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An instrument integrating thermal desorption (TD) to selected ion flow tube mass spectrometry (SIFT-MS) is presented, and its application to analyze volatile organic compounds (VOCs) in human breath is demonstrated for the first time. The rationale behind this development is the need to analyze breath samples in large-scale multicenter clinical projects involving thousands of patients recruited in different hospitals. Following adapted guidelines for validating analytical techniques, we developed and validated a targeted analytical method for 21 compounds of diverse chemical class, chosen for their clinical and biological relevance. Validation has been carried out by two independent laboratories, using calibration standards and real breath samples from healthy volunteers. The merging of SIFT-MS and TD integrates the rapid analytical capabilities of SIFT-MS with the capacity to collect breath samples across multiple hospitals. Thanks to these features, the novel instrument has the potential to be easily employed in clinical practice.

Seizure unpredictability plays a major role in disability and decreased quality of life in people with epilepsy. Dogs have been used to assist people with disabilities and have shown promise in detecting seizures. There have been reports of trained seizure-alerting dogs (SADs) successfully detecting when a seizure is occurring or indicating imminent seizures, allowing patients to take preventative measures. Untrained pet dogs have also shown the ability to detect seizures and provide comfort and protection during and after seizures. Dogs' exceptional olfactory abilities and sensitivity to human cues could contribute to their seizure-detection capabilities. This has been supported by studies in which dogs have distinguished between epileptic seizure and non-seizure sweat samples, probably though the detection of volatile organic compounds (VOCs). However, the existing literature has limitations, with a lack of well-controlled, prospective studies and inconsistencies in reported timings of alerting behaviours. More research is needed to standardize reporting and validate the results. Advances in VOC profiling could aid in distinguishing seizure types and developing rapid and unbiased seizure detection methods. In conclusion, using dogs in epilepsy management shows considerable promise, but further research is needed to fully validate their effectiveness and potential as valuable companions for people with epilepsy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The endogenous cannabinoid system (ECS), including the endocannabinoids (eCBs), anandamide (AEA), and 2-arachidonoylglycerol (2-AG), plays an integral role in psychophysiological functions. Although frequent cannabis use is associated with adaptations in the ECS, the impact of acute smoked cannabis administration on circulating eCBs, and the relationship between cannabis effects and circulating eCBs are poorly understood. Methods: This study measured the plasma levels of AEA, 2-AG, and Δ-9-tetrahydrocannabinol (THC), subjective drug-effects ratings, and cardiovascular measures at baseline and 15-180 min after cannabis users ( n =26) smoked 70% of a cannabis cigarette (5.6% THC). Results: Cannabis administration increased the ratings of intoxication, heart rate, and plasma THC levels relative to baseline. Although cannabis administration did not affect eCB levels relative to baseline, there was a significant positive correlation between baseline AEA levels and peak ratings of "High" and "Good Drug Effect." Further, baseline 2-AG levels negatively correlated with frequency of cannabis use (mean days/week) and with baseline THC metabolite levels. Conclusions: In a subset of heavy cannabis smokers: (1) more frequent cannabis use was associated with lower baseline 2-AG, and (2) those with lower AEA got less intoxicated after smoking cannabis. These findings contribute to a sparse literature on the interaction between endo- and phyto-cannabinoids. Future studies in participants with varied cannabis use patterns are needed to clarify the association between circulating eCBs and the abuse-related effects of cannabis, and to test whether baseline eCBs predict the intoxicating effects of cannabis and are a potential biomarker of cannabis tolerance.

Urinary volatolomics offers a noninvasive approach for disease detection and monitoring. Herein we present an improved methodology for global volatolomic profiling. Wide coverage was achieved by utilizing a multiphase sorbent for volatile organic compound (VOC) extraction. A single, midpolar column gas chromatography (GC) assay yielded substantially higher numbers of monitored VOCs compared to our previously reported single-sorbent method. Multidimensional GC (GC×GC) enhanced further biomarker discovery while data analysis was simplified by using a tile-based approach. At the same time, the required urine volume was reduced 5-fold from 2 to 0.4 mL. The applicability of the methodology was demonstrated in a pancreatic ductal adenocarcinoma cohort where previous findings were confirmed while a series of additional VOCs with diagnostic potential were discovered.

