Alessia Fazzini

Validation of an LC–MS/MS salivary assay for glucocorticoid status assessment: Evaluation of the diurnal fluctuation of cortisol and cortisone and of their association within and between serum and saliva

Salivary steroid testing represents a valuable source of biological information; however, the proper measurement of low salivary levels is challenging for direct immunoassays, lacking adequate sensitivity and specificity and causing poor inter-laboratory reproducibility. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has overcome previous analytical limits, often providing results deviating from previous knowledge. Nowadays, LC-MS/MS is being introduced in clinical laboratories for salivary cortisol testing; however, so far only a few studies have reported thorough biological validation based on LC-MS/MS data. In this study, we provide a thorough analytical, pre-analytical and biological validation of an LC-MS/MS method for the measurement of salivary cortisol (F) and of its inactive metabolite cortisone (E). Analytes were extracted from 50μl of saliva, were then separated in 7.5min LC-gradient and detected by negative electrospray ionization-multiple reaction monitoring. The reliability of a widely diffused collection device, Salivette(®), was assessed and the overall procedure was validated. The diurnal cortisol and cortisone fluctuation in saliva and serum was described by a four paired collection protocol (8 am, 12 am, 4 pm and 8 pm) in 19 healthy subjects. The assay allowed the quantitation of F and E down to 39.1 and 78.1pg/ml, with an imprecision range of 5.5-9.5%, 3.9-14.1% and 2.6-14.4%, and an accuracy range of 105.5-113.1%, 88.5-98.7% and 90.7-96.7% for both analytes at low, medium and high levels, respectively. Salivette(®) provided comparable results and better precision (CV<1.0%) as referred to direct spitting (CV<13.0%). A parallel diurnal rhythm in saliva and serum was observed for cortisol and cortisone, with values lowering from the morning to the evening time points (P<0.0001). While salivary E linearly correlated to total serum F (R(2)=0.854, P<0.001), salivary F showed an exponential relationship (R(2)=0.903, P<0.001) with serum F reflecting the free circulating fraction. A non linear association between E and F was observed in saliva (R(2)=0.941, p<0.001) consistent with the type II 11β-HSD activity. We concluded that our LC-MS/MS method allowed a sensitive evaluation of salivary levels of cortisol and cortisone. The simultaneous determination of both hormones in saliva allowed the differential estimation of the active and of the total glucocorticoid exposure over the daytime. The assay could provide further insight into the comprehension of normal and dysfunctional glucocorticoid circadian rhythm.

Sleep restriction alters plasma endocannabinoids concentrations before but not after exercise in humans

Following binding to cannabinoid receptors, endocannabinoids regulate a variety of central nervous system processes including appetite and mood. Recent evidence suggests that the systemic release of these lipid metabolites can be altered by acute exercise and that their levels also vary across the 24-h sleep-wake cycle. The present study utilized a within-subject design (involving 16 normal-weight men) to determine whether daytime circulating endocannabinoid concentrations differ following three nights of partial sleep deprivation (4.25-h sleep opportunity, 2:45-7a.m. each night) vs. normal sleep (8.5-h sleep opportunity, 10:30p.m.-7a.m. each night), before and after an acute bout of ergometer cycling in the morning. In addition, subjective hunger and stress were measured. Pre-exercise plasma concentrations of 2-arachidonoylglycerol (2AG) were 80% higher 1.5h after awakening (vs. normal sleep, p<0.05) when participants were sleep-deprived. This coincided with increased hunger ratings (+25% vs. normal sleep, p<0.05). Moreover, plasma 2AG was elevated 15min post-exercise (+44%, p<0.05). Sleep duration did not however modulate this exercise-induced rise. Finally, subjective stress was generally lower on the day after three nights of short sleep vs. normal sleep, especially after exercise (p<0.05). Given that activation of the endocannabinoid system has been previously shown to acutely increase appetite and mood, our results could suggest that behavioral effects of acute sleep loss, such as increased hunger and transiently improved psychological state, may partially result from activation of this signaling pathway. In contrast, more pronounced exercise-induced elevations of endocannabinoids appear to be less affected by short sleep duration.

Clomiphene citrate effect in obese men with low serum testosterone treated with metformin due to dysmetabolic disorders: A randomized, double-blind, placebo-controlled study

