Advanced analysis of the steroids profile for the identification of predictors of cardiovascular risk in patients with adrenal incidentaloma by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)

La valutazione del profilo steroideo mediante cromatografia liquida abbinata alla spettrometria di massa (LC-MS/MS è di grande utilità nella diagnosi e tipizzazione delle masse del surrene. Abbiamo analizzato il profilo steroideo basale e post test di soppressione al desametasone, in 302 pazienti con incidentaloma surrenalico, mediante un metodo LC-MS/MS per la quantificazione di 11 steroidi. Abbiamo poi valutato le associazioni con la salute cardiovascolare durante il periodo di follow-up. Lo scopo secondario prevedeva lo studio della steroidogenesi intra-tumore ed il confronto con il tessuto surrenalico normale corrispondente, mediante lo sviluppo di un nuovo metodo LC-MS/MS per la caratterizzazione di un pannello di 22 steroidi surrenalici. Da tale studio è emerso che i soggetti con adenoma unilaterale e secrezione disregolata di cortisolo avevano valori basali più elevati di cortisolo, 11-desossicortisolo e corticosterone e livelli ridotti di DHEA rispetto ai pazienti con adenoma non funzionante. I pazienti con secrezione disregolata hanno mostrato la mancata soppressione di cortisolo, 11-desossicortisolo e corticosterone post test al desametasone indipendentemente dalla morfologia della lesione. I livelli di cortisolo e corticosterone post test al desametasone erano inoltre associati con una prevalenza più elevata del peggioramento dell’ipertensione. Pazienti con adenoma unilaterale e secrezione disregolata avevano un’incidenza più elevata del peggioramento dell’ipertensione e per l’insorgenza di nuovi eventi cardiovascolari rispetto ai non secernenti, con il cortisolo post desametasone (Hazard Ratio 1.02, 95% CI 1.01-1.03, P<0.001) ed il corticosterone basale (Hazard Ratio 1.06, 95% CI 1.01-1.12, P<0.031) come maggiori predittori. Dallo studio della sterodogenesi tissutale è emerso il potenziale valore informativo di alcuni steroidi non tradizionali, le cui variazioni erano frequentemente riscontrate nel tessuto tumorale rispetto al tessuto surrenalico normale. I pazienti con incidentaloma surrenalico hanno mostrato un profilo steroideo differente in relazione allo status funzionale ed alla morfologia dei surreni che si associava a differenti livelli di rischio cardiovascolare.The assessment of steroid profile by liquid-chromatography tandem mass spectrometry has proved to be of great usefulness in the diagnosis and characterization of the adrenal masses. We analyzed the circulating steroid profile in 302 patients with adrenal incidentaloma, by an LC-MS/MS method for the quantification of 11 steroids. We then assessed the associations with cardiovascular health during the follow-up period (median 39 months). The secondary aim was the exploratory study of intra-tumor steroidogenesis and the comparison with the corresponding normal tissue, through the development of a new LC-MS/MS method for the characterization of a panel of 22 adrenal steroids. We found that subjects with unilateral adenoma and dysregulated cortisol secretion had higher basal values of cortisol, 11-deoxychortisol and corticosterone and reduced DHEA levels compared to patients with non-functioning adenoma. Moreover, subjects with hyperplasia and dysregulated cortisol secretion had high cortisol and reduced androgen levels compared to non-functioning hyperplasia. Patients with dysregulated secretion showed no suppression of cortisol, 11-deoxycortisol and corticosterone post dexamethasone-test regardless of lesion morphology. After suppression-test, cortisol and corticosterone levels were also associated with higher prevalence of worsening hypertension. Patients with unilateral adenoma and dysregulated secretion had higher incidence of worsening hypertension and of onset of new cardiovascular events than non-secreting, with post-dexamethasone cortisol (Hazard Ratio 1.02, 95%CI 1.01-1.03, P<0.001) and basal corticosterone (Hazard Ratio 1.06, 95%CI 1.01-1.12, P<0.031) as major predictors. The study of tissue sterodogenesis revealed the usefulness of non-classical steroids, such as the metabolites of cortisol, progesterone, 16-hydroxyprogesterone and some 11-oxidized C19-androgens such as 11-hydroxydrostenedione, 11-ketoadrostenedione and 11-hydroxytestosterone whose variations were frequently found in adrenal lesions compared to normal adrenal tissue. We concluded that patients with adrenal incidentaloma showed a different steroid profile in relation to the functional status and morphology of the adrenals, which was associated with different levels of cardiovascular risk

