Aim: This study aims to compare the anti-drug antibody (ADA) interference in four pharmacokinetic (PK) assays across different platforms (AlphaLISA, Gyrolab, LC-MS/MS) and to devise a strategy for ADA interference mitigation to improve the accuracy of measured drug in total PK assays. Materials & methods: Spiked test samples, created to achieve different ADA concentrations in human serum also containing an insulin analogue, were analyzed alongside pooled clinical samples using four assays. Results & conclusion: Interference was observed in all platforms. A novel approach using the Gyrolab mixing CD, including acid dissociation in the PK assay, significantly reduced interference and thereby improved relative error from >99% to <= 20% yielding measurements well within the acceptance criteria. Clinical sample results reinforced findings from the test samples.

Aim: With microsamples of blood, full pharmacokinetic profiles from individual animals can be obtained as an alternative to the sparse-sampling approach, where conventional volume samples from several animals are required. However, microsamples require assays that are more sensitive. Methods: The sensitivity of the LC-MS assay was increased 47-fold using microflow LC-MS. Results & conclusion: By analyzing both microsamples and conventional samples from the same animals, it is demonstrated that sparse-sampling profiles can be nonrepresentative of the full profiles. This bias can affect the tested treatment by increasing or reducing its apparent effect. Microsampling enables unbiased results compared with sparse-sampling. An increase in assay sensitivity to balance the low sample volumes was achievable by microflow LC-MS.

Extended half-life (EHL) factor VIII (FVIII)-replacement therapies enable patients with haemophilia A to maintain higher activity levels with fewer injections. N8-GP (turoctocog alfa pegol; Esperoct®) is an EHL product derived from conjugation of polyethylene glycol (PEG) to a recombinant FVIII protein. Upon activation, PEG is released from the active protein and excreted in urine and faeces. While PEG levels are expected to reach steady state with repeated dosing, there has been some discussion regarding whether abnormal accumulation of PEG in plasma and tissues may occur. Our objective was to examine plasma PEG concentrations in rats and humans repeatedly treated with N8-GP for periods of up to 5 years. PEG levels were measured using liquid chromatography-tandem mass spectrometry in plasma samples from rats treated with N8-GP as part of a 52-week toxicity study. Human plasma samples from children, adolescents and adults treated with N8-GP as part of the pathfinder programme were also examined (NCT01731600; NCT01480180). These data were compared with steady-state PEG levels predicted by pharmacokinetic modelling of single-dose rat data. PEG levels reached steady state in plasma in both rats and humans after repeated dosing. The timing and degree of PEG increase to steady state were in line with or below model predictions, confirming the utility of the pharmacokinetic model and indicating that rat data can be used to estimate human plasma PEG levels. Steady-state PEG levels were reached in plasma from rats and humans repeatedly treated with N8-GP. No unexpected increase in PEG was observed.

An isotope-dilution TurboFlow-LC-MS/MS method for simultaneous quantification of the ten steroid metabolites dehydroepiandrosterone sulfate (DHEAS), progesterone, 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), corticosterone, 11-deoxycortisol, cortisol, cortisone, testosterone (T), and estrone 3-sulfate (E1-S) in serum was developed and validated. Limits of quantification, variability (inter- and intra-day), analytical range and linearity were all found to be acceptable for clinical use. Furthermore, sample stability was evaluated including the influence of freeze-thaw cycles and the effects of temperature and storage time. The method was applied to 391 serum samples from healthy, Danish boys 10-18years old. The concentration ranges of the included steroid metabolites for this population are presented. Concentrations of DHEAS, 17-OHP, Adione and T in the 391 serum samples were furthermore compared to results obtained using an existing LC-MS/MS method in our laboratory. Excellent agreement was found between the methods. Furthermore, the improved sensitivity of the new method allowed for quantification of a number of samples found to be below the LOQs of the existing method. Thus, the two instruments and their associated methods were validated as possible back-ups for each other, which we consider an extremely important issue in high-throughput laboratories analyzing clinical samples on a regular basis. The ten analytes included can be analyzed simultaneously but it is also possible only to include some of these analytes for specific diagnostic purposes which make the new method an extremely useful tool in the clinical laboratory.

