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Objective: Accurate measurement of steroid hormones remains challenging. Mass spectrometry affords a reliable means for quantitating steroid profiles accurately. Our objective was to establish and define (1) the extent of diurnal fluctuations in steroid concentrations that potentially necessitate strict adherence to time of sample acquisition and (2) time-dependent steroid reference intervals. Design: Nine steroid markers were examined in couplets in males and females. Methods: Using isotope dilution high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis, we developed a multi-steroid profile requiring only a minimal volume of serum (0.1 mL). Couplet (AM and PM) measurements of steroid hormones for 120 healthy females (F) and 62 healthy males (M) were obtained. Patients were recruited from several participating centers. Results: The following diurnal values were noted to be significantly different in both females and males: cortisone, cortisol, corticosterone, 11 deoxycortisol (11 DOC), androstenedione, 17a-hydroxyprogesterone (17 OHP) and dehydroepiandrosterone (DHEA). Testosterone was only found to have significant diurnal variance in males. Progesterone showed no significant difference in AM and PM values for either groups and thus may provide an internal control. Conclusions: When diagnosing endocrine disorders, it is imperative to acknowledge the 24-h diurnal variation of the biochemical steroid markers. We highlight the importance of standardization of collection times and appropriate implementation of reference intervals. Precis: We identify diurnal fluctuations in steroid concentrations with time of day and emphasize the importance of adhering to firm time of sample acquisition. National Center for Advancement of Translational Science (UL1TR001409) United States Department of Health & Human Services National Institutes of Health (NIH) - USA (NCT00428987) National Center for Advancement of Translational Science NIH Intramural Research Grant United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) (ZIEHD008737) United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Center for Advancing Translational Sciences (NCATS) (UL1TR001409) CLINICAL CENTER (ZIACL010355)

Background With liquid chromatography–tandem mass spectrometry (LC-MS/MS) increasingly being used for the quantification of steroid hormones , there is a need for studies that re-establish reference intervals and biological variation in well-defined cohorts. Methods A plasma steroid hormone profiling method using LC-MS/MS for quantification of progesterone , 17-hydroxyprogesterone , androstenedione , testosterone and dihydrotestosterone was developed and validated. For reference interval assessment, 280 well-characterized healthy subjects from the LifeLines cohort were selected, including 40 women using oral contraceptive pills (OCP). The biological variation was examined in 30 healthy individuals. Samples were collected over a period of 4 months with 4 week intervals. Results The developed method proved to be robust and sensitive. The reference interval levels in men are higher, whereas in women the levels tend to decrease with increasing age. In addition, women using OCP had lower levels of 17-OH-progesterone and androstenedione. The biological variation is generally higher in women compared to men, especially with regard to the inter-individual variation. Conclusions The gender-specific determination of the reference intervals, together with the observation that the biological variation demonstrated a high degree of variation, allows interpretation of data on individual and group level for improved biochemical characterization of patients in clinical practice.

A simple analytical procedure for the simultaneous determination of eight endogenous steroids (testosterone, androstenedione, 17β-estradiol, estrone, pregnenolone, progesterone, dihydroandrostenedione, and dihydrotestosterone) in aquatic molluscs by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC–MS) has been developed. After a microwave-assisted extraction, samples were further extracted and purified using two successive SPE (EnviChrom-P and NH2) cartridges. Steroids were derivatized with a mixture of N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA)/mercaptoethanol/ammonium iodide (NH4I) and determined by GC–MS in selective ion monitoring mode. Recoveries were in the range 85–114%, although slightly lower for dihydrotestosterone, and the repeatability of the procedure, expressed as the coefficient of variation, was lower than 16%. The limits of detection determined in digestive glands of mussels were in the range 0.1–0.4 ng g−1 wet weight for all the steroids. The developed procedure was then applied to the monitoring of steroid profiles in the digestive glands of mussels from the Arcachon Bay (France) during two reproductive cycles. In parallel, two physiological parameters (lipid content and the condition index of mussels) were also monitored, as well as the seawater temperature and salinity. Only progesterone and pregnenolone were detected in the digestive glands of mussels, and the seasonal variations of progesterone levels seemed to be related to the spawning periods of Mytilus sp. in the Bay. The current challenge for the determination of natural steroids in aquatic invertebrates is also briefly discussed.