The emergence of metabolomics and quantification approaches is revealing new biomarkers applied to drug discovery. In this context, tandem mass spectrometry is the method of choice, requiring a specific validation process for preclinical and clinical applications. Research on the two classes of lipid mediators, steroids and cannabinoids, has revealed a potential interaction in cannabis addiction and metabolism-related disorders. Here we present the development of GC-MS/MS and LC-MS/MS methods for routine quantification of targeted steroids and cannabinoids, respectively. The methods were developed using an isotopic approach, including validation for linearity, selectivity, LLOQ determination, matrix effect, carryover, between- and within-run accuracy and precision, and stability tests to measure 11 steroids and seven cannabinoids in human plasma. These methods were satisfactory for most validity conditions, although not all met the acceptance criteria for all analytes. A comparison of calibration curves in biological and surrogate matrices and in methanol showed that the latter condition was more applicable for our quantification of endogenous compounds. In conclusion, the validation of our methods met the criteria for GLP-qualified rather than GLP-validated methods, which can be used for routine analytical studies for dedicated preclinical and clinical purposes, by combining appropriate system suitability testing, including quality controls in the biological matrix.

Volatolomics offers an opportunity for noninvasive detection and monitoring of human disease. While gas chromatography-mass spectrometry (GC-MS) remains the technique of choice for analyzing volatile organic compounds (VOCs), barriers to wider adoption in clinical practice still exist, including: sample preparation and introduction techniques, VOC extraction, throughput, volatolome coverage, biological interpretation, and quality control (QC). Therefore, we developed a complete pipeline for untargeted urinary volatolomic profiling. We optimized a novel extraction technique using HiSorb sorptive extraction, which exhibited high analytical performance and throughput. We achieved a broader VOC coverage by using HiSorb coupled with a set of complementary chromatographic methods and time-of-flight mass spectrometry. Furthermore, we developed a data preprocessing strategy by evaluating internal standard normalization, batch correction, and we adopted strict QC measures including removal of nonlinearly responding, irreproducible, or contaminated metabolic features, ensuring the acquisition of high-quality data. The applicability of this pipeline was evaluated in a clinical cohort consisting of pancreatic ductal adenocarcinoma (PDAC) patients ( n = 28) and controls ( n = 33), identifying four urinary candidate biomarkers (2-pentanone, hexanal, 3-hexanone, and p -cymene), which can successfully discriminate the cancer and noncancer subjects. This study presents an optimized, high-throughput, and quality-controlled pipeline for untargeted urinary volatolomic profiling. Use of the pipeline to discriminate PDAC from control subjects provides proof of principal of its clinical utility and potential for application in future biomarker discovery studies.

The interest around analysis of volatile organic compounds (VOCs) within breath has increased in the last two decades. Uncertainty remains around standardisation of sampling and whether VOCs within room air can influence breath VOC profiles. To assess the abundance of VOCs within room air in common breath sampling locations within a hospital setting and whether this influences the composition of breath. A secondary objective is to investigate diurnal variation in room air VOCs. Room air was collected using a sampling pump and thermal desorption (TD) tubes in the morning and afternoon from five locations. Breath samples were collected in the morning only. TD tubes were analysed using gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF-MS). A total of 113 VOCs were identified from the collected samples. Multivariate analysis demonstrated clear separation between breath and room air. Room air composition changed throughout the day and different locations were characterized by specific VOCs, which were not influencing breath profiles. Breath did not demonstrate separation based on location, suggesting that sampling can be performed across different locations without affecting results., (© 2022. The Author(s).)