SCOPE Resveratrol has a diverse array of healthful effects on metabolic parameters in different experimental paradigms but has also potential to inhibit steroidogenesis in rodent adrenals. The aim of the present study was to characterize the effects of resveratrol on human fetal adrenal steroidogenesis at gestational weeks (GW) 9-12. METHODS AND RESULTS Adrenals from aborted fetuses (GW10-12) were used to prepare primary cultures of human fetal adrenocortical cells (HFAC). HFAC were treated in the presence or absence of ACTH (10 ng/mL) with or without resveratrol (10 μM) for 24 h. The production of steroids by HFAC was analyzed by gas and liquid chromatography coupled to tandem/mass spectrometry. The expression of steroidogenic enzymes at GW 9-12 was quantified by automated Western blotting. We observed that resveratrol significantly suppressed synthesis of dehydroepiandrosterone (DHEA), androstenedione and 11-deoxicortisol by ACTH-activated and unstimulated HFAC, which was associated with inhibition of the activities and expression of cytochromes 17α-hydroxylase/17,20 lyase (CYP17) and 21-hydroxylase (CYP21) in these fetal adrenocortical cells. CONCLUSION Our in vitro findings on the sensitivity of human fetal adrenal steroidogenesis to resveratrol at GW9-12 suggest that intake of this polyphenol at high doses by women who are at early stages of pregnancy is undesirable.

We report reference ranges based on LC-MS/MS for testosterone (T), free testosterone (FT) and its precursors, i.e. 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEAS) and androstenedione (Adione), in relation to different health markers and lifestyle factors.The study was based on 304 healthy men aged 30-61 years participating in a population-based cross-sectional study (Health2008). Examination program consisted of a clinical examination, completion of a self-administered questionnaire and blood sampling. Steroid metabolites were measured by a validated and sensitive LC-MS/MS method. Older age-groups were significantly associated with decreased concentrations of DHEA, DHEAS, Adione, and FT, while no significant associations with age were shown for 17-OHP or T. Participants with BMI >= 30 kg/m(2) had lower age-related steroid metabolite z-scores compared to participants with BMI < 30 kg/m(2), i.e. 17-OHP: -0.51 vs. 0.08 (p < 0.001); DHEA: -0.27 vs. 0.09 (p = 0.014); Adione: -0.29 vs. 0.09 (p = 0.012); T: -0.99 vs. 0.14 (p < 0.001); and FT -0.55 vs. 0.05 (p < 0.001), respectively.In conclusion, this large study on serum steroid metabolites and concomitant assessment of health markers in healthy men provides age-related reference ranges, and furthermore evaluates the impact of lifestyle factors and metabolic syndrome on androgen metabolite levels. (C) 2016 Elsevier B.V. All rights reserved.

BACKGROUND Recently, LC-MS/MS was stated to be the method of choice to measure sex steroids. Because information on the mutual agreement of LC-MS/MS methods is scarce, we compared 7 published LC-MS/MS methods for the simultaneous measurement of testosterone, androstenedione, and dehydroepiandrosterone (DHEA). METHODS We used 7 published LC-MS/MS methods to analyze in duplicate 55 random samples from both men and women. We performed Passing-Bablok regression analysis and calculated Pearson correlation coefficients to assess the agreement of the methods investigated with the median concentration measured by all methods, and we calculated the intraassay CV of each method derived from duplicate results and the CVs between the methods. RESULTS Median concentrations of testosterone were 0.22-1.36 nmol/L for women and 8.27-27.98 nmol/L for men. Androstenedione and DHEA concentrations were 0.05-5.53 and 0.58-18.04 nmol/L, respectively. Intraassay CVs were 2.9%-10%, 1.2%-8.8%, 2.7%-13%, and 4.3%-16% for testosterone in women, testosterone in men, androstenedione, and DHEA. Slopes of the regression lines calculated by Passing-Bablok regression analysis were 0.92-1.08, 0.92-1.08, 0.90-1.13, and 0.91-1.41 for all testosterone values, testosterone in women, androstenedione, and DHEA. Intermethod CVs were 14%, 8%, 30%, and 22% for testosterone in women, testosterone in men, androstenedione, and DHEA. CONCLUSIONS In general, the LC-MS/MS methods investigated show reasonable agreement. However, some of the assays show differences in standardization, and others show high variation.