Context Low testosterone (T) levels are often found in obese men with impaired glucose tolerance (IGT) and overt type 2 diabetes (T2DM); however, the mechanisms underlying this condition and its correct therapy are still under debate. Objective To evaluate the effectiveness of clomiphene citrate (CC) in increasing endogenous T levels in obese men with low serum T and with IGT or T2DM treated with metformin (MET). Design Cross-over, randomized, double-blind, placebo-controlled study. Methods 24 obese men, aged 47.3 ±. 6.3 (range 35–55 years), with low T level (≤3 ng/mL) and naïve diagnosis of IGT or T2DM were included. Subjects were randomized to CC 25 mg/day or placebo (Plac) with MET 2 g/day for 3 months. After a 6-week wash-out period, subjects were moved to the alternative arm for additional 3 months. Clinical evaluation and blood exams performed prior to and at the end of treatment. Results Of 24 randomized, 21 were evaluable, classified as IGT (n = 11) or T2DM (n = 10). Compared to baseline levels, T levels increased significantly after 3 months of CC treatment (3.03±0.80 to 5.99±1.67 ng/mL P<0.001) but not after the Plac treatment (2.87±0.78 to 3.09±0.84 ng/mL P<0.001 between the treatments). T changes were similar in IGT and T2DM subjects. Gonadotropins as well raised significantly after CC treatment (LH 3.83±1.45 to 8.53±6.40 mU/mL; FSH 4.84±1.67 to 10.15±5.08 mU/mL P<0.001 respectively), whereas no changes for LH (3.51±1.59 to 3.63±1.39 mU/mL) but a smooth increased for FSH (4.61±2.49 to 5.39±2.65 mU/mL; P = 0.004) were shown after Plac treatment (LH P = 0.001 and FSH P = 0.002 between treatments). Furthermore, fasting glucose (106.8±23.2 to 101.1±25.7 mg/dL; P = 0.004), insulin (19.3±12.1 to 15.6±10.1 μU/mL; P = 0.010) and HOMA-IR (4.94±2.89 to 3.69±2.12; P = 0.001) decreased significantly during the CC treatment period, whereas no significant changes were observed in any of these parameters in the Plac treatment. Conclusions A low dose of CC therapy was able to significantly increase serum T levels in all participants with mild modifications of clinical and metabolic parameters. Trial registration EudraCT 2011-000439-10

Parallel diurnal fluctuation of testosterone, androstenedione, dehydroepiandrosterone and 17OHprogesterone as assessed in serum and saliva: validation of a novel liquid chromatography-tandem mass spectrometry method for salivary steroid profiling

Abstract 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.

Salivary cortisol and cortisone responses to short-term psychological stress challenge in late adolescent and young women with different hyperandrogenic states

Hyperandrogenic disorders have been associated with psychological distress, reduced quality of life, anxiety and depression. The hypothalamic-pituitary-adrenal (HPA) axis plays a pivotal role in the adaptive response to stressor events. Salivary cortisol (SalF) and cortisone (SalE) testing have been proven to be useful in the evaluation of HPA-axis activity. This study investigated whether SalF and SalE responses to two putative stressor levels differed between the hyperandrogenic states in late adolescent and young women, thus measuring the HPA-axis adaptive response to acute stress events. We selected 161 drug-free females aged 16-19 years from a large population previously enrolled in a cross-sectional epidemiological study. Saliva was collected in the morning before and after two putative stressor events consisting in a self-filled questionnaire (weaker stressor) and in a structured interview plus physical examination by an endocrinologist (stronger stressor). SalF and SalE, as well as blood steroids, were assessed by liquid chromatography-tandem mass spectrometry. Subjects were subdivided into different groups according to the presence of: isolated menstrual irregularities (MI, oligo-amenorrhea; n = 22), isolated hirsutism (HIR, modified Ferriman-Gallwey score ≥ 8; n = 26), isolated hyperandrogenaemia (HT, testosterone >0.438 ng/mL; n = 14), and polycystic ovary syndrome (PCOS, MI with HIR and/or HT, n = 16). The remaining 83 apparently healthy subjects were used as controls. SalF and SalE significantly decreased after the weaker stressor, following the physiologic diurnal loss, in all the groups except for isolated HIR, where they remained unchanged (P = 0.091 and P = 0.118, respectively). In contrast, SalF and SalE remained unchanged after the stronger stressor in isolated MI, isolated HT and controls, whereas SalF increased significantly in isolated HIR (P = 0.011), and SalE increased significantly both in isolated HIR (P = 0.005) and in PCOS (P = 0.011) groups. SalF percentage variation in response to the stronger stressor was positively associated with systolic blood pressure in PCOS (P = 0.018), and both SalF and SalE percentage variations were positively associated with diastolic blood pressure in the isolated HIR group (P = 0.010 and P = 0.006, respectively). In addition, in the isolated HIR group, the SalF percentage variation was negatively associated with HDL cholesterol levels (P = 0.005). Finally, SalF and SalE percentage variations were positively associated with circulating androstenedione (P = 0.031 and P = 0.011, respectively) and DHEA (P = 0.020 and P = 0.003, respectively) in the isolated HIR group. In conclusion, this study demonstrates that hirsute and PCOS adolescent and young women are characterized by HPA-axis overactivity in response to stressful stimuli, as detectable by salivary glucocorticoid measurements. These data also indicate that the higher the HPA-axis activity, the higher the adrenal androgen output and the worse the metabolic profile.