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Two sisters with hirsutism presented with mild hirsutism and isolated, grossly elevated (> 34.9 nmol/L) serum concentrations of androstenedione measured by competitive, homogeneous immunoassay. The clinically discordant laboratory results prompted us to look for assay interference. In this immunoassay, horseradish peroxidase (HRP)-conjugated androstenedione competes with endogenous androstenedione for binding with the solid-phase polyclonal rabbit IgG antibodies. After a wash step, the amount of signal generated by the bound HRP conjugate is inversely proportional to the androstenedione concentration. Alternative analysis by tandem mass spectrometry (a good first line option for troubleshooting) and repeating the competitive immunoassay after polyethylene glycol treatment returned androstenedione concentrations within reference limits. These findings suggested that the original result was spuriously elevated due to assay interference. Additionally, the patient samples were pre-incubated with heterophile blocking reagents, normal rabbit IgG antibodies and HRP-conjugated normal goat IgG antibodies, followed by repeat measurement using the immunoassay. Only samples pre-incubated with HRP-conjugate returned significantly lower androstenedione (9.5 and 12.5 nmol/L, respectively), implying neutralisation of the interfering antibodies. Androstenedione remained grossly elevated in the other experiments. This deductive exercise showed that the interference is due to autoantibodies against the HRP label used in the immunoassay. Another immunoassay using HRP label (5α-dihydrotestosterone) also produced gross elevation that was normal by tandem mass spectrometry analysis. Assay interferences, though not uncommon, are frequently overlooked. Laboratory results discordant with clinical features should prompt consideration of assay interference to avoid unnecessary investigations and treatment. This is the first report of autoantibodies against the HRP label used in immunoassay. [ABSTRACT FROM AUTHOR]

Background Liquid-chromatography tandem mass spectrometry (LC–MS/MS) has become the method of choice in steroid hormone measurement. However, little information on the mutual agreement of LC–MS/MS methods is available. We compared eight routine unpublished LC–MS/MS methods for the simultaneous measurement of testosterone and androstenedione. Methods Sixty random serum samples from male and female volunteers were analysed in duplicate by eight routine LC–MS/MS methods. We performed Passing–Bablok regression analyses and calculated Pearson's correlation coefficients to assess the agreement of the methods investigated with one published method known to be accurate. Intra-assay CV of each method was calculated from duplicate results, recoveries for each method were calculated from six spiked samples. Furthermore, a CV between the investigated methods was calculated. Results The concentrations ranged from 0.05–1.26 nmol/L, 6.15–24.44 nmol/L and 0.15–4.78 nmol/L for testosterone in females, testosterone in males and androstenedione, respectively. The intra-assay CVs were between 3.7–16.0%, 0.9–5.2% and 1.2–9.5% for testosterone in females, testosterone in males and androstenedione, respectively. The slopes of the regression lines ranged between 0.90–1.25, 0.87–1.24 and 0.94–1.31 for testosterone concentrations in females, all testosterone values and androstenedione, respectively. Inter-method CVs were 24%, 14% and 29% for testosterone for concentrations in females and males and androstenedione, respectively. These compare unfavourably to the variation found earlier in published methods. Conclusion Although most routine LC–MS/MS methods investigated here showed a reasonable agreement, some of the assays showed a high variation. The observed differences in standardization should be taken into account when applying reference values, or should, preferably, be solved. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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, 17α-hydroxyprogesterone, Δ4-androstenedione and testosterone in serum from pre-pubertal children. Run time was 10.75min. Limits of quantification were as follows: DHEA, 0.88nM; DHEAS, 48nM; 17α-hydroxyprogesterone, 0.19nM; Δ4-androstenedione, 0.18nM and testosterone, 0.10nM. 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, Δ4-androstenedione and testosterone. DHEAS gave overall similar results but with several outliers, while levels of Δ4-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. [Copyright &y& Elsevier]

Abstract: Background: Salivary steroid measurements have the potential to provide a non-invasive assessment of serum free steroid concentrations. Salivary testing has been proposed as a possible substitute for serum testing partly because of easy sample collection. Methods: We developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for simultaneous measurement of testosterone and androstendione in saliva. We spiked 100μL of saliva with isotope-labeled internal standards and performed extraction with diethyl ether. After evaporation both steroids were analyzed by LC–MS/MS operating in the positive mode electrospray ionization (ESI) after separation on a reversed-phase column. Results: The calibration curves for analysis of testosterone and androstendione exhibited consistent linearity and reproducibility in the range of 20–1000pmol/L. The limit of quantitation (LOQ) was 10pmol/L for both steroids. The mean recovery of the analytes added to saliva ranged from 97 to 102%. Between-run CVs were 3.6–4.0% for testosterone and 4.8–8.0% for androstendione. The upper limit (the 97.5 percentile) of testosterone and androstendione reference ranges for males was 356pmol/L and 344pmol/L and that for females 39pmol/L and 349pmol/L, respectively. Conclusions: This LC–MS/MS method has the required lower limit of quantification for analysis of salivary concentrations of testosterone and androstendione. The assay is rapid, reliable and simple to perform with a routine LC–MS/MS spectrometer with required sensitivity. [Copyright &y& Elsevier]

