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

Revisiting hyper- and hypo-androgenism by tandem mass spectrometry.

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

Female and male serum reference intervals for challenging sex and precursor steroids by liquid chromatography - tandem mass spectrometry

Measuring some sex and precursor steroids is still challenging even by liquid chromatography \u2013 tandem mass spectrometry (LC-MS/MS), and few normal values are available. We developed a LC-MS/MS method for estradiol, estrone, dihydrotestosterone and 17-hydroxypregnenolone measurement, compared it with direct immunoassays, and generated sex, age, menopausal and menstrual status specific reference intervals. Liquid-liquid extraction was optimized on 300 \u3bcL serum spiked with isotopic internal standards. A 2D-LC system allowed on-line purification and separation in 11 min run. Electrospray ionization was enhanced by ammonium fluoride. MS-detection was obtained by multiple reaction monitoring. Direct ECLIA for estradiol (n = 80) and RIA for estrone (n = 41) were compared with LC-MS/MS. Reference values were estimated in healthy, lean women in reproductive age (n = 118), menopausal women (n = 33) and men (n = 159). The assay showed satisfying imprecision, trueness, recovery and selectivity. Adequate functional sensitivity was achieved for measuring estrone (18.1 pmol/L) and 17-hydroxypregnenolone (117 pmol/L) in all subjects, and estradiol (35.9 pmol/L) and dihydrotestosterone (134 pmol/L) in women in reproductive age and men, but not in menopausal women. Compared with LC-MS/MS, immunoassays showed good agreement for estradiol but severe disagreement for estrone. Estrogens exhibited sex, menopausal and menstrual variations. Dihydrotestosterone and 17-hydroxypregnenolone depended on sex and menopause, the latter also declining with age in men. Strictly defined reference intervals in the adult female and male population were generated for challenging steroids such as estrogens, dihydrotestosterone and 17-hydroxypregnenolone by a novel LC-MS/MS method. Our achievement can be used to deepen the comprehension of several endocrine diseases

Report from the HarmoSter study: Impact of calibration on comparability of LC-MS/MS measurement of circulating cortisol, 17OH-progesterone and aldosterone

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. 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. 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. 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

Impact of age, body weight and metabolic risk factors on steroid reference intervals in men

Objective: To evaluate the independent impact of age, obesity and metabolic risk factors on 13 circulating steroid levels; to generate reference intervals for adult men. Design: Cross-sectional study. Methods: Three hundred and fifteen adults, drug-free and apparently healthy men underwent clinical and biochemical evaluation. Thirteen steroids were measured by LC-MS/MS and compared among men with increasing BMI. Moreover, the independent impact of age, BMI and metabolic parameters on steroid levels was estimated. Upper and lower reference limits were generated in steroid-specific reference sub-cohorts and compared with dysmetabolic subcohorts. Results: We observed lower steroid precursors and testosterone and increase in estrone levels in men with higher BMI ranges. By multivariate analysis, 17-hydroxyprogesterone and dihydrotestosterone decreased with BMI, while cortisol decreased with waist circumference. Estrone increased with BMI and systolic blood pressure. Testosterone decreased with worsening insulin resistance. 17-hydroxypregnenolone and corticosterone decreased with increasing total/HDL-cholesterol ratio. Age-related reference intervals were estimated for 17-hydroxypregnenolone, DHEA, 17-hydroxyprogesterone, corticosterone, 11-deoxycortisol, cortisol and androstenedione, while age-independent reference intervals were estimated for progesterone, 11-deoxycorticosterone, testosterone, dihydrotestosterone, estrone and estradiol. Testosterone lower limit was 2.29 nmol/L lower (P = 0.007) in insulin resistant vs insulin sensitive men. Furthermore, the upper limits for dihydrotestosterone (-0.34 nmol/L, P = 0.045), cortisol (-87 nmol/L, P = 0.045-0.002) and corticosterone (-10.1 nmol/L, P = 0.048-0.016) were lower in overweight/obese, in abdominal obese and in dyslipidaemic subjects compared to reference sub-cohorts, respectively. Conclusions: Obesity and mild unmedicated metabolic risk factors alter the circulating steroid profile and bias the estimation of reference limits for testosterone, dihydrotestosterone, cortisol and corticosterone. Applying agedependent reference intervals is mandatory for steroid precursors and corticosteroids