03). The median DHEA-S concentration among 18 term newborns treated with lopinavir-ritonavir was 9242 ng/mL (IQR, 1347-25,986 ng/mL) compared with 484 ng/mL (IQR, 218-1308 ng/mL) among 17 term controls (P < .001). The <em>17OHP</em> and DHEA-S concentrations were positively correlated (r = 0.53; P = .001). All term newborns treated with lopinavir-ritonavir were asymptomatic, although 3 premature newborns experienced life-threatening symptoms compatible with adrenal insufficiency, including hyponatremia and hyperkalemia with, in 1 case, cardiogenic shock. All symptoms resolved following completion of the lopinavir-ritonavir treatment.
CONCLUSIONS
Among newborn children of HIV-1-infected mothers exposed in utero to lopinavir-ritonavir, postnatal treatment with a lopinavir-ritonavir-based regimen, compared with a zidovudine-based regimen, was associated with transient adrenal dysfunction.
Publication
Journal: Clinical Chemistry
August/29/2006
Abstract
BACKGROUND
Congenital adrenal hyperplasia is a group of autosomal recessive disorders caused by a deficiency of 1 of 4 enzymes required for the synthesis of glucocorticoids, mineralocorticoids, and sex hormones. Analysis of 11-deoxycortisol (11DC), 17-hydroxyprogesterone (<em>17OHP</em>), 17-hydroxypregnenolone (<em>17OHP</em>r), and pregnenolone (Pr) in blood allows detection of these enzyme defects.
METHODS
The steroids were extracted from 200 microL of serum or plasma by solid-phase extraction, derivatized to form oximes, and extracted again with methyl t-butyl ether. Instrumental analysis was performed on an API 4000 tandem mass spectrometer with electrospray ionization in positive mode and multiple reaction-monitoring acquisition.
RESULTS
The limits of detection were 0.025 microg/L for 11DC, <em>17OHP</em>, and Pr and 0.10 microg/L for <em>17OHP</em>r. The method was linear to 100 microg/L for 11DC, <em>17OHP</em>, and Pr, respectively, and to 40 microg/L for <em>17OHP</em>r. Within- and between-run (total) imprecision (CVs) were <7.1% and 11%, respectively. Reference intervals for children in Tanner stages 1 through 5 and adult males and females for <em>17OHP</em>, 11DC, Pr, and <em>17OHP</em>r were established. Prepared samples were stable for >72 h.
CONCLUSIONS
The detection limit and selectivity of this method and its small sample volume requirement allow analysis of endogenous concentrations of adrenal steroids in serum or plasma from children and adults. The method thus has an important potential role in the evaluation of the status of 4 of the enzymes involved in adrenal steroid biosynthesis.
Publication
Journal: Journal of Clinical Endocrinology and Metabolism
July/3/1997
Abstract
Polycystic ovary syndrome (PCOS) is associated with chronic anovulation, hyperandrogenemia, insulin resistance (IR)/hyperinsulinemia, and a high incidence of obesity. Thus, PCOS serves as a useful model to assess the role of IR and chronic endogenous insulin excess on leptin levels. Thirty-three PCOS and 32 normally cycling (NC) women of similar body mass index (BMI) were studied. Insulin sensitivity (S(I)) was assessed by rapid ivGTT in a subset of 28 PCOS and 29 NC subjects; percent body fat was determined by dual-energy x-ray absorptiometry (DEXA) in 14 PCOS and 17 NC. Fasting (0800 h) and 24-h mean hourly insulin levels were 2-fold higher (P < 0.0001), and S(I) was 50% lower (P = 0.005) in PCOS than in NC, while serum androstenedione (A), testosterone (T), 17-alpha hydroxyprogesterone (<em>17OHP</em>), and estrone (E1) levels were elevated (P < 0.0001), and sex hormone-binding globulin (SHBG) levels were decreased (P < 0.01). Twenty-four hour LH pulse frequency, mean pulse amplitude, and mean LH levels were elevated in PCOS (P < 0.001) as compared with NC. Serum leptin levels for PCOS (24.1 +/- 2.6 ng/mL) did not differ from NC (21.5 +/- 3.5 ng/mL) and were positively correlated with BMI (r = 0.81) and percent body fat (r = 0.91) for the two groups (both P < 0.0001). Leptin levels for PCOS and NC correlated positively with fasting and 24-h mean insulin levels (r = 0.81, P < 0.0001 for both PCOS and NC) and negatively with S(I) and SHBG levels. Leptin concentrations for PCOS, but not NC, correlated positively with 24-h mean glucose levels and inversely with 24-h mean LH levels and 24-h mean LH pulse amplitude. Leptin levels were not correlated with estrogen or androgen levels for either PCOS or NC, although leptin levels were positively related to the ratios of E1/SHBG and E2/SHBG for both PCOS and NC and to the ratio of T/SHBG for PCOS only. In stepwise multivariate regression with forward selection, only 24-h mean insulin levels contributed significantly (P < 0.01) to leptin levels independent of BMI and percent body fat for both PCOS and NC. Given this relationship and the presence of 2-fold higher 24-h mean insulin levels in PCOS, the expected elevation of leptin levels in PCOS was not found. This paradox may be explained by the presence of adipocyte IR specific to PCOS, which may negate the stimulatory impact of hyperinsulinemia on leptin secretion, a proposition requiring further study.
