RESUMO
OBJECTIVES: 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. METHODS: 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. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2â¯% for androstenedione, 1.6-10.8â¯% for DHEAS, and 4.3-8.7â¯% and 2.6-7.1â¯% for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20â¯%. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6â¯% for androstenedione (p<0.001), 7.2 vs. 4.9â¯% for DHEAS (p<0.001), 6.4 vs. 7.6â¯% for female testosterone (p<0.001) and 6.8 and 7.4â¯% for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5â¯% and -4.9 to 18.7â¯% for androstenedione, -10.9 to 4.8â¯% and -3.4 to 3.5â¯% for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6â¯% for testosterone in females, and -7.0 to 8.5â¯% and -7.5 to 11.8â¯% for testosterone in males, respectively. CONCLUSIONS: 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.
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Androstenodiona , Sulfato de Desidroepiandrosterona , Testosterona , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Androstenodiona/sangue , Androstenodiona/análise , Calibragem , Sulfato de Desidroepiandrosterona/sangue , Sulfato de Desidroepiandrosterona/análise , Sulfato de Desidroepiandrosterona/normas , Espectrometria de Massa com Cromatografia Líquida/métodos , Espectrometria de Massa com Cromatografia Líquida/normas , Espectrometria de Massas em Tandem/normas , Espectrometria de Massas em Tandem/métodos , Testosterona/sangue , Testosterona/análise , Testosterona/normasRESUMO
OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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Cortisona , Humanos , Cromatografia Líquida/métodos , Cortodoxona , Corticosterona , Espectrometria de Massas em Tandem/métodos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Obesity and related co-morbidities represent a major health challenge nowadays, with a rapidly increasing incidence worldwide. The gut microbiome has recently emerged as a key modifier of human health that can affect the development and progression of obesity, largely due to its involvement in the regulation of food intake and metabolism. However, there are still few studies that have in-depth explored the functionality of the human gut microbiome in obesity and even fewer that have examined its relationship to eating behaviors. METHODS: In an attempt to advance our knowledge of the gut-microbiome-brain axis in the obese phenotype, we thoroughly characterized the gut microbiome signatures of obesity in a well-phenotyped Italian female cohort from the NeuroFAST and MyNewGut EU FP7 projects. Fecal samples were collected from 63 overweight/obese and 37 normal-weight women and analyzed via a multi-omics approach combining 16S rRNA amplicon sequencing, metagenomics, metatranscriptomics, and lipidomics. Associations with anthropometric, clinical, biochemical, and nutritional data were then sought, with particular attention to cognitive and behavioral domains of eating. RESULTS: We identified four compositional clusters of the gut microbiome in our cohort that, although not distinctly associated with weight status, correlated differently with eating habits and behaviors. These clusters also differed in functional features, i.e., transcriptional activity and fecal metabolites. In particular, obese women with uncontrolled eating behavior were mostly characterized by low-diversity microbial steady states, with few and poorly interconnected species (e.g., Ruminococcus torques and Bifidobacterium spp.), which exhibited low transcriptional activity, especially of genes involved in secondary bile acid biosynthesis and neuroendocrine signaling (i.e., production of neurotransmitters, indoles and ligands for cannabinoid receptors). Consistently, high amounts of primary bile acids as well as sterols were found in their feces. CONCLUSIONS: By finding peculiar gut microbiome profiles associated with eating patterns, we laid the foundation for elucidating gut-brain axis communication in the obese phenotype. Subject to confirmation of the hypotheses herein generated, our work could help guide the design of microbiome-based precision interventions, aimed at rewiring microbial networks to support a healthy diet-microbiome-gut-brain axis, thus counteracting obesity and related complications.
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Microbioma Gastrointestinal , Humanos , Feminino , Microbioma Gastrointestinal/genética , RNA Ribossômico 16S/genética , Multiômica , Obesidade/genética , Dieta , Comportamento Alimentar/fisiologia , Fezes/microbiologiaRESUMO
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 < 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 < 0.0001). The prevalence of biochemical hypogonadism was higher with LC-MS/MS than CI, both for TT (5.1% vs 3.2%, p < 0.0001) or cFT (9.5% vs 7%, p < 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.