Abstract: Background: Newborn screening for congenital adrenal hyperplasia (CAH) is commonly accomplished by measurement of 17-α-hydroxyprogestrone (17-OHP) using enzyme immunoassay (EIA). EIA contributes a significant number of false positives. Therefore, second-tier steroid profile by liquid chromatography–tandem mass spectrometry (LC–MS/MS) is warranted. Methods: Dried blood spots (DBS) were extracted with a mixture of methanol and water containing the deuterium labeled internal standards of d8-17-OHP, d7-androstenedione, and d4-cortisol. The final extracts were analyzed for 17-OHP, androstenedione and cortisol by LC-MS/MS in the multiple reaction monitoring (MRM) mode. Results: Mean recoveries of the target analytes, 17-OHP, androstenedione and cortisol, were between 97 and 115% with an average intra- and inter-assay CVs ranging from 3.9–9.9% to 3.6–10.1%, respectively. The high efficiency of this method enabled us to test 11,598 specimens, identified as indeterminate by EIA in ~6 years; resulting in 809 presumptive positives reducing the false positives rate by 93%. Conclusions: The three steroid profile provided better screening outcomes of CAH than 17-OHP concentration alone. Our sample preparation allowed high throughput using common laboratory chemicals. Using three internal standards significantly improved method precision and accuracy. The reduction in false positives significantly reduces anxiety for newborns and their families. [Copyright &y& Elsevier]

Deficiency of 17β-hydroxysteroid dehydrogenase type3 (17β-HSD3) isoenzyme which catalyzes the synthesis of testosterone from Δ4-androstenedione, is the cause of 46, XY disorders of sex development (DSD). 17β-HSD3 deficiency is a rare autosomal recessive disorder, which is caused by mutations in the HSD17B gene found on chromosome 9q22. Up to now, almost 33 mutations in the HSD17B3 gene have been reported. Here, we report a patient with a novel mutation in HSD17B3 gene leading to 17β-HSD3 deficiency. The patient was admitted because of primary amenorrhea and signs of virilization at puberty. The chromosome analysis showed a 46, XY karyotype. Hormonal evaluation revealed a high Δ4-androstenedione level with a low serum testosterone/androstenedione (T/A) ratio. Sequence analysis of HSD17B3 gene revealed the presence of a homozygous missense mutation in exon 11 resulting in a premature stop codon (p.Y287). Gonadectomy was performed after the molecular diagnosis and estrogen replacement therapy was initiated. With this report, we emphasize that 17β-HSD3 deficiency should be considered in virilized female patients at puberty if the T/A ratio is less than 0.8, and the molecular analysis should be performed for the precise diagnosis and genetic counseling. [ABSTRACT FROM AUTHOR]

The adrenal and gonadal androgens, testosterone, androstenedione and dehydroepiandrosterone (DHEA) play an important role in sexual development as well as in other processes. We developed a method for simultaneous quantitative analysis of serum and plasma testosterone, androstenedione and DHEA levels using Isotope-Dilution Liquid-Chromatography Tandem Mass Spectrometry (ID-LC–MS/MS). Samples underwent liquid–liquid extraction and were analyzed on an Acquity 2D-UPLC-System and a Xevo TQ-S tandem mass spectrometer (Waters). The intra-assay and inter-assay coefficients of variation were < 4.0%, < 6.3% and < 7.0% and < 6.0%, < 8.1% and < 7.7% for testosterone, androstenedione and DHEA, respectively. Inter-assay CVs at the lower limit were 10.6%, 16.9% and 9.0% for testosterone (0.10 nmol/L), androstenedione (0.10 nmol/L) and DHEA (1.0 nmol/L), respectively. Recoveries of spiked analytes were 93–107%. The present testosterone method compared well (y = 1.00x − 0.04; r = 0.998) to a published ID-LC–MS/MS method for testosterone in our lab. The latter method being concordant with a published reference method (Bui et al., 2013). The present method compared well to a published ID-LC–MS/MS method (Kushnir et al., 2010) (y = 1.06x − 0.06; r = 0.996 for testosterone; y = 1.04x − 0.04; r = 0.995 for androstenedione and y = 1.03x + 0.01; r = 0.991 for DHEA). In conclusion, we developed a sensitive and accurate ID-LC–MS/MS method to simultaneously measure serum testosterone, androstenedione and DHEA in serum and plasma. [ABSTRACT FROM AUTHOR]