The analysis of volatile organic compounds (VOCs) can provide important clinical information (entirely non-invasively); however, the exact extent to which VOCs from human skin can be signatures of health and disease is unknown. This systematic review summarises the published literature concerning the methodology, application, and volatile profiles of skin VOC studies. An online literature search was conducted in accordance with the preferred reporting items for systematic reviews and meta-analysis, to identify human skin VOC studies using untargeted mass spectrometry (MS) methods. The principal outcome was chemically verified VOCs detected from the skin. Each VOC was cross-referenced using the CAS number against the Human Metabolome and KEGG databases to evaluate biological origins. A total of 29 studies identified 822 skin VOCs from 935 participants. Skin VOCs were commonly sampled from the hand ( n = 9) or forearm ( n = 7) using an absorbent patch ( n = 15) with analysis by gas chromatography MS ( n = 23). Twenty-two studies profiled the skin VOCs of healthy subjects, demonstrating a volatolome consisting of aldehydes (18%), carboxylic acids (12%), alkanes (12%), fatty alcohols (9%), ketones (7%), benzenes and derivatives (6%), alkenes (2%), and menthane monoterpenoids (2%). Of the VOCs identified, 13% had putative endogenous origins, 46% had tentative exogenous origins, and 40% were metabolites from mixed metabolic pathways. This review has comprehensively profiled the human skin volatolome, demonstrating the presence of a distinct VOC signature of healthy skin, which can be used as a reference for future researchers seeking to unlock the clinical potential of skin volatolomics. As significant proportions of identified VOCs have putative exogenous origins, strategies to minimise their presence through methodological refinements and identifying confounding compounds are discussed.

Rationale: Chronic obstructive pulmonary disease (COPD) is a condition punctuated by acute exacerbations commonly triggered by viral and/or bacterial infection. Early identification of exacerbation triggers is important to guide appropriate therapy, but currently available tests are slow and imprecise. Volatile organic compounds (VOCs) can be detected in exhaled breath and have the potential to be rapid tissue-specific biomarkers of infection etiology. Objectives: To determine whether volatile organic compound measurement could distinguish viral from bacterial infection in COPD. Methods: We used serial sampling within in vitro and in vivo studies to elucidate the dynamic changes that occur in VOC production during acute respiratory viral infection. Highly sensitive gas chromatography-mass spectrometry techniques were used to measure VOC production from infected airway epithelial-cell cultures and in exhaled breath samples from healthy subjects experimentally challenged with rhinovirus (RV)-A16 and from subjects with COPD with naturally occurring exacerbations. Measurements and Main Results: We identified a novel VOC signature comprising decane and other long-chain alkane compounds that is induced during RV infection of cultured airway epithelial cells and is also increased in the exhaled breath from healthy subjects experimentally challenged with RV and from patients with COPD during naturally occurring viral exacerbations. These compounds correlated with the magnitude of antiviral immune responses, viral burden, and exacerbation severity but were not induced by bacterial infection, suggesting that they represent a specific virus-inducible signature. Conclusions: Our study highlights the potential for measurement of exhaled breath VOCs as rapid, noninvasive biomarkers of viral infection. Further studies are needed to determine whether measurement of these signatures could be used to guide more targeted therapy with antibiotic/antiviral agents for COPD exacerbations.

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., (© 2021 John Wiley & Sons Ltd.)

The analysis of volatile organic compounds (VOCs) within breath for noninvasive disease detection and monitoring is an emergent research field that has the potential to reshape current clinical practice. However, adoption of breath testing has been limited by a lack of standardization. This protocol provides a comprehensive workflow for online and offline breath analysis using selected ion flow tube mass spectrometry (SIFT-MS). Following the suggested protocol, 50 human breath samples can be analyzed and interpreted in <3 h. Key advantages of SIFT-MS are exploited, including the acquisition of real-time results and direct compound quantification without need for calibration curves. The protocol includes details of methods developed for targeted analysis of disease-specific VOCs, specifically short-chain fatty acids, aldehydes, phenols, alcohols and alkanes. A procedure to make custom breath collection bags is also described. This standardized protocol for VOC analysis using SIFT-MS is intended to provide a basis for wider application and the use of breath analysis in clinical studies.