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Infecções por HIV , Hipogonadismo , Globulina de Ligação a Hormônio Sexual/análise , Cromatografia Líquida , Estudos Transversais , Infecções por HIV/complicações , Humanos , Hipogonadismo/complicações , Imunoensaio , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Espectrometria de Massas em Tandem , TestosteronaRESUMO
OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. METHODS: Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. RESULTS: Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. CONCLUSIONS: Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.
Assuntos
Hidrocortisona , Progesterona , Aldosterona , Calibragem , Cromatografia Líquida/métodos , Humanos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
OBJECTIVE: Endocannabinoids have been implicated in the pathophysiology of Major Depressive Disorder (MDD) and might represent potential targets for therapeutic intervention. Objectives of the study were: (1) to measure plasma levels of endocannabinoids in a group of antidepressant-free depressed outpatients; (2) to explore their relationship with the severity of depressive symptoms as subjectively perceived by the patients; and (3) to investigate the effect of the selective serotonin reuptake inhibitor escitalopram on endocannabinoid levels. METHODS: We measured plasma levels of the two major endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anadamide), in 12 drug-free outpatients diagnosed with MDD and in 12 matched healthy controls. In the patient group, endocannabinoids plasma levels were assessed at baseline and after 2 months of treatment with escitalopram. RESULTS: Baseline plasma levels of the two endocannabinoids did not differ between depressed patients and healthy controls. However, there was a significant inverse correlation between 2-arachidonoylglycerol levels and the severity of subjectively perceived depressive symptoms. Treatment with escitalopram did not change endocannabinoid levels in depressed patients, although it caused the expected improvement of depressive symptoms. CONCLUSIONS: Our results suggest that 2-arachidonylglycerol, the most abundant endocannabinoid in the central nervous system, might act to mitigate depressive symptoms, and raise the interesting possibility that 2-arachidonylglycerol and anandamide are differentially regulated in patients affected by MDD. Also, our data suggest but do not prove that the endocannabinoid system is not regulated by serotonergic transmission, at least in depressed patients.
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Transtorno Depressivo Maior , Ácidos Araquidônicos , Depressão , Transtorno Depressivo Maior/tratamento farmacológico , Endocanabinoides , Escitalopram , Glicerídeos , HumanosRESUMO
BACKGROUND: Clomiphene citrate (CC) has been shown to restore the hypothalamic-pituitary-gonadal (HPG) axis by increasing testosterone (T) levels to physiological levels in patients with dysmetabolic conditions such as obesity, metabolic syndrome and type 2 diabetes mellitus (T2DM). However, the data are unclear regarding the effects on Sertoli cell (SC) function. AIM: To study SC function by assessing Inhibin B (IB) and anti-Mullerian hormone (AMH) levels at baseline and after 3 months of CC treatment. MATERIALS AND METHODS: This is an ancillary study of a cross-over, randomised, double-blind, placebo-controlled trial performed to evaluate androgen response to CC treatment in dysmetabolic obese subjects with low T levels treated with metformin. We evaluated SC function by assessing IB and AMH levels at baseline and after 3 months of each treatment in ten dysmetabolic obese subjects with low T levels. In all subjects, the influence of the clinical characteristics, metabolic and hormonal baseline parameters on SC and Leydig (LC) function, evaluated respectively with AMH, IB, follicle-stimulating hormone (FSH) and T levels, was tested. RESULTS: No significant changes were observed for IB and AMH concentrations after each treatment period. Whereas T and oestradiol (E2) levels were shown to be significantly higher in the CC plus metformin phase (CC/Met) only. No clinical, metabolic or hormonal parameters showed significant effects on serum AMH at baseline or after treatments. However, baseline T, dihydrotestosterone (DHT) and E2 positively affected IB levels during CC/Met therapy (P = .003, P = .038 and P = .049, respectively). Baseline leptin and FSH had a negative (P = 031) and positive (P = .048) respectively role on T levels during CC/Met, as they were statistically significant compared to the placebo period (Plac/Met). CONCLUSION: Unlike the LC activity, CC was unable to influence SC function, as shown by the lack of IB and AMH serum modifications, thus suggesting an intrinsic nonreversible defect of SC cells in patients with dysmetabolic conditions.
