Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.

Supplementary key words endocannabinoids • validation • reference intervalsThe endocannabinoids (ECs) are bioactive lipid mediators derived from membrane phospholipids. Since the discovery of the fi rst lipid mediator of the endocannabinoid system (ECS), arachidonoyl-ethanolamide (AEA), also called anandamide ( 1 ), several molecules belonging to this family were identifi ed, the most important being 2-arachidonoyl-glycerol (2AG) and its isomer 1AG among MAGs ( 2, 3 ), palmitoyl-ethanolamide (PEA) and oleoylethanolamide (OEA) among the NAEs, to which AEA belongs ( 4 ). Both 2AG and AEA act on cannabinoid receptor type 1 (CB1) and type 2 (CB2), whereas PEA and OEA act by infl uencing AEA metabolism and binding the peroxisome proliferator-activated receptor ␣ ( 5, 6 ), thus defi ned endocannabinoid related compounds (ERC ).Abstract The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefi niteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/ MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoylethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by online purifi cation was chosen, allowing high-throughput and reliability. We estimated gender-specifi c reference intervals on 121 healthy normal weight subjects fulfi lling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas signifi cantly higher MAG levels were found in males compared with females. MAGs also signifi cantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of defi nitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research fi eld into the clinical fi eld. -Fanelli,F

ContextLow 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.ObjectiveTo 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).DesignCross-over, randomized, double-blind, placebo-controlled study.Methods24 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.ResultsOf 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.ConclusionsA 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 registrationEudraCT 2011-000439-10

ObjectiveThe endocannabinoid system hypertonicity features obesity. Excess circulating 2-arachidonoylglycerol was variously associated with obesity-related metabolic impairment; however, unstandardized experimental and analytical settings have clouded its usefulness as a dysmetabolism biomarker. We aimed at assessing the influence of body mass index (BMI), menopause in women, and aging in men on 2-arachidonoylglycerol relationship with metabolic parameters.MethodsAdult, unmedicated women (premenopausal (preMW): n = 103; menopausal (MW): n = 81) and men (n = 144) were stratified in normal weight (NW; BMI: 18.5–24.9 kg/m2), overweight (OW; BMI: 25.0–29.9 kg/m2), and obese (OB; BMI ≥ 30.0 kg/m2) classes. Anthropometric and metabolic parameters were determined. Plasma 2-arachidonoylglycerol was measured by a validated liquid chromatography-mass spectrometry assay.Results2-arachidonoylglycerol level was raised by menopause (P < 0.001) and by obesity in preMW (P < 0.001) and in men (P = 0.019). In the overall cohorts, 2-arachidonoylglycerol displayed BMI-independent relationships with dyslipidemia (preMW, MW and men), insulin resistance (MW and men), and hypertension (men), but not with waist circumference. Within preMW BMI classes, 2-arachidonoylglycerol correlations were found with triglycerides (P = 0.020) and total cholesterol (TC; P = 0.040) in OB women. In MW, 2-arachidonoylglycerol correlation with triglycerides was found in NW (P = 0.001) and OW (P = 0.034), but not in OB class. Moreover, we found 2-arachidonoylglycerol correlations with TC (P = 0.003), glucose (P < 0.001), and HOMA-IR (P = 0.035) specific for NW MW class. In men, 2-arachidonoylglycerol correlated with triglycerides in NW, OW (both P < 0.001), and OB (P = 0.029), with SBP (P = 0.023) and diastolic BP (DBP; P = 0.048) in OB, and with TC (P < 0.001) in OW class. In NW class 2-arachidonoylglycerol correlations were found with insulin (P = 0.003) and HOMA-IR (P = 0.001), both enhanced by aging (both P = 0.004), and with glucose (P = 0.015) and HDL (P = 0.004).ConclusionsPlasma 2AG is a biomarker of clustering metabolic dysfunctions, especially in lean men and menopausal women, and could be of help in identifying subjects with elevated cardiometabolic risk despite a healthy anthropometric appearance.

The HEART score is a simple scoring system, ranging from 0 to 10, specifically developed for risk stratification of patients with undifferentiated chest pain. It has been validated for the conventional troponin, but not for high-sensitive troponin. We assess a modified version of the HEART score using a single high-sensitivity troponin T dosage at presentation, regardless of symptom duration, and with different ECG criteria to evaluate if the patients with a low HEART score could be safely discharged early. The secondary aim was to confirm a statistically significant difference in each HEART score group (low 0-3, intermediate 4-6, high 7-10) in the occurrence of major adverse cardiac events at 30 and 180 days. We retrospectively analyzed the HEART score of 1597 consecutive patients admitted to the Emergency Department of our Hospital for chest pain between January 1 and June 30, 2014. Of these, 190 did not meet the inclusion criteria and 29 were lost to follow-up. None of the 512 (37.2 %) patients with a low HEART score had an event within 180 days. The difference between the cumulative incidences of events in the three HEART score groups was statistically significant (P < 0.0001). We demonstrate that it might be possible to safely discharge Emergency Department chest pain patients with a low modified HEART score after an initial determination of high-sensitive troponin T, without a prolonged observation period or an additional cardiac testing.

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 sub-cohorts. 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 age-dependent reference intervals is mandatory for steroid precursors and corticosteroids.

Background: Clomiphene citrate (CC) has been shown to restore the hypothalamicpituitary-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, doubleblind, 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. K E Y W O R D SAMH, Inhibin B, obesity, testosterone | 39 PELUSI Et aL.

No abstract