Green method for 17-hydroxyprogesterone extraction and determination using PDMS stir bar sorptive extraction coupled with HPLC: optimization by response surface methodology.

Quantifying small amounts of the 17-hydroxyprogesterone in various matrix is crucial for different purposes. In this study, a commercial polydimethylsiloxane stir bar was used to extract hormone from water and urine samples. Analysis was performed by high-performance liquid chromatography using a UV detector. The response surface methodology was used to optimize the desorption and extraction steps, with predicted optimal point relative errors of 1.25% and 6.40%, respectively. The optimized method was validated with a linear range of 1.21-1000.00 for aqueous and 2.43-2000.00 ng mL-1 for urine samples. The coefficient of determination was 0.9998 and 0.9967, and the detection limit of the proposed method was obtained to be 0.40 and 0.80 ng mL-1 for aqueous and urine samples, respectively. The recovery percentage and relative standard deviation within a day and between three days after the addition of three different concentration levels of the standard to the control sample were 87-103% and 0.4-3.6% for aqueous and 87.5-101% and 0.1-5.2% for urine samples, respectively. The results show that the proposed method can be appropriate and cost-effective for extracting and analyzing this hormone. In addition, using three different tools, the greenness of the proposed method was proven.

Reduced activity of 11ß-hydroxylase accounts for elevated 17α-hydroxyprogesterone in preterms.

OBJECTIVE: To characterize the urinary steroid metabolome of neonates and infants born either at term or preterm. STUDY DESIGN: We retrospectively analyzed urinary steroid hormone metabolites determined by gas chromatography-mass spectrometry of 78 neonates and infants born at term and 83 neonates and infants born preterm (median 34 weeks of gestational age). The subjects' 11ß-hydroxylase and 21-hydroxylase activities were assessed on the basis of urinary metabolite substrate-to-product ratios. RESULTS: Preterm neonates and infants had elevated urinary concentrations of 17α-hydroxyprogesterone (17OHP) metabolites (P<.001) but lower urinary concentrations of the 21-deoxycortisol metabolite pregnanetriolone (PTO) (P<.01). One reason was lower 11ß-hydroxylase activity in preterms. We could demonstrate a correlation between low 11ß-hydroxylase activity and high urinary concentrations of 17OHP metabolites (r=0.51, P<.001) but low urinary concentrations of the 21-deoxycortisol metabolite PTO (r=-0.24, P=.03) in preterms. CONCLUSIONS: Low 11ß-hydroxylase activity may explain increased 17OHP but decreased 21-deoxycortisol metabolite excretion in preterms. Our analysis clarifies, first, why preterms have higher 17OHP levels and thus higher rates of false-positive screening results for congenital adrenal hyperplasia than do term infants, and, second, why 21-deoxycortisol or its urinary metabolite PTO is more specific than 17OHP for the diagnosis of 21-hydroxylase deficiency.

Non-targeted metabolomic approach reveals urinary metabolites linked to steroid biosynthesis pathway after ingestion of citrus juice.

Citrus juice intake has been highlighted because of its health-promoting effects. LC-MS based metabolomics approaches are applied to obtain a better knowledge on changes in the concentration of metabolites due to its dietary intake and allow a better understanding of involved metabolic pathways. Eight volunteers daily consumed 400 mL of juice for four consecutive days and urine samples were collected before intake and 24h after each citrus juice intake. Urine samples were analysed by nanoHPLC-q-TOF, followed by principal component analysis (PCA) and Student's t-test (p<0.05). PCA showed a separation between two groups (before and after citrus juice consumption). This approach allowed the identification of four endocrine compounds (tetrahydroaldosterone-3-glucuronide, cortolone-3-glucuronide, testosterone-glucuronide and 17-hydroxyprogesterone), which belonged to the steroid biosynthesis pathway as significant metabolites upregulated by citrus juice intake. Additionally, these results confirmed the importance of using the non-targeted metabolomics technique to identify new endogenous metabolites, up- or down-regulated as a consequence of food intake.

Clinicopathological features, biochemical and molecular markers in 5 patients with adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a very rare malignant tumor with poor prognosis. To gain insight into the pathogenic significance of ACC, we studied clinicopathological features and gene expression profile in ACC. We analyzed five ACC cases (two men and three women) with the median age of 45-year-old who underwent adrenalectomy at our institute. Endocrine studies revealed that two cases had subclinical Cushing's syndrome (SCS) and one with concomitant estrogen-secreting tumor, while the rest of three cases had non-functioning tumors. Analysis of urinary steroids profile by gas chromatography/mass spectrometry showed increased metabolites of corticosteroid precursors, such as 17-OH pregnenolone, 17-OH progesterone, dehydroepiandorosterone (DHEA), and 11-deoxycortisol in all five cases. The pathological diagnosis of ACC was based on Weiss's criteria with its score ≥ 3. The mean size of the resected tumors was 87 mm and Ki67/MIB1 labeling index, a proliferative marker, was 3-27%. Immunohistochemical analysis revealed a disorganized expression of several steroidogenic enzymes, such as 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase, and DHEA-sulfotransferase. Among several genes determined by RT-PCR, insulin-like growth factor (IGF)-II mRNA was consistently and abundantly expressed in all 5 tumor tissues. Postoperatively, two cases with SCS developed local recurrence and liver metastasis. The present study suggests that the disorganized expression of steroidogenic enzymes and the overexpression of IGF-II by the tumor are hallmarks of ACC, which could be used as biochemical and molecular markers for ACC.