Assuntos
Hormônio Antimülleriano/sangue , Clomifeno/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Antagonistas de Estrogênios/farmacologia , Hipoglicemiantes/farmacologia , Hipogonadismo/tratamento farmacológico , Inibinas/efeitos dos fármacos , Síndrome Metabólica/tratamento farmacológico , Metformina/farmacologia , Obesidade/tratamento farmacológico , Células de Sertoli/efeitos dos fármacos , Testosterona/sangue , Adulto , Clomifeno/administração & dosagem , Estudos Cross-Over , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Di-Hidrotestosterona/sangue , Método Duplo-Cego , Quimioterapia Combinada , Estradiol/sangue , Antagonistas de Estrogênios/administração & dosagem , Seguimentos , Humanos , Hipogonadismo/sangue , Hipogonadismo/etiologia , Inibinas/sangue , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/complicações , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/complicaçõesRESUMO
STUDY QUESTION: What are the consequences of ageing on human Leydig cell number and hormonal function? SUMMARY ANSWER: Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age. WHAT IS KNOWN ALREADY: There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer. STUDY DESIGN, SIZE, DURATION: This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsies were obtained from 15 heart beating organ donors (age range: 19-85 years) and 24 patients (age range: 19-45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age. MAIN RESULTS AND THE ROLE OF CHANCE: Increasing age was negatively associated with Leydig cell number (R = -0.49; P < 0.01) and concomitantly with the Sertoli cell population size (R= -0.55; P < 0.001). A positive correlation (R = 0.57; P < 0.001) between Sertoli cell and Leydig cell numbers was detected at all ages, indicating that somatic cell attrition is a relevant cellular manifestation of human testis status during ageing. INSL3 mRNA expression (R= -0.52; P < 0.05) changed in parallel with Leydig cell number and age. Importantly, steroidogenic capacity of Leydig cells in cultured testis tissue fragments from young and old donors did not differ. Consistently, age did not influence the mRNA expression of steroidogenic enzymes. The described changes in Leydig cell phenotype with ageing are strengthened by the fact that the different age-related effects were mostly evaluated in tissue from the same men. LIMITATIONS, REASONS FOR CAUTION: In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables. WIDER IMPLICATIONS OF THE FINDINGS: This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.
Assuntos
Células Intersticiais do Testículo , Espectrometria de Massas em Tandem , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatografia Líquida , Humanos , Insulina , Masculino , Pessoa de Meia-Idade , Proteínas , Células de Sertoli , Espermatogênese , Testículo , Adulto JovemRESUMO
Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family-palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF; n = 41), polycythemia vera (PV; n = 9), and essential thrombocythemia (ET; n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC-MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.
Assuntos
Endocanabinoides/sangue , Etanolaminas/sangue , Transtornos Mieloproliferativos/sangue , Policitemia Vera/sangue , Mielofibrose Primária/sangue , Trombocitemia Essencial/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Amidas/sangue , Ácidos Araquidônicos/sangue , Cromatografia Líquida/métodos , Feminino , Glicerídeos/sangue , Humanos , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , Ácidos Oleicos/sangue , Ácidos Palmíticos/sangue , Policitemia Vera/diagnóstico , Policitemia Vera/genética , Alcamidas Poli-Insaturadas/sangue , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/genética , Espectrometria de Massas em Tandem/métodos , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/genéticaRESUMO
Putative genetic markers have been associated with ETEC F4 (Mucine 4 [MUC4]; MUC4GG;CG as susceptible; MUC4CC as resistant) and F18 (Fucosyltransferase 1 [FUT1]; FUT1GG;AG as susceptible; FUT1AA as resistant) resistances respectively. In this study, 71 post-weaning pigs were followed from d0 (35 days old) to d42 (77 days of age) to investigate the effect of MUC4 or FUT1 genotypes on the mid-jejunal microbiota composition, pigs expression of genes related to inflammation (IL8, GPX2, REG3G, TFF3, CCL20 and LBPI) and glycomic binding pattern profile (Ulex europaeus agglutinin I [UEA] fucose-binding lectin and peanut agglutinin [PNA] galactose-specific), and on blood plasma targeted metabolomics profile, faecal score and performance parameters of growing healthy pigs. The MUC4 and FUT1 resistant genotypes improved the pigs' growth performance and had firmed faecal score susceptible genotypes in d0-d21 period. Pigs with MUC4GG genotype had a higher jejunal expression of genes relate to immune function (CCL20 and REG3G) than MUC4CG and MUC4CC pigs (p < 0.05). MUC4CG pigs had higher expression of TFF3 (implicated in mucosal integrity) than MUC4GG and MUC4CC (p < 0.05). FUT1 influenced the alpha- and beta-jejunal microbial indices. The FUT1AA group had a higher number of operational taxonomic units (OTUs) belonging to Lactobacillus genus, while FUT1GG group had a higher number of OTUs belonging to Veillonella genus. MUC4CC pigs had lower scores for UEA on brush borders and goblet cells in villi than MUC4GG (p < 0.05). FUT1AA pigs had lower UEA positivity and higher PNA positivity on brush borders and goblet cells than FUT1AG and FUT1GG (p < 0.05). Both FUT1 and MUC4 influenced the metabolic profile of healthy pigs. Results highlight the role of MUC4 and FUT1 on pig intestinal homoeostasis and improved the knowledge regarding the potential interaction between host genetics, gut microbiota composition and host metabolism in a healthy status.