Publication
Journal: Human Reproduction
March/25/2012
Abstract
BACKGROUND
Polycystic ovary syndrome (PCOS) patients typically have 17-hydroxyprogesterone (<em>17OHP</em>) hyperresponsiveness to GnRH agonist (GnRHa) (PCOS-T). The objective of this study was to determine the source of androgen excess in the one-third of PCOS patients who atypically lack this type of ovarian dysfunction (PCOS-A).
METHODS
Aged-matched PCOS-T (n= 40), PCOS-A (n= 20) and controls (n= 39) were studied prospectively in a General Clinical Research Center. Short (4 h) and long (4-7 day) dexamethasone androgen-suppression tests (SDAST and LDAST, respectively) were compared in subsets of subjects. Responses to SDAST and low-dose adrenocorticotropic hormone (ACTH) were then evaluated in all.
RESULTS
Testosterone post-SDAST correlated significantly with testosterone post-LDAST and <em>17OHP</em> post-GnRHa (r = 0.671-0.672), indicating that all detect related aspects of ovarian dysfunction. An elevated dehydroepiandrosterone peak in response to ACTH, which defined functional adrenal hyperandrogenism, was similarly prevalent in PCOS-T (27.5%) and PCOS-A (30%) and correlated significantly with baseline dehydroepiandrosterone sulfate (DHEAS) (r = 0.708). Functional ovarian hyperandrogenism was detected by subnormal testosterone suppression by SDAST in most (92.5%) PCOS-T, but significantly fewer PCOS-A (60%, P< 0.01). Glucose intolerance was absent in PCOS-A, but present in 30% of PCOS-T (P < 0.001). Most of the PCOS-A cases with normal testosterone suppression in response to SDAST (5/8) lacked evidence of adrenal hyperandrogenism and were obese.
CONCLUSIONS
Functional ovarian hyperandrogenism was not demonstrable by SDAST in 40% of PCOS-A. Most of these cases had no evidence of adrenal hyperandrogenism. Obesity may account for most hyperandrogenemic anovulation that lacks a glandular source of excess androgen, and the SDAST seems useful in making this distinction.
Publication
Journal: Pediatrics
January/30/2002
Abstract
OBJECTIVE
To evaluate whether congenital adrenal hyperplasia (CAH) patients can be detected by newborn screening before the occurrence of life-threatening salt wasting and whether the prevalence, specificity, and sensitivity are adequate enough for a routine screening procedure.
METHODS
From 1998, a 2-year regional pilot screening for CAH was performed. In 1998, cutoff levels for <em>17OHP</em> were primarily based on birth weight, and in 1999 on gestational age. In addition, nationwide, all newly diagnosed patients with CAH were reported to the Dutch Pediatric Surveillance Unit to compare screened CAH patients with CAH patients in the area without screening.
RESULTS
In 2 years, 176 684 newborns were screened and 15 CAH patients (7 males/8 females) were detected. Therapy was started at the median age of 7 days. In the area without screening, 223 307 infants were born and 19 CAH patients (10 males/9 females) were reported to the Dutch Pediatric Surveillance Unit. Therapy was started at the median age of 14 days. The mean (standard deviation) serum sodium concentration was 134.5 (3.4) mmol/L in the area of screening versus 124.5 (10.8) mmol/L in the area without screening. The overall prevalence was 1:11 764. In 1998 and 1999, the specificity was 99.76% and 99.97%, respectively. The positive predictive value was 4.5% and 16%, respectively. To date, no false-negative cases have been detected.
CONCLUSIONS
Severe salt wasting can be prevented by neonatal screening. The prevalence, specificity, and sensitivity allowed addition of screening for CAH to the routinely performed national neonatal screening program.
Publication