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.

Cytochrome P450 oxidoreductase deficiency in three patients initially regarded as having 21-hydroxylase deficiency and/or aromatase deficiency: diagnostic value of urine steroid hormone analysis.

In this study, we report on three Japanese patients with cytochrome P450 oxidoreductase (POR) deficiency (PORD). Case one was a 46,XY patient who was found to have mildly increased 17alpha-hydroxyprogesterone (17-OHP) by the neonatal mass screening. There was no maternal virilization during pregnancy, and he had no skeletal or genital abnormality. Thus, he was initially diagnosed as having nonclassical 21-hydroxylase deficiency (21-OHD). Cases two and three were 46,XX patients who were identified because of severely virilized external genitalia and maternal virilization during pregnancy. In case two, the neonatal mass screening was normal, and she had no skeletal abnormality except for mild adduction of bilateral third toes. Thus, she was initially diagnosed as having aromatase deficiency. In case three, the neonatal mass screening showed moderately increased 17-OHP, and no skeletal lesion other than rigid second metacarpophalangeal joints was identified in early infancy. Thus, she was initially suspected as having 21-OHD and/or aromatase deficiency. Subsequently, endocrine studies including urine steroid hormone analysis were performed for the assessment of glucocorticoid treatment in case one and for the virilized genitalia in cases two and three, showing adrenal and/or gonadal dysfunction characteristic of PORD. Thus, molecular analysis of POR was carried out, demonstrating homozygosity for R457H in cases one through three. The results imply that clinical features in PORD can be similar to those in 21-OHD or aromatase deficiency, and that comprehensive assessment of the pregnant course, physical examination, and adrenal and gonadal function studies is essential for the precise diagnosis of PORD.

The role of luteinizing hormone in the pathogenesis of hyperadrenocorticism in neutered ferrets.

Four studies were performed to test the hypothesis that gonadotrophic hormones, and particularly luteinizing hormone (LH) play a role in the pathogenesis of ferrets: (I) adrenal glands of ferrets with hyperadrenocorticism were studied immunohistochemically to detect LH-receptors (LH-R); (II) gonadotrophin-releasing hormone (GnRH) stimulation tests were performed in 10 neutered ferrets, with measurement of androstenedione, 17alpha-hydroxyprogesterone and cortisol as endpoints; (III) GnRH stimulation tests were performed in 15 ferrets of which 8 had hyperadrenocorticism, via puncture of the vena cava under anesthesia; and (IV) urinary corticoid/creatinine (C/C) ratios were measured at 2-week intervals for 1 year in the same ferrets as used in study II. Clear cells in hyperplastic or neoplastic adrenal glands of hyperadrenocorticoid ferrets stained positive with the LH-R antibody. Plasma androstenedione and 17alpha-hydroxyprogesterone concentrations increased after stimulation with GnRH in 7 out of 8 hyperadrenocorticoid ferrets but in only 1 out of 7 healthy ferrets. Hyperadrenocorticoid ferrets had elevated urinary C/C ratios during the breeding season. The observations support the hypothesis that gonadotrophic hormones play a role in the pathogenesis of hyperadrenocorticism in ferrets. This condition may be defined as a disease resulting from the expression of LH-R on sex steroid-producing adrenocortical cells.

The diagnosis of congenital adrenal hyperplasia in the newborn by gas chromatography/mass spectrometry analysis of random urine specimens.

Definitive neonatal diagnosis of congenital adrenal hyperplasia (CAH) is frequently complicated by normal 17-hydroxyprogesterone levels in 21-hydroxylase-deficient patients, residual maternal steroids, and other interfering substances in neonatal blood. In an effort to improve the diagnosis, we developed a gas chromatography/mass spectrometry method for simultaneous measurement of 15 urinary steroid metabolites as early as the first day of life. Furthermore, we developed 11 precursor/product ratios that diagnose and clearly differentiate the four enzymatic deficiencies that cause CAH. Random urine samples from 31 neonatal 21-hydroxylase-deficient patients and 59 age-matched normal newborns were used in the development. Additionally, samples from two 11 beta-hydroxylase-deficient patients and one patient each for 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase deficiencies were used. The throughput for one bench-top gas chromatography/mass spectrometry instrument is 20 samples per day. Thus, this method affords an accurate, rapid, noninvasive means for the differential diagnosis of CAH in the newborn period without the need for invasive testing and ACTH stimulation.

[Effects of different loading exercises on endogenous sex hormones and their metabolites].

Small loading and strengthening exercises were performed by 22 athletes(11 males and 11 females). The levels of serum hormones and their urinary metabolites were detected. The results showed that the serum levels of testosterone(T), free testosterone(FT) and 17-OH-progesterone(17-OHP) in male athletes exhibited no conspicuous changes. The excretion of their urinary androsteroids was significantly increased after exercises(P < 0.05), but significantly decreased in the recovery period(P < 0.05). After small loading exercises, their serum levels of T and FT revealed no obvious changes except those of 17-OHP which evidently increased in female athletes(P < 0.05); whereas the excretion of their urinary steroid hormones declined promptly after the exercises and in the recovery period (all P < 0.05). After strengthening exercises, the serum levels of T, FT and 17-OHP decreased distinctly in female athletes (all P < 0.05), the excretion of their urinary steroid hormones raised clearly after training and in the recovery period(P < 0.05). Therefore, it is suggested that the secretion and metabolism of sex hormones in the organism are influenced by the loading quality of sports during training, and sexual difference exists simultaneously.