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Fucosiltransferases/metabolismo , Microbioma Gastrointestinal/fisiologia , Variação Genética , Homeostase/genética , Mucina-4/metabolismo , Suínos/genética , Animais , Bactérias/classificação , Feminino , Fucosiltransferases/genética , Regulação da Expressão Gênica , Genótipo , Homeostase/fisiologia , Masculino , Mucina-4/genética , Suínos/microbiologia , Suínos/fisiologia , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Similarities in neural activation patterns in obese and substance-dependent subjects led to the food addiction concept, but studies exploiting this issue for obesity stratification are missing. We assessed brain activation in response to food cues using 18 F-2-fluoro-2-deoxy-glucose-PET in 36 overweight women, stratified by low or high food addiction groups according to the Yale Food Addiction Scale (YFAS). Assessments were repeated after a 3-month diet. We found greater activation in thalamus, hypothalamus, midbrain, putamen, and occipital cortex (reward), but not in prefrontal and orbitofrontal cortices (control/reward receipt) in the high-YFAS versus low-YFAS group. In high-YFAS subjects, orbitofrontal responsiveness was inversely related to YFAS severity and hunger rating, and positive associations were observed between regional brain activation and lipid intake. A 3-month diet abolished group differences in brain activation. Our data suggest that food addiction distinguishes an overweight phenotype that can be reversed by diet, opening to personalized strategies in obesity treatment.
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Encéfalo/fisiologia , Restrição Calórica , Dependência de Alimentos/fisiopatologia , Sobrepeso/dietoterapia , Sobrepeso/fisiopatologia , Adulto , Sinais (Psicologia) , Feminino , Alimentos , Dependência de Alimentos/diagnóstico , Humanos , Fenótipo , Escalas de Graduação Psiquiátrica , Resultado do TratamentoRESUMO
BACKGROUND: Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones regulating development and reproductive functions by acting on the same receptor (LHCGR). We compared the LH and hCG activity in gonadal cells from male mouse in vitro, i.e. primary Leydig cells, which is a common tool used for gonadotropin bioassay. Murine Leydig cells are naturally expressing the murine LH receptor (mLhr), which binds human LH/hCG. METHODS: Cultured Leydig cells were treated by increasing doses of recombinant LH and hCG, and cell signaling, gene expression and steroid synthesis were evaluated. RESULTS: We found that hCG is about 10-fold more potent than LH in cAMP recruitment, and slightly but significantly more potent on cAMP-dependent Erk1/2 phosphorylation. However, no significant differences occur between LH and hCG treatments, measured as activation of downstream signals, such as Creb phosphorylation, Stard1 gene expression and testosterone synthesis. CONCLUSIONS: These data demonstrate that the responses to human LH/hCG are only quantitatively and not qualitatively different in murine cells, at least in terms of cAMP and Erk1/2 activation, and equal in activating downstream steroidogenic events. This is at odds with what we previously described in human primary granulosa cells, where LHCGR mediates a different pattern of signaling cascades, depending on the natural ligand. This finding is relevant for gonadotropin quantification used in the official pharmacopoeia, which are based on murine, in vivo bioassay and rely on the evaluation of long-term, testosterone-dependent effects mediated by rodent receptor.
