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

This is the first study providing LC-MS/MS-based, menstrual phase-specific reference intervals for the circulating androgen profile in young females. We identified a subgroup of anovulatory healthy females characterized by androgen imbalance.

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.