Adrenarche is characterized by steadily rising levels of adrenal androgen metabolites from 4–6 years of age. We recently described marked gender-specific differences in circulating ratios between selected adrenal androgen metabolites in a cross-sectional study. This may suggest gender differences in steroidogenic enzyme activities. We therefore aimed at verifying these findings in a prospective, longitudinal study of healthy boys and girls who were examined during pubertal transition.A longitudinal study of 20 healthy children from the COPENHAGEN Puberty Study, followed every 6 months for 5 years. Clinical examinations were conducted and serum concentrations of Androstenedione (Adione), 17-hydroxyprogesterone (17-OHP), testosterone (T), dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) were quantified by a newly developed LC-MS/MS method. DHEA, DHEAS, Adione, 17-OHP and T increase with age. Boys had higher levels of DHEAS from 10.5 years of age, whereas girls had higher levels of Adione from 13 years of age compared to boys. Interestingly, we observed significantly higher ratios of DHEAS/DHEA (sulfotransferase activity) in boys before and after pubertal onset compared to girls, whereas Adione/17-OHP (CYP 17 activity) appeared to increase more in pubertal girls compared to boys. This suggests that adrenal steroidogenic enzyme activities show developmental as well as gender-specific changes in healthy children.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Background: Accurate and selective assessment of testosterone requires use of a sensitive LC-MS/MS method, especially at low levels as those seen in young children. Methods: The present longitudinal study of 20 healthy children from the Copenhagen Puberty Study followed every 6 months for 5 years evaluates the longitudinal increase in serum testosterone before, during and after pubertal onset quantified by a newly developed LC-MS/MS method in comparison with immunoassay. Testosterone concentrations in serum samples (n = 177) were determined by LC-MS/MS (detection limit 0.1 nmol/l) and by immunoassay (detection limit 0.23 nmol/l). Results: Serum concentrations of testosterone increased gradually with age by both methods. However, serum testosterone was quantifiable in 9/10 girls prior to pubic hair development measured with LC-MS/MS, and in 2/10 girls measured with immunoassay. In boys, testosterone was quantifiable in 10/10 boys 1 year prior to pubic hair development measured with LC-MS/MS, and only in 1/10 boys measured with immunoassay. Serum testosterone levels were quantifiable 1.5 years (range 0.5-2.5) earlier using LC-MS/MS. Conclusion: Assessment of longitudinal circulating levels of serum testosterone using a selective LC-MS/MS method proved to be more sensitive in predicting early peripubertal changes in healthy children compared to levels determined by immunoassay.

The influence of sex, age, pubertal development and oral contraceptives on dehydroepiandrosterone (DHEA), DHEA sulfate (DHEAS), 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), testosterone (T), calculated free testosterone (fT), free androgen index (FAI) and selected ratios in 1798 serum samples from healthy children, adolescents and young adults was evaluated. Samples were analyzed by Turboflow-LC-MS/MS. Sex hormone-binding globulin was analyzed by immunoassay. All steroid metabolite concentrations were positively associated with age and pubertal development in both sexes and generally higher in males than in females except for Adione. The pubertal rise in T in males was more pronounced compared to females, reflecting contribution from the testes. Ratios between steroid metabolites varied and depended on sex and age. All ratios were lower during infancy compared to later in life. Use of oral contraceptives significantly lowered serum concentrations of all steroid metabolites, fT, FAI, the 17-OHP/Adione, the Adione/T and the DHEA/Adione ratios, but not the DHEA/DHEAS ratio. We provide reference ranges for DHEA, DHEAS, 17-OHP, Adione, T, fT, FAI and selected ratios in relation to sex, age and pubertal development. Use of oral contraceptives strongly influences adrenal steroidogenesis and should be considered when diagnosing and monitoring treatment of patients with disorders of sex development.