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 Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 5α and 5α,3α 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 chromatography-tandem 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&nbsp;µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100&nbsp;×&nbsp;3&nbsp;mm, 3&nbsp;µm column, with 100&nbsp;µM 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 &lt;&nbsp;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 ≤&nbsp;3.3 in 5α and 5α,3α steroids. In conclusion, our LC-MS/MS method allows exploring the Leydig steroidogenesis flow according to multiple pathways. Beside the expected stimulation of the canonical pathway, hCG increased progesterone metabolism and, to a low extent, the backdoor route

Neuroactive Steroids in First-Episode Psychosis: A Role for Progesterone?

Neuroactive steroids may play a role in the pathophysiology of psychotic disorders, but few studies examined this issue. We compared serumlevels of cortisol, testosterone, dehydroepiandrosterone, and progesterone between a representative sample of firstepisode psychosis (FEP) patients and age- and gender-matched healthy subjects. Furthermore, we analyzed the associations between neuroactive steroids levels and the severity of psychotic symptom dimensions.Male patients had lower levels of progesterone than controls

Primary, secondary and compensated male biochemical hypogonadism in people living with HIV (PLWH): relevance of sex hormone-binding globulin (SHBG) measurement and comparison between liquid chromatography-tandem mass spectrometry (LC-MS/MS) and chemiluminescent immunoassay for sex steroids assay

Background: Data about classification of hypogonadism and estrogen deficiency in male people living with HIV (PLWH) are scanty. Aim: To investigate the prevalence and characterization of biochemical hypogonadism and relative estrogen deficiency in male PLWH aged &lt; 50 comparing liquid chromatography-tandem mass spectrometry (LC-MS/MS) with chemiluminescent immunoassay (CI), and combining gonadotropin, sex hormone-binding globulin (SHBG) and serum estradiol (E2) measurements. Methods: Prospective, cross-sectional, observational study. Serum total testosterone (TT), E2, gonadotropins, SHBG were measured by CI. TT and E2 were also assessed by LC-MS/MS. Free testosterone (cFT) was calculated by Vermeulen equation. Results: A total of 316 PLWH (45.3 ± 5.3 years) were enrolled. TT and cFT by LC-MS/MS were lower compared to CI (p &lt; 0.0001). The prevalence of biochemical hypogonadism was higher with LC-MS/MS than CI, both for TT (5.1% vs 3.2%, p &lt; 0.0001) or cFT (9.5% vs 7%, p &lt; 0.0001). The prevalence of hypogonadism (overt + compensated) was 17.1% for cFT using LC-MS/MS. Secondary form of hypogonadism was more prevalent than primary. The prevalence of relative estrogen deficiency was of 30.0% among hypogonadal patients and 15.5% among eugonadal. Conclusions: The prevalence of male hypogonadism results underestimated by CI compared to LC-MS/MS in PLWH, both for TT and cFT. SHBG and gonadotropins are essential for detecting T deficiency.Background: Data about classification of hypogonadism and estrogen deficiency in male people living with HIV (PLWH) are scanty. Aim: To investigate the prevalence and characterization of biochemical hypogonadism and relative estrogen deficiency in male PLWH aged &lt; 50 comparing liquid chromatography-tandem mass spectrometry (LC-MS/MS) with chemiluminescent immunoassay (CI), and combining gonadotropin, sex hormone-binding globulin (SHBG) and serum estradiol (E2) measurements. Methods: Prospective, cross-sectional, observational study. Serum total testosterone (TT), E2, gonadotropins, SHBG were measured by CI. TT and E2 were also assessed by LC-MS/MS. Free testosterone (cFT) was calculated by Vermeulen equation. Results: A total of 316 PLWH (45.3 ± 5.3 years) were enrolled. TT and cFT by LC-MS/MS were lower compared to CI (p &lt; 0.0001). The prevalence of biochemical hypogonadism was higher with LC-MS/MS than CI, both for TT (5.1% vs 3.2%, p &lt; 0.0001) or cFT (9.5% vs 7%, p &lt; 0.0001). The prevalence of hypogonadism (overt + compensated) was 17.1% for cFT using LC-MS/MS. Secondary form of hypogonadism was more prevalent than primary. The prevalence of relative estrogen deficiency was of 30.0% among hypogonadal patients and 15.5% among eugonadal. Conclusions: The prevalence of male hypogonadism results underestimated by CI compared to LC-MS/MS in PLWH, both for TT and cFT. SHBG and gonadotropins are essential for detecting T deficiency