Assuntos
Gonadotropina Coriônica/farmacologia , Células Intersticiais do Testículo/metabolismo , Hormônio Luteinizante/farmacologia , Transdução de Sinais/fisiologia , Testosterona/biossíntese , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacosRESUMO
We present a case report of an atypical giant pituitary adenoma secreting follicle-stimulating hormone (FSH). A 55-year-old patient presented for erectile dysfunction, loss of libido and fatigue. The biochemical evaluation showed very high FSH serum levels in the presence of central hypogonadism. Neither testicular enlargement nor increased sperm count was observed, thus a secretion of FSH with reduced biological activity was supposed. The histological examination after neuro-surgery showed an atypical pituitary adenoma with FSH-positive cells. Hypogonadism persisted and semen analyses impaired until azoospermia in conjunction with the reduction in FSH levels suggesting that, at least in part, this gonadotropin should be biologically active. Thus, we hypothesized a concomitant primary testicular insufficiency. The patient underwent short-term treatment trials with low doses of either recombinant luteinizing hormone (LH) or human chorionic gonadotropin (hCG) in three consecutive treatment schemes, showing an equal efficacy in stimulating testosterone (T) increase. This is the first case of atypical, giant FSH-secreting pituitary adenoma with high FSH serum levels without signs of testicular hyperstimulation, in presence of hypogonadism with plausible combined primary and secondary etiology. Hypophysectomized patients may represent a good model to assess both pharmacodynamics and effective dose of LH and hCG in the male.
Assuntos
Adenoma/complicações , Gonadotropina Coriônica/uso terapêutico , Hormônio Foliculoestimulante/metabolismo , Hipogonadismo/etiologia , Hormônio Luteinizante/uso terapêutico , Neoplasias Hipofisárias/complicações , Adenoma/diagnóstico por imagem , Adenoma/metabolismo , Adenoma/cirurgia , Disfunção Erétil , Hormônio Foliculoestimulante/sangue , Humanos , Células Intersticiais do Testículo/metabolismo , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/diagnóstico por imagem , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/cirurgiaRESUMO
BACKGROUND: Salivary androgen testing represents a valuable source of biological information. However, the proper measurement of such low levels is challenging for direct immunoassays, lacking adequate accuracy. In the last few years, many conflicting findings reporting low correlation with the serum counterparts have hampered the clinical application of salivary androgen testing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it possible to overcome previous analytical limits, providing new insights in endocrinology practice. METHODS: Salivary testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA) and 17OHprogesterone (17OHP) were extracted from 500µL of saliva, separated in 9.5 min LC-gradient and detected by positive electrospray ionization - multiple reaction monitoring. The diurnal variation of salivary and serum androgens was described by a four paired collection protocol (8 am, 12 am, 4 pm and 8 pm) in 19 healthy subjects. RESULTS: The assay allowed the quantitation of T, A, DHEA and 17OHP down to 3.40, 6.81, 271.0 and 23.7 pmol/L, respectively, with accuracy between 83.0 and 106.1% for all analytes. A parallel diurnal rhythm in saliva and serum was observed for all androgens, with values decreasing from the morning to the evening time points. Salivary androgen levels revealed a high linear correlation with serum counterparts in both sexes (T: R>0.85; A: R>0.90; DHEA: R>0.73 and 17OHP: R>0.89; p<0.0001 for all). CONCLUSIONS: Our LC-MS/MS method allowed a sensitive evaluation of androgen salivary levels and represents an optimal technique to explore the relevance of a comprehensive androgen profile as measured in saliva for the study of androgen secretion modulation and activity in physiologic and pathologic states.
