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7α-Hydroxy-4-cholesten-3-one (C4) is the common intermediary of both primary bile acids. C4 is recommended by the British Society of Gastroenterology for the investigation of bile acid diarrhoea (BAD) in patients with chronic diarrhoea. This project aimed to develop and validate an assay to quantitate C4 in serum and assess the stability of C4 in unseparated blood.Accuracy was underpinned by calibrating to quantitative nuclear magnetic resonance analysis. C4 was analysed in a 96-well plate format with a deuterated C4 internal standard and liquid-liquid extraction. Validation followed the 2018 Food and Drug Administration guidelines. To assess C4 stability, healthy volunteers (n=12) donated 8 fasted samples each. Samples were incubated at 20 °C for up to 72 h and retrieved, centrifuged, aliquoted and frozen for storage at different time points prior to C4 analysis.The C4 method demonstrated excellent analytical performance and passed all validation criteria. The method was found to be accurate, precise, free from matrix effects and interference. After 72 h of delayed sample separation, C4 concentration gradually declined by up to 14 % from baseline. However, the change was not significant for up to 12 h.We present a robust method of analysing serum C4, offering a convenient alternative to 75SeHCAT for BAD investigation. C4 was found to decline in unseparated blood over time; however, after 12 h the mean change was <5 % from baseline. Our results suggest C4 is suitable for collection from both primary and secondary care prior to gastroenterology referral. [ABSTRACT FROM AUTHOR]

Recent studies suggest that vitamin D-deficiency is linked to increased risk of common human health problems. To define vitamin D 'status' most routine analytical methods quantify one particular vitamin D metabolite, 25-hydroxyvitamin D3 (25OHD3). However, vitamin D is characterized by complex metabolic pathways, and simultaneous measurement of multiple vitamin D metabolites may provide a more accurate interpretation of vitamin D status. To address this we developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse multiple vitamin D analytes, with particular emphasis on the separation of epimer metabolites. A supportive liquid-liquid extraction (SLE) and LC-MS/MS method was developed to quantify 10 vitamin D metabolites as well as separation of an interfering 7α-hydroxy-4-cholesten-3-one (7αC4) isobar (precursor of bile acid), and validated by analysis of human serum samples. In a cohort of 116 healthy subjects, circulating concentrations of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3), 24,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3), and 25-hydroxyvitamin D2 (25OHD2) were quantifiable using 220μL of serum, with 25OHD3 and 24R,25(OH)2D3 showing significant seasonal variations. This high-throughput LC-MS/MS method provides a novel strategy for assessing the impact of vitamin D on human health and disease.

The most suitable biochemical markers for therapy adjustment in patients with congenital adrenal hyperplasia are controversial. 11-Oxygenated androgens are a promising new approach. The objective of this study was to investigate the diurnal rhythm of 11-ketotestosterone in children and adolescents in saliva and to correlate it with salivary 17-hydroxyprogesterone. Fifty-one samples of steroid day-profiles from 17 patients were additionally analysed for 11-ketotestosterone, retrospectively. All patients were treated in our university outpatient clinic for paediatric endocrinology between 2020 and 2022. Steroid day-profiles of 17 patients could be examined. The cohort showed a balanced sex ratio. The median age was 13 years. The measurements for 17-hydroxyprogesterone were carried out during routine care by immunoassay. The measurements of 11-ketotestosterone were performed from frozen saliva samples using an implemented in-house protocol for liquid chromatography–tandem mass spectrometry (LC-MS/MS). The most important outcome were the absolute values for 11-ketotestosterone, their diurnal rhythmicity and the correlation with 17-hydroxyprogesterone. Both steroids show a circadian diurnal rhythm. 17-hydroxyprogesterone and 11-ketotestosterone correlate significantly. 11-Ketotestosterone showed a positive correlation with BMI at all times of the day. 11-Ketotestosterone shows circadian rhythmicity in our cohort and correlates with 17-hydroxyprogesterone. These findings serve as an important basis for prospective research into 11-oxygenated androgens as therapeutic markers in paediatrics. However, 11-ketotestosterone appears to be very dependent on BMI. [ABSTRACT FROM AUTHOR]

Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. Intra-laboratory CVs ranged between 4.2–13.2 % for androstenedione, 1.6–10.8 % for DHEAS, and 4.3–8.7 % and 2.6–7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were −13.3 to 20.5 % and −4.9 to 18.7 % for androstenedione, −10.9 to 4.8 % and −3.4 to 3.5 % for DHEAS, −2.7 to 6.5 % and −11.3 to 6.6 % for testosterone in females, and −7.0 to 8.5 % and −7.5 to 11.8 % for testosterone in males, respectively. Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability. [ABSTRACT FROM AUTHOR]

