Fluorescence polarization immunoassay for rapid determination of dehydroepiandrosterone in human urine.

In this paper, five fluorescein-labeled dehydroepiandrosterone (DHEA) derivatives (tracers) with different chain lengths between the fluorescein and hapten were synthesized and featured so as to establish a fluorescence polarization immunoassay (FPIA) for DHEA detection in human urine samples with previously prepared polyclonal antibody against DHEA. The outcomes of the structure of tracer on FPIA sensitivity were investigated. Under the optimal condition, the fluorescence polarization value (FP) decreases linearly in DHEA concentration, ranging from 1.6 to 243.3 ng mL-1, with the limit of detection of 1.1 ng mL-1 and IC50 value of 25.1 ng mL-1. Moreover, the developed FPIA was time-saving as it could complete the detection within 3 min. FPIA and commercial enzyme-linked immunosorbent assay kit were both applied to analyze the spiked human urine samples with DHEA. Excellent recoveries (92.1-108.0%) and satisfactory correlation coefficient (R2 = 0.98) were acquired with the two methods, indicating that the developed FPIA was a fast and efficient screening immunoassay with accuracy and sensitivity for DHEA detection in human urine samples. Graphical abstract.

Suppression of adrenal function by low-dose prednisone: assessment with 24-hour urinary steroid hormone profiles--a review of five cases.

The impact of the synthetic glucocorticoid prednisone on adrenal steroid hormone production was examined using 24-hour urinary steroid hormone profiling. Five women, who were chronically taking low-dose prednisone, were tested, and the relevant literature was reviewed. As expected, adrenal glucocorticoid production, measured by urinary terminal cortisol and cortisone metabolites, was markedly suppressed compared to reference range values (p=0.03). Urinary cortisol and cortisone, reflecting circulating glucocorticoids, were decreased to a lesser extent than their terminal metabolites. Urinary dehydroepiandrosterone (DHEA) excretion was dramatically suppressed (p=0.03), while the downstream androgen metabolites androsterone and etiocholanolone were suppressed to a lesser extent. Aldosterone and tetrahydrocorticosterone production demonstrated modest suppression after prednisone administration, but allo-tetrahydrocorticosterone, which is highly sensitive to adrenocorticotropic hormone (ACTH) secretion, was suppressed to a greater extent. Prednisone administration results in a decrease in ACTH secretion by the anterior pituitary, suppressing synthesis of glucocorticoids, DHEA, and DHEA metabolites. Decreased glucocorticoid synthesis is adaptive, because prednisone is active at the glucocorticoid receptor, but suppression of DHEA synthesis is not mitigated by prednisone. DHEA is an important sex hormone precursor, neurosteroid, and endocrine and immune modulator; therefore, DHEA depletion may have significant adverse consequences in terms of sex hormone production, bone health, endocrine and immune system function, and neuropsychiatric status. Studies of DHEA replacement in patients taking prednisone for lupus demonstrate amelioration of some of these adverse effects.

Assessment of 5 alpha-reductase activity in hirsute women: comparison of serum androstanediol glucuronide with urinary androsterone and aetiocholanolone excretion.

OBJECTIVE: Recent evidence suggests that androstanediol glucuronide (AG), a metabolite of dihydrotestosterone (DHT) formed in skin, is frequently elevated in hirsute women, presumably reflecting enhanced 5 alpha-reductase activity. An alternative method of demonstrating 5 alpha-reductase activity is the androsterone (A)/aetiocholanolone (E) ratio in urine. A and E are the 5 alpha- and 5 beta-reduced metabolites, respectively, of androstenedione, which is the principal metabolite of dehydroepiandrosterone (D). Although serum AG and the urinary A/E ratio have both been considered valid methods for assessing 5 alpha-reductase activity, the two have not been previously compared in hirsute women. The present study was undertaken to assess 5 alpha-reductase activity in hirsute patients as determined by these two different methods. PATIENTS AND MEASUREMENTS: We surveyed 47 untreated women (ages 17-33) with various degrees of hirsutism. Serum testosterone, bioavailable testosterone, dehydroepiandrosterone sulphate, and AG were determined. Additionally, A, E and D were measured in 24-hour collections of urine. RESULTS: For the 47 women, 37 had elevated blood levels of AG (17.4 +/- 2.2, mean +/- SEM; normal < 8 nmol/l), but only 18 of these had an increased urinary A/E ratio (> 1.5). All but one of the remainder had elevated urinary and/or serum androgen levels. Overall, no significant correlation between AG and A/E was observed. There was a highly significant correlation between AG in serum and A in urine (r = 0.82, P < 0.001). AG was also positively related to dehydroepiandrosterone sulphate (r = 0.64; P < 0.005), bioavailable testosterone (r = 0.6; P < 0.001), aetiocholanolone (r = 0.58; P < 0.001) and total testosterone (r = 0.52; P < 0.01). In contrast, A/E was not significantly related to androgen production. CONCLUSIONS: There is a poor correlation between AG and the A/E ratio in hirsute women. Although AG may be raised by increased 5 alpha-reductase activity, it is probably also affected by the presence of elevated androgens regardless of 5 alpha-reductase activity.

Fractionation of urinary 17-ketosteroids by gas chromatography: a neglected procedure in the assessment of hyperandrogenicity in the hirsute female.

17-Ketosteroids were determined by gas chromatography in twenty four-hour urine samples from 62 hirsute females. The method permitted the determination of androsterone (A), aetiocholanolone (E) and dehydroepiandrosterone (D). Elevated concentrations of one or more of these metabolites were detected in 81% of the samples. Two main patterns of hyperandrogenicity were observed: 1) Hyper A + E (27%) and 2) Hyper A (26%). Elevated AD, AED, D, ED or E were less common, but in total these patterns comprised another 28%. The plasma testosterone and total urinary 17-ketosteroid concentrations were elevated in only 21% and 23% of the samples, respectively. Thirty two out of 33 patients with elevated urine metabolites showed significant suppression following dexamethasone administration (2 mg/day during 6 days). Thus, dexamethasone suppressable hyperandrogenicity was predominant in this group of hirsute females. Elevations of urinary androsterone and aetiocholanolone are probably contingent on the relative activities of 5 alpha- and 5 beta-reductases) in the presence of increased androstenedione secretion. Elevations of urinary dehydroepiandrosterone suggest decreased adrenal cortical 3 beta-hydroxysteroid dehydrogenase) activity. Thus, fractionation of urinary 17-ketosteroids seems to be an effective test in the evaluation of hirsutism.