Several studies have been performed during recent years to investigate the existence of a possible endocrine cause for premenstrual syndrome (PMS); the results reported are often discordant. Great interest has been raised around allopregnanolone, which could be involved in the determination of mood disorders reported by PMS patients. During the luteal phase, lower levels of this hormone have been detected in PMS patients. The aim of our study was to evaluate estradiol, progesterone, dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), androstenedione, total and free testosterone, cortisol, pregnenolone and allopregnanolone levels in 20 patients suffering from PMS and to compare them with those found in 20 fertile healthy women in the follicular and the luteal phases. Adrenocorticotropic hormone (ACTH) tests after dexamethasone suppression were performed in 10 patients of each group during the follicular and the luteal phases. In the PMS group, significantly lower allopregnolone levels were found in the luteal phase, while progesterone was lower in the PMS group in both phases. In the PMS group, higher free testosterone levels were found during the luteal phase and higher DHEA levels in both the follicular and the luteal phases. The present data confirm reduced allopregnanolone levels in the luteal phase in PMS patients, together with higher levels of DHEA and free testosterone. It is possible to conclude that, in addition to the previously described reduced luteal secretion of allopregnanolone, the adrenal gland production of this steroid in PMS sufferers is also impaired in the luteal phase. Considering the specific actions of these hormones on the control of mood and behavior, this specific hormonal milieu may contribute to the cyclic occurrence of anxiety, aggressiveness and irritability reported by PMS patients.

After the incubation of minced mammary tissues from non-lactating/non-pregnant (NL/NP), nonlactating/pregnant (NL/P), fully lactating (FL) and late-lactating (LL) cows with [14C]-labelled pregnenolone or progesterone and dehydroepiandrosterone (DHEA), the following metabolites were identified at all stages: 20alpha-dihydropregnenolone, progesterone (from pregnenolone), 5alpha-pregnanedione, 5alpha-pregnan-3beta-ol-20-one, 20alpha- and 20beta-dihydroprogesterone (from progesterone), 5-androstene-3beta,17beta-diol, 5alpha-androstanedione, 5alpha-androstan-3beta-ol-17-one, androstenedione, testosterone and DHEA acyl ester (from DHEA). These products indicate the occurrence of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase, 17beta-hydroxysteroid oxidoreductase (17beta-HOR), 20alpha- and 20beta-hydroxysteroid dehydrogenases, steroid 5alpha-reductase and acyl transferase activities. Incubation of mammary tissue homogenates with [1,2,6,7-(3)H]androstenedione and testosterone confirmed the presence of a 17beta-HOR acting prevalently in a reductive way but failed to show evidence of any aromatase activity beyond background level. When total RNA from mammary tissues of NL/NP and LL cows was reverse-transcribed and amplified by polymerase chain reaction (PCR) with three sets of primers specific for bovine P450scc, P450c17 and P450arom cDNAs, no fragment of the expected size could be detected on gel. Southern analysis with corresponding digoxigenin-labelled ovarian probes, however, gave a positive signal for P450arom cDNA in five out of eight samples of LL mammary tissue. These data indicate that the bovine mammary gland has very limited steroidogenic capabilities that are essentially compatible with the terminal activation of circulating steroids from steroidogenic endocrines. It is uncertain, however, whether this conclusion applies to anestrous or ovariectomized lactating cows as well.

In vitro biosynthesis of steroids from progesterone in ovaries and pyloric ceca of Asterias rubens has been investigated. The biosynthesis of 17α-hydroxyprogesterone, androstenedione, testosterone, 20α-dihydroprogesterone, 11-desoxycorticosterone, and 5α-pregnane-3,20-dione could be demonstrated to take place in the tissues of both organs by using [1,2-3H]progesterone as a precursor. The yields of intermediates of the Δ4-pathway and of 11-desoxycorticosterone are small, being higher in the ovaries than in the pyloric ceca. The yields of 20α-dihydroprogesterone are low, those of 5α-pregnane-3,20-dione are high. In both cases the yields in the pyloric ceca exceed those in the ovaries. The results indicate the presence of the following biosynthetic enzyme systems in ovaries and pyloric ceca of Asterias rubens: 17α-hydroxylase, C17---C20-lyase, 17β-HSD, 20β-HSD, 21-hydroxylase, and 5α-reductase. The importance of these enzymes for the metabolism of progesterone, i.e., the biosynthesis of C19-steroids, 20α-dihydroprogesterone, and corticosteroids, will be discussed.

1. 1. Homogenates of ovaries of 3- and 12-month-old guppies were incubated with pregnenolone-7α- 3 H and progesterone-4- 14 C, and with androstenedione-1,2- 3 H, respectively. 2. 2. From the double-labeled experiment, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione and testosterone were isolated and identified by paper- and thin-layer chromatography, derivative formation and by recrystallization to constant specific activity. 3. 3. In the ovary of the guppy the delta-5 pathway in steroidogenesis predominates, as may be concluded from the 3 H/ 14 C ratios of the identified metabolites. 4. 4. From the androstenedione-1,2- 3 H incubation, ovaries of 12-month-old guppies appeared to be able to synthesize steroids with functional groups in the 11-position (11-ketotestosterone and 11β-hydroxytestosterone). 5. 5. The androgen synthesis in ovaries of older guppies may be considered as a possible explanation of the sex conversion occasionally observed.