Volatile breath metabolites serve as potential disease biomarkers. Online mass spectrometry (MS) presents real-time quantification of breath volatile organic compounds (VOCs). The study aims to assess the relationship between two online analytical mass spectrometry techniques in the quantification of target breath metabolites: selected ion flow tube mass spectrometry (SIFT-MS) and proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS). The two following techniques were employed: (i) direct injection with bag sampling using SIFT-MS and PTR-ToF-MS and (ii) direct injection and thermal desorption (TD) tube comparison using PTR-ToF-MS. The concentration of abundant breath metabolites, acetone and isoprene, demonstrated a strong positive linear correlation between both mass spectrometry techniques ( r = 0.97, r = 0.89, respectively; p < 0.001) and between direct injection and TD tube ( r = 0.97, r = 0.92, respectively; p < 0.001) breath sampling techniques. This was reflected for the majority of short chain fatty acids and alcohols tested ( r > 0.80, p < 0.001). Analyte concentrations were notably higher with the direct injection of a sampling bag compared to the TD method. All metabolites produced a high degree of agreement in the detection range of VOCs between SIFT-MS and PTR-ToF-MS, with the majority of compounds falling within 95% of the limits of agreement with Bland-Altman analysis. The cross platform analysis of exhaled breath demonstrates strong positive correlation coefficients, linear regression, and agreement in target metabolite detection rates between both breath sampling techniques. The study demonstrates the transferability of using data outputs between SIFT-MS and PTR-ToF-MS. It supports the implementation of a TD platform in multi-site studies for breath biomarker research in order to facilitate sample transport between clinics and the laboratory.

This paper comprises an updated version of the 2014 review which reported 1846 volatile organic compounds (VOCs) identified from healthy humans. In total over 900 additional VOCs have been reported since the 2014 review and the VOCs from semen have been added. The numbers of VOCs found in breath and the other bodily fluids are: blood 379, breath 1488, faeces 443, milk 290, saliva 549, semen 196, skin 623 and urine 444. Compounds were assigned CAS registry numbers and named according to a common convention where possible. The compounds have been included in a single table with the source reference(s) for each VOC, an update on our 2014 paper. VOCs have also been grouped into tables according to their chemical class or functionality to permit easy comparison. Careful use of the database is needed, as a number of the identified VOCs only have level 2-putative assignment, and only a small fraction of the reported VOCs have been validated by standards. Some clear differences are observed, for instance, a lack of esters in urine with a high number in faeces and breath. However, the lack of compounds from matrices such a semen and milk compared to breath for example could be due to the techniques used or reflect the intensity of effort e.g. there are few publications on VOCs from milk and semen compared to a large number for breath. The large number of volatiles reported from skin is partly due to the methodologies used, e.g. by collecting skin sebum (with dissolved VOCs and semi VOCs) onto glass beads or cotton pads and then heating to a high temperature to desorb VOCs. All compounds have been included as reported (unless there was a clear discrepancy between name and chemical structure), but there may be some mistaken assignations arising from the original publications, particularly for isomers. It is the authors' intention that this work will not only be a useful database of VOCs listed in the literature but will stimulate further study of VOCs from healthy individuals; for example more work is required to confirm the identification of these VOCs adhering to the principles outlined in the metabolomics standards initiative. Establishing a list of volatiles emanating from healthy individuals and increased understanding of VOC metabolic pathways is an important step for differentiating between diseases using VOCs., (© 2021 IOP Publishing Ltd.)

Objectives: To examine the feasibility and acceptability of breath research in primary care., Design: Non-randomised, prospective, mixed-methods cross-sectional observational study., Setting: Twenty-six urban primary care practices., Participants: 1002 patients aged 18-90 years with gastrointestinal symptoms., Main Outcome Measures: During the first 6 months of the study (phase 1), feasibility of patient enrolment using face-to-face, telephone or SMS-messaging (Short Message Service) enrolment strategies, as well as processes for breath testing at local primary care practices, were evaluated. A mixed-method iterative study design was adopted and outcomes evaluated using weekly Plan-Do-Study-Act cycles, focus groups and general practitioner (GP) questionnaires.During the second 6 months of the study (phase 2), patient and GP acceptability of the breath test and testing process was assessed using questionnaires. In addition a 'single practice' recruitment model was compared with a 'hub and spoke' centralised recruitment model with regards to enrolment ability and patient acceptability.Throughout the study feasibility of the collection of a large number of breath samples by clinical staff over multiple study sites was evaluated and quantified by the analysis of these samples using mass spectrometry., Results: 1002 patients were recruited within 192 sampling days. Both 'single practice' and 'hub and spoke' recruitment models were effective with an average of 5.3 and 4.3 patients accrued per day, respectively. The 'hub and spoke' model with SMS messaging was the most efficient combined method of patient accrual. Acceptability of the test was high among both patients and GPs. The methodology for collection, handling and analysis of breath samples was effective, with 95% of samples meeting quality criteria., Conclusions: Large-scale breath testing in primary care was feasible and acceptable. This study provides a practical framework to guide the design of Phase III trials examining the performance of breath testing in primary care., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography-mass spectrometry (GC-MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC-MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples.