Human exposure to chemicals may be estimated by back-calculating urinary concentrations resulting from biomonitoring studies if knowledge of the chemical's toxicokinetic properties is available. In this paper, available toxicokinetic data for back-calculating urinary concentrations into daily intake values for bisphenol A (BPA), phthalates, parabens, and triclosan (TCS) are reviewed and knowledge gaps are identified. Human data is evaluated and presented with relevant animal data. Focus is on the recovery of the administered dose, the route of administration, and differences between humans and animals. Two human toxicokinetic studies are currently used to conclude that an oral dose of BPA is recoverable in urine and that no free BPA is present in plasma in spite of several contradicting biominotoring studies. Urinary recovery of an oral dose of phthalates in humans is complicated to assess due to extensive metabolism. In animals using 14 C-marked phthalates, near-complete recovery is observed. An oral dose of 14 C-marked parabens is also almost completely recovered in animals. In both humans and animals, however, two unspecific metabolites are formed, which complicates the back-calculation of parabens in humans. The recovery of both oral and dermal TCS in humans has been studied, but due to background levels of TCS, the back-calculation is difficult to perform. In conclusion, due to limited data, reasonable estimates of daily intake values based on urinary data are often not possible to obtain. Several knowledge gaps are identified and new studies are suggested. The route of administration used in toxicokinetic studies often does not match realistic scenarios.

SummaryBackgroundAdrenal disorders such as congenital adrenal hyperplasia result in abnormal adrenal size and morphology, but little is known about the clinical value of magnetic resonance imaging (MRI) in determining adrenal volume.ObjectiveTo evaluate the potential usefulness of MR methodology, to estimate adrenal size in healthy children and to evaluate determinants of adrenal volume such as age, gender, body size, pubic hair stage and serum levels of adrenal androgens.DesignTwo hundred and thirty‐five healthy children (116 girls and 119 boys) (age range 10·0–14·8 years) were examined by MRI. Clinical examinations (anthropometry and pubertal staging) were performed, and five androgen metabolites were measured in blood samples by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS).ResultsIt was possible to determine adrenal volume in 115/235 children using MRI. The adrenals were not measurable in 51% of children due to breathing and moving artefacts. The median volume of the right adrenal gland was 0·46 ml in girls and 0·46 ml in boys. The median volume of the left adrenal gland was 0·34 ml in girls and 0·40 ml in boys. Adrenal size was positively associated with body surface area (estimate B = 0·34 ml/year, P = 0·003), age (estimate B = 0·05 ml/year, P = 0·021) and pubic hair stage (estimate B = 0·05 ml/stage, P = 0·075). No associations between adrenal size and serum levels of adrenal androgens were observed.ConclusionIt was possible to determine adrenal volume by MRI in only 50% of healthy children aged 10–15 years. Adrenal volume increased with age and Tanner stage of pubic hair. Future studies will unravel whether adrenal MRI is useful when evaluating children with adrenal diseases.