Assuntos
17-alfa-Hidroxiprogesterona/sangue , Androstenodiona/sangue , Ritmo Circadiano , Desidroepiandrosterona/sangue , Saliva/química , Espectrometria de Massas em Tandem/normas , Testosterona/sangue , Adulto , Idoso , Análise Química do Sangue/normas , Cromatografia Líquida/normas , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE: Studies in humans suggest that consumption of low-carbohydrate, high-fat diets (LC-HF) could be detrimental for growth and bone health. In young male rats, LC-HF diets negatively affect bone health by impairing the growth hormone/insulin-like growth factor axis (GH/IGF axis), while the effects in female rats remain unknown. Therefore, we investigated whether sex-specific effects of LC-HF diets on bone health exist. METHODS: Twelve-week-old male and female Wistar rats were isoenergetically pair-fed either a control diet (CD), "Atkins-style" protein-matched diet (LC-HF-1), or ketogenic low-protein diet (LC-HF-2) for 4 weeks. In females, microcomputed tomography and histomorphometry analyses were performed on the distal femur. Sex hormones were analysed with liquid chromatography-tandem mass spectrometry, and endocrine parameters including GH and IGF-I were measured by immunoassay. RESULTS: Trabecular bone volume, serum IGF-I and the bone formation marker P1NP were lower in male rats fed both LC-HF diets versus CD. LC-HF diets did not impair bone health in female rats, with no change in trabecular or cortical bone volume nor in serum markers of bone turnover between CD versus both LC-HF diet groups. Pituitary GH secretion was lower in female rats fed LC-HF diet, with no difference in circulating IGF-I. Circulating sex hormone concentrations remained unchanged in male and female rats fed LC-HF diets. CONCLUSION: A 4-week consumption of LC-HF diets has sex-specific effects on bone health-with no effects in adult female rats yet negative effects in adult male rats. This response seems to be driven by a sex-specific effect of LC-HF diets on the GH/IGF system.
Assuntos
Osso e Ossos/fisiologia , Dieta com Restrição de Carboidratos , Dieta Hiperlipídica , Fatores Sexuais , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Glicemia/metabolismo , Dieta com Restrição de Proteínas , Estradiol/sangue , Feminino , Hormônios Esteroides Gonadais/sangue , Hormônio do Crescimento/sangue , Fator de Crescimento Insulin-Like I/metabolismo , Leptina/sangue , Lipídeos/sangue , Masculino , Osteogênese , Fragmentos de Peptídeos/sangue , Pró-Colágeno/sangue , Ratos , Ratos Wistar , Albumina Sérica/metabolismoRESUMO
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.
Assuntos
Androstenodiona/sangue , Cromatografia Líquida/normas , Desidroepiandrosterona/sangue , Espectrometria de Massas em Tandem/normas , Testosterona/sangue , Adulto , Calibragem , Feminino , Humanos , Masculino , Variações Dependentes do Observador , Análise de Regressão , Reprodutibilidade dos Testes , Fatores SexuaisRESUMO
OBJECTIVE: The psychosocial implications of polycystic ovary syndrome (PCOS) phenotypes have been investigated in different patient populations, but little attention was paid to adolescent and young women. The aim of this study was to evaluate the psychosocial correlates of PCOS and other hyperandrogenic states in a population of late adolescent and young females and to provide a psychological characterization of specific clinical hyperandrogenic phenotypes. DESIGN: This was a cross-sectional study. PATIENTS: High school female students, aged 16-19 years. MEASUREMENTS: The study protocol was designed with three possible levels of participation characterized by an increased level of commitment. For the specific purposes of this investigation, we focused on the subsamples of students whose clinical state was assessed by medical examination (n = 835) and for whom additional laboratory tests were available (n = 394). Psychological evaluation encompassed psychological distress, levels of stress, well-being, illness behaviour and quality of life (as measured by the Symptom Questionnaire, the Psychosocial Index and the Psychological Well-Being scales). RESULTS: Significantly higher levels of psychological distress and impaired well-being and quality of life were found among late adolescent and young women with isolated clinical hyperandrogenism compared to their normal counterparts. Furthermore, females with PCOS showed significantly greater hostility/irritability compared to healthy control subjects. CONCLUSIONS: These findings highlight the importance of early recognizing and adequately managing psychological distress in such patients.
