Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel

The canonical androgen synthesis in Leydig cells involves Delta 5 and Delta 4 steroids. Besides, the backdoor pathway, eompassing 5 alpha and 5 alpha,3 alpha steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatographytandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500 mu L supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 x 3 mm, 3 mu m column, with 100 mu M NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and <= 3.3 in 5 alpha and 5 alpha,3 alpha steroids

Short term Leydig cell stimulation by LH and hCG in man with central hypogonadism

Short term Leydig cell stimulation by LH and hCG in man with central hypogonadis

Low Serum Testosterone (T) Is Associated with Poor Health Status in Young to Middle-Aged Human Immunodeficiency Virus (HIV)-Infected Men.

BACKGROUND: The relationship between health status, defined by frailty and comorbidities, and serum T levels has been widely demonstrated in general population, while only one previous retrospective study has explored it in HIV-infected men1. AIM: To investigate the association between frailty and go- nadal status by assessing serum total T (TT) with Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) in a cohort of HIV-infected men. METHODS: Prospective, cross-sectional, observational study on HIV-infected men (age <50 years) with on- going Highly Active Antiretroviral Therapy. Serum TT was assessed by the gold standard ID-LC-MS/MS. Sex hormone-binding globulin (SHBG) was measured by chem- iluminescent immunoassay. Calculated free T (cFT) was obtained by Vermeulen equation. Multimorbidity was de- fined as at least 3 comorbid conditions, including: hyperten- sion, diabetes, cardiovascular disease, chronic obstructive pulmonary disease, chronic kidney disease, osteoporosis, chronic viral hepatitis and cancers. Frailty was calculated through the validated 37-item frailty index (FI)2. Patients with FI>0.21 were considered frail. Statistical ana- lysis: Mann-Whitney U test was used to compare contin- uous variables. Correlations were performed using linear regression models. RESULTS: 315 consecutive HIV-infected men were enrolled (mean age 45.3\ub15.3 years; average duration of HIV-infection 16.3\ub18.8 years). 128 patients (40.5%) were co- morbid and 207 (64.9%) were frail. Either cFT (p=0.001) or TT (p<0.001) were lower in comorbid patients than others. FT was inversely related to the number of comorbidities (p<0.001, R2=0.045). Accordingly, cFT (p=0.003) and TT (p<0.001) were significantly lower in frail patients.Frailty score was inversely correlated with cFT (p<0.001, R2=0.058), TT (p=0.041, R2=0.014) and SHBG (p=0.003, R2=0.029). However, after adjustment for age and duration of HIV-infection, cFT, TT and SHBG were excluded from the regression model. CONCLUSIONS: Low cFT and TT levels are associated with multimorbidity and poor health status in HIV infected men. The bidirectional nature of this relationship leads to the figuration of an intriguing vicious circle where T de- ficiency triggers the onset of comorbidities or, vice versa, poor health status induces hypogonadism. At the same time, notwithstanding the inverse relation between FT and frailty, it seems that other stronger predictive factors, and in particular the duration of infection, are involved in de- termining the health outcome in this clinical setting

Free testosterone (FT) is inversely related to frailty in human immunodeficiency virus (HIV)-infected men.

BACKGROUND HIV-infection is associated to several age-related comorbidities, such as a premature decline of serum testosterone (T). There is evidence about the relationship between health status, represented by frailty and comorbidities, and serum T levels in general population, while only one previous retrospective study investigated it in HIV-infected men. AIM To investigate the association between frailty and gonadal status by assessing serum total T (TT) with Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) in a cohort of HIV-infected men. METHODS Prospective, cross-sectional, observational study on HIV-infected men (age years) with ongoing Highly Active Antiretroviral Therapy (HAART). Serum TT was assessed by the gold standard ID- LC-MS/MS. Sex hormone-binding globulin (SHBG) was measured by chemiluminescent immunoassay. Free T (FT) was calculated by Vermeulen equation. Frailty was calculated through -items multimorbidity frailty index. Saca aa Parameters were not normally distributed and Mann- Whitney U test was used to compare continuous variables. Correlations were performed using linear regression models. RESULTS consecutive HIV-infected men were enrolled (mean age .. years; average duration of HIV-infection .. years). patients (.) had TT below ng/dL and patients (.) had calculated FT below pg/mL. Overall, patients (.) had T deficiency defined by low TT levels and/or low FT. patients (.) showed SHBG above the normal range (. nmol/L). Frailty score (p.), age (p.), duration of HIV-infection and of HAART (p.) significantly differed between eugonadic and hypogonadic patients, while no difference was found for BMI (p.). FT inversely correlated with frailty score (p., R.), while TT did not (p.). At stepwise multivariate regression analysis, FT showed an inverse relation with age (p.,R.), years of infection (-.,p.,R.) and years of HAART (-.,p.,R.), but not with frailty score and BMI of patients. CONCLUSIONS To the best of our knowledge, this is the first properly-designed prospective study aiming to investigate the relationship between general health status and gonadal function in a cohort of HIV-infected men. FT is inversely related to frailty score, suggesting an impairment of gonadal function in those patients affected by more multimorbidities in this setting as well as in general population. At the same time, the age of patient and the duration of HIV-infection seem to be more potent predictive factors for serum FT levels than frailty score. In clinical practice it is important to check for testosterone in these patients due to frequent alterations of SHBG

Report from the HarmoSter study: inter-laboratory comparison of LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization

Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males

Background: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. Methods: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. Findings: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing). Interpretation: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats. Funding: MIUR project "Dipartimenti di Eccellenza 2018-2020" to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and "Bando Ricerca COVID-19 Toscana" project to Azienda Ospedaliero-Universitaria Senese. Private donors for COVID-19 research and charity funds from Intesa San Paolo

Biomass sustainability criteria:greenhouse gas accounting issues for biogas and biomethane facilities

Biomass sustainability criteria were introduced in the UK following the EU Renewable Energy Directive. Criteria are now applicable to solid biomass and biogas, however because it is not mandatory criteria can be adapted by member states with the risk of different interpretation. Operators are required to report greenhouse gas (GHG) emissions for every MJ of energy produced. This paper provides a rigorous analysis of the current GHG emissions accounting methodology for biogas facilities to assess expected compliance for producers. This research uses data from operating CHP and biomethane facilities to calculate GHG emissions using the existing methodology and Government calculator. Results show that whilst many biogas facilities will meet GHG thresholds, as presently defined by Government, several operators may not comply due to methodological uncertainties and chosen operating practices. Several GHG accounting issues are identified which need to be addressed so the biogas industry achieves its reporting obligations and is represented objectively with other bioenergy technologies. Significant methodological issues are highlighted; including consignment definition, mass balance allocation, measurement of fugitive methane emissions, accounting for digestate co-products, fossil fuel comparators, and other accounting problems. Recommendations are made to help address the GHG accounting issues for policy makers and the biogas industry