Insulin-like factor 3 (INSL3) is a promising marker of Leydig cell function with potentially high clinical relevance. Limited data of INSL3 levels in relation to other reproductive hormones in healthy pubertal boys exist. In this study, we aimed to evaluate longitudinal serum changes in INSL3 compared with LH, FSH, testosterone, inhibin B, and anti-Müllerian hormone (AMH) during puberty in healthy boys. Ten boys were included from the longitudinal part of the COPENHAGEN Puberty Study. Pubertal evaluation, including testicular volume, was performed and blood samples were drawn every 6 months for 5 years. Serum concentrations of testosterone were determined by a newly developed LC-MS/MS method, and serum concentrations of INSL3, AMH, inhibin B, FSH, and LH respectively were determined by validated immunoassays. The results showed that serum INSL3 levels increased progressively with increasing age, pubertal onset, and testicular volume. In six of the ten boys, LH increased before the first observed increase in INSL3. In the remaining four boys, the increase in LH and INSL3 was observed at the same examination. The increases in serum concentrations of LH, testosterone, and INSL3 were not parallel or in ordered succession and varied interindividually. We demonstrated that INSL3 concentrations were tightly associated with pubertal onset and increasing testicular volume. However, the pubertal increases in LH, INSL3, and testosterone concentrations were not entirely parallel, suggesting that INSL3 and testosterone may be regulated differently. Thus, we speculate that INSL3 provides additional information on Leydig cell differentiation and function during puberty compared with traditional markers of testicular function.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Diagnosis and management of infants and children with sex steroid disorders require fast and simultaneous assessment of several sex steroid metabolites in serum at low concentrations and on small sample volumes. Therefore, we developed a sensitive and selective TurboFlow-LC-MS/MS method for quantification of DHEA, DHEAS, 17 alpha-hydroxyprogesterone, Delta 4-androstenedione and testosterone in serum from pre-pubertal children. Run time was 10.75 min. Limits of quantification were as follows: DHEA, 0.88 nM; DHEAS, 48 nM; 17 alpha-hydroxyprogesterone, 0.19 nM; Delta 4-androstenedione, 0.18 nM and testosterone, 0.10 nM. Intra-day relative standard deviation ranged from 4.6 to 13.8% and inter-day relative standard deviation ranged from 5.7 to 15.7%. Steroid concentrations in 38 serum samples from pre-pubertal children were compared with results obtained by immunoassays for DHEAS, Delta 4-androstenedione and testosterone. DHEAS gave overall similar results but with several outliers, while levels of Delta 4-androstenedione were found to be much lower when analysed by LC-MS/MS. Testosterone was not detected in any of the samples analysed using a sensitive immunoassay, while 30 of 38 samples were quantifiable using the current LC-MS/MS method. The presented method is suitable in a clinical setting for simultaneous quantification of five steroids important for management of children with disorders of sex development and steroid biosynthesis defects. (C) 2013 Elsevier B.V. All rights reserved.

Human risk assessment of chemicals is traditionally presented as the ratio between the actual level of exposure and an acceptable level of exposure, with the acceptable level of exposure most often being estimated by appropriate authorities. This approach is generally sound when assessing the risk of individual chemicals. However, several chemicals may concurrently target the same receptor, work through the same mechanism or in other ways induce the same effect(s) in the body. In these cases, cumulative risk assessment should be applied. The present study uses biomonitoring data from 129 Danish children and adolescents and resulting estimated daily intakes of four different phthalates. These daily intake estimates are used for a cumulative risk assessment with anti-androgenic effects as the endpoint using Tolerable Daily Intake (TDI) values determined by the European Food Safety Authorities (EFSA) or Reference Doses for Anti-Androgenicity (RfD AA) determined by Kortenkamp and Faust [Int J Androl 33 (2010) 463] as acceptable levels of exposure. United States Environmental Protection Agency Reference Doses (US EPA RfD) could not be used as none of them identifies anti-androgenic effects as the most sensitive endpoint for the phthalates included in this article. Using the EFSA TDI values, 12 children exceeded the hazard quotient for the sum of di-n-butyl phthalate and di-iso-butyl phthalate (∑DBP((i+n))) and one child exceeded the hazard quotient for di-(2-ethylhexyl)phthalate (DEHP). Nineteen children exceeded the cumulated hazard index for three phthalates. Using the RfD AA values, one child exceeded the hazard quotient for DEHP and the same child exceeded the cumulated hazard index for four phthalates. The EFSA TDI approach thus is more restrictive and identifies ∑DBP((i+n)) as the compound(s) associated with the greatest risk, while DEHP is the compound associated with the greatest risk when using the RfD AA approach.