Assuntos
Hiperandrogenismo/psicologia , Adolescente , Adulto , Estudos Transversais , Feminino , Humanos , Masculino , Síndrome do Ovário Policístico/psicologia , Qualidade de Vida , Inquéritos e Questionários , Adulto JovemRESUMO
BACKGROUND: Various glucocorticoid replacement therapies (GRTs) are available for adrenal insufficiency (AI). However, their effectiveness in restoring glucocorticoid rhythm and exposure lacks adequate biochemical markers. We described the diurnal salivary cortisol (SalF) and cortisone (SalE) rhythm among different GRTs and analysed the associations between saliva-derived parameters and life quality questionnaires. METHODS: Control subjects (CSs, n = 28) and AI patients receiving hydrocortisone (HC, n = 9), cortisone acetate (CA, n = 23), and dual-release hydrocortisone once (DRHC-od, n = 10) and twice a day (DRHC-td, n = 6) collected 9 saliva samples from 07:00 to 23:00. Patients compiled Pittsburgh Sleep Quality Index, Hospital Anxiety and Depression Scale, and Addison disease-specific quality-of-life questionnaires. SalE and SalF were measured by liquid chromatography-mass spectrometry. Exposure was monitored using SalE for HC and DRHC and SalF for CA. Area under the curve (AUC) was computed. Different GRTs were compared by Z-scores calculated from saliva-derived parameters. Questionnaire results predictors were evaluated with multiple regression analysis. RESULTS: Compared with controls, all GRTs resulted in glucocorticoid overexposure in the morning. Hydrocortisone, CA, and DRHC-td caused overexposure also in afternoon and evening. Compared with other treatments, CA determined increased Z-score-07:00 (P < .001), DRHC-td determined increased Z-score-AUC07:00â14:00 (P = .007), and DRHC-od induced lower Z-score-AUC14:00â23:00 (P = .015). Z-scores-AUC14:00â16:00 ≥ .619 best predicted questionnaire scores. CONCLUSIONS: None of the GRTs mimics normal glucocorticoid rhythmicity and exposure. SalE, SalF, and Z-score may be useful markers for monitoring and comparing different GRTs. Excess glucocorticoid in early afternoon best associated with depressive symptoms and worse life and sleep quality.
Assuntos
Insuficiência Adrenal , Cortisona , Humanos , Glucocorticoides/efeitos adversos , Hidrocortisona/análise , Projetos Piloto , Insuficiência Adrenal/induzido quimicamente , Insuficiência Adrenal/diagnóstico , Insuficiência Adrenal/tratamento farmacológico , Cortisona/uso terapêutico , Cortisona/análise , Saliva/químicaRESUMO
Modern endocrinology is living a critical age of transition as far as laboratory testing and biochemical diagnosis are concerned. Novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for steroid measurement in biological fluids have abundantly demonstrated their analytical superiority over immunometric platforms that until now have dominated the world of steroid hormones determination in clinical laboratories. One of the most useful applications of LC-MS/MS is in the hypogonadism and hyperandrogenism field: LC-MS/MS has proved particularly suitable for the detection of low levels of testosterone typical of women and children, and in general more reliable in accurately determining hypogonadal male levels. This technique also offers increased informative power by allowing multi-analytical profiles that give a more comprehensive picture of the overall hormonal asset. Several LC-MS/MS methods for testosterone have been published in the last decade, some of them included other androgen or more comprehensive steroid profiles. LC-MS/MS offers the concrete possibility of achieving a definitive standardization of testosterone measurements and the generation of widely accepted reference intervals, that will set the basis for a consensus on the diagnostic value of biochemical testing. The present review is aimed at summarizing technological advancements in androgen measurements in serum and saliva. We also provide a picture of the state of advancement of standardization of testosterone assays, of the redefinition of androgen reference intervals by novel assays and of studies using LC-MS/MS for the characterization and diagnosis of female hyperandrogenism and male hypogonadism.
Assuntos
Cromatografia Líquida/métodos , Hiperandrogenismo/diagnóstico , Hipogonadismo/diagnóstico , Espectrometria de Massas em Tandem/métodos , Androgênios/análise , Androgênios/sangue , Humanos , Hiperandrogenismo/sangue , Hiperandrogenismo/metabolismo , Hipogonadismo/sangue , Hipogonadismo/metabolismo , Testosterona/análise , Testosterona/sangueRESUMO
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 µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 × 3 mm, 3 µm column, with 100 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 < 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 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.