A study published in the British Journal of Dermatology examined the adherence to treatment in patients with psoriasis using serum methotrexate (MTX) levels as a measure. The study found that 39.3% of patients surveyed showed biochemical nonadherence to MTX, suggesting that nonadherence may be common in psoriasis treatment. The study emphasized the need for objective measures of adherence, as self-reported adherence may be overestimated. However, the study had limitations such as a small sample size and the inability to collect patient demographics and clinical response data. Further research is needed to understand the impact of nonadherence on treatment outcomes in psoriasis. The study was supported by the National Institute for Health and Care Research and Versus Arthritis, and the authors disclosed conflicts of interest related to research grants and fees from pharmaceutical companies. [Extracted from the article]

To develop a sensitive liquid chromatography-tandem mass spectrometry method capable of measuring serum methotrexate in patients with rheumatoid arthritis to assess adherence to drug treatment. Isotopically labelled internal standard and deionised water were added to sample prior to solid phase extraction using a Waters Oasis Max ion-exchange 96-well plate. Following extraction, samples were analysed by LC-MS/MS on a TQS-micro mass spectrometer. Mean recovery was 107 % for four different concentrations of methotrexate spiked into seven patient samples, whilst post extraction spiking gave a mean recovery of 100 %. Between-batch and within-batch CVs were ≤6 % at three different concentrations of methotrexate in fresh frozen plasma. Mean bias was <5 % for between-batch and within batch analysis at three different weighed in concentrations of methotrexate certified reference material. The lower limit of quantification of the assay was 0.1 nmol/L with linearity up to approximately 100 nmol/L. Dilution linearity studies were used to validate the dilution of patient samples prior to analysis. There was no significant interference in the method from lipaemia, haemolysis or icterus. A sensitive LC-MS/MS assay for methotrexate has been developed and validated. The method has been used to measure methotrexate adherence in patient samples from clinical trials and could be used in future research to assess the ability of the assay as a biofeedback intervention to improve adherence to methotrexate therapy. [ABSTRACT FROM AUTHOR]

Context Quality of life (QoL) has been inconsistently reported in children and young people (CYP) with congenital adrenal hyperplasia (CAH). Objective Assess QoL in CYP with CAH in the UK alongside biometric and androgen profiles. Design To define the evidence base for health care delivery, we conducted a cross-sectional study in CYP with CAH in the UK. Questionnaire results were compared with normative data and between groups, and modelled for association with sex, height, weight, body mass index, or steroid biomarkers of CAH control. Setting Tertiary care in 14 UK centers. Patients Results from 104 patients, 55% female, mean age 12.7 years (SD 3.0), paired responses from parents. Interventions Strengths and Difficulties questionnaire (SDQ) and pediatric QoL questionnaire. Main Outcome Measure Total QoL scores as assessed by SDQ and a pediatric QoL questionnaire in comparison to normative data. Results Total scores were worse in parents than normative data, but similar in patients. Patient QoL was rated better in social functioning but worse in emotional, school, and peer domains by patients, and worse in total scores and domains of peer problems, and psychosocial, emotional, and school functioning by parents. Parents consistently scored QoL of their children lower than their child. Larger height-SD score and lower weight-SD score were associated with better QoL. Girls with lower steroid biomarkers had worse SDQ scores. Conclusions In CYP with CAH, reduced height, increased weight, and hormonal biomarkers consistent with overtreatment were associated with worse QoL; addressing these problems should be prioritized in clinical management. Clinical Trials Registration Number: SCH/15/088 [ABSTRACT FROM AUTHOR]

Spermatogenesis is regulated by the 2 pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). This process is considered impossible without the absolute requirement of LH-stimulated testicular testosterone (T) production. The role of FSH remains unclear because men and mice with inactivating FSH receptor (FSHR) mutations are fertile. We revisited the role of FSH in spermatogenesis using transgenic mice expressing a constitutively strongly active FSHR mutant in a LH receptor-null (LHR-null) background. The mutant FSHR reversed the azoospermia and partially restored fertility of [Lhr.sup.-/-] mice. The finding was initially ascribed to the residual Leydig cell T production. However, when T action was completely blocked with the potent antiandrogen flutamide, spermatogenesis persisted. Hence, completely T-independent spermatogenesis is possible through strong FSHR activation, and the dogma of T being a sine qua non for spermatogenesis may need modification. The mechanism for the finding appeared to be that FSHR activation maintained the expression of Sertoli cell genes considered androgen dependent. The translational message of our findings is the possibility of developing a new strategy of high- dose FSH treatment for spermatogenic failure. Our findings also provide an explanation of molecular pathogenesis for Pasqualini syndrome (fertile eunuchs; LH/T deficiency with persistent spermatogenesis) and explain how the hormonal regulation of spermatogenesis has shifted from FSH to T dominance during evolution., Introduction It is textbook knowledge that spermatogenesis is impossible without luteinizing hormone-stimulated (LH-stimulated) testicular testosterone (T) production (1, 2). Human mutations and genetically modified mice provide further proof for this, [...]