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

Profession, Professionalität, Professionalisierung

Alignment of LH receptor amino acid sequences obtained from the UniProt database ( http://www.uniprot.org ). Homo sapiens LHCGR (UniProt identifier: P22888), Mus musculus Lhr (P30730) and Rattus norvegicus Lhr (P16235) sequences were aligned by the UniProt online tool Clustal Omega 1.2.1 ( http://www.uniprot.org/align ). Boxes indicate sequence divergence; :=conservation of strong groups;. = conservation of weak groups or no consensus. (DOC 92 kb

Normative Basal Values of Hormones and Proteins of Gonadal and Adrenal Functions from Birth to Adulthood

In clinical practice, it is fundamental to compare the results of hormonal examinations obtained in the laboratory with reliable reference values. This is particularly difficult when faced with rare conditions, such as disorders of sex development, where not routinely assayed peptide hormones as well as intermediate steroid metabolites are often needed and local reliable reference values are not available. There are considerable differences among techniques and assays used in clinical and research laboratories. In fact, laboratory hormonology is undergoing a critical transition between techniques for quantitative determination: established immunoassays and mass spectrometry. Harmonizing results from different laboratories is a major challenge along the path leading to the establishment of consensus reference intervals for steroid hormones. Most of the efforts are being concentrated on testosterone, with very encouraging results being provided by the harmonization of liquid chromatography-tandem mass spectrometry results. However, this goal is still far from being achieved for the other steroid and small-molecule hormones, and a much more challenging perspective is foreseeable for protein hormones. In addition to technical issues, the importance of the definition and of the characterization of the reference population as well as sampling and processing methodology should not be underestimated, as these aspects may impact on hormonal axis and compound stability. The aim of the present review is to provide a comprehensive overview of the circulating reference values in basal condition of the hormones and proteins involved in sex development reported to date in the peer-reviewed literature. We present a series of tables where we have collected the reference intervals for each specific hormone and protein

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&nbsp;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

Defining hyperandrogenism in women with polycystic ovary syndrome: A challenging perspective

Objective: This study was designed to assess the steroid profiling by liquid chromatography coupled with tandem mass spectrometry in PCOS women with different phenotypes. Design: Cross-sectional study. Setting: University hospital of Bologna, Italy. Patients and Methods: A total of 156 PCOS women and 141 controls comparable for age were investigated. All underwent a steroid profiling by liquid chromatography coupled with tandem mass spectrometry. Metabolic parameters were also investigated and hirsutism was measured by the modified Ferriman-Gallwey (mF-G) score. Results: Three distinct phenotypes were initially defined according to the combination of hirsutism (mF-G ≥ 8) and/or high testosterone (T) (HA), oligo-amenorrhea (OA), and polycystic ovarian morphology (PCOm); OA + PCOm (n = 43), HA + OA (n = 65), and HA + OA + PCOm (n = 45). T, androstenedione (A), and free androgen index (FAI) levels progressively increased in the 3 PCOS phenotypes with respect to the controls, with the highest values in the HA + OA + PCOm phenotype. The various combinations of hirsutism, high T, high A, and high FAI made it possible to categorize the 3 original phenotypes into 8 hyperandrogenic subgroups, characterized by divergent additional steroid profile and metabolic pattern. A total of 90% of patients with PCOS thus proved hyperandrogenic. Interestingly, half the PCOS women originally classified as having the OA-PCOm phenotype were categorized in a hyperandrogenic subgroup. No significant correlation was found between T, A, and the mF-G score. In contrast, significant correlation was found between A and both T and FAI. Conclusions: This study provides evidence that, by including a steroid profile in the definition of hyperandrogenemia, the majority of women with PCOS are hyperandrogenic, although a clinical and biochemical heterogeneity exists. In addition, these data demonstrate that hirsutism and high androgen levels cannot be used indifferently to define hyperandrogenism