The analysis of urinary volatile organic compounds (VOCs) is a promising field of research with the potential to discover new biomarkers for cancer early detection. This systematic review aims to summarise the published literature concerning cancer-associated urinary VOCs. A systematic online literature search was conducted to identify studies reporting urinary VOC biomarkers of cancers in accordance with the recommendations of the Cochrane Library and Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines. Thirteen studies comprising 1266 participants in total were included in the review. Studies reported urinary VOC profiles of five cancer subtypes: prostate cancer, gastrointestinal cancer, leukaemia/lymphoma, lung cancer, and bladder cancer. Forty-eight urinary VOCs belonging to eleven chemical classes were identified with high diagnostic performance. VOC profiles were distinctive for each cancer type with limited cross-over. The metabolic analysis suggested distinctive phenotypes for prostate and gastrointestinal cancers. The heterogenicity of study design, methodological and reporting quality may have contributed to inconsistencies between studies. Urinary VOC analysis has shown promising performance for non-invasive diagnosis of cancer. However, limitations in study design have resulted in inconsistencies between studies. These limitations are summarised and discussed in order to support future studies.

Rationale: The analysis of volatile organic compounds (VOCs) within exhaled breath potentially offers a non-invasive method for the detection and surveillance of human disease. Oral contamination of exhaled breath may influence the detection of systemic VOCs relevant to human disease. This study aims to assess the impact of oral cleansing strategies on exhaled VOC levels in order to standardise practice for breath sampling., Methods: Ten healthy volunteers consumed a nutrient challenge followed by four oral cleansing methods: (a) water, (b) saltwater, (c) toothbrushing, and (d) alcohol-free mouthwash. Direct breath sampling was performed using selected ion flow tube mass spectrometry after each intervention., Results: Proposed reactions suggest that volatile fatty acid and alcohol levels (butanoic, pentanoic acid, ethanol) declined with oral cleansing interventions, predominantly after an initial oral rinse with water. Concentrations of aldehydes and phenols (acetaldehyde, menthone, p-cresol) declined with oral water rinse; however, they increased after toothbrushing and mouthwash use, secondary to flavoured ingredients within these products. No significant reductions were observed with sulphur compounds., Conclusions: Findings suggest that oral rinsing with water prior to breath sampling may reduce oral contamination of VOC levels, and further interventions for oral decontamination with flavoured products may compromise results. This intervention may serve as a simple and inexpensive method of standardisation within breath research., (© 2019 John Wiley & Sons, Ltd.)

Breath analysis is highly acceptable to patients and health care professionals, but its implementation in clinical practice remains challenging. Clinical trials and routine practice require a robust system for collection, storage, and processing of large numbers of samples. This work describes a platform based upon the hyphenation of thermal desorption (TD) with proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS), coupled by means of an original modification of the TD interface. The performance of TD-PTR-ToF-MS was tested against seven oxygenated volatile organic compounds (VOCs), belonging to three chemical classes (i.e., fatty acids, aldehydes, and phenols), previously identified as possible biomarkers of colorectal and esophago-gastric adenocarcinoma. Limits of detection and quantification were on the order of 0.2-0.9 and 0.3-1.5 parts per billion by volume (ppbV), respectively. Analytical recoveries from TD tubes were 80% or higher, linear response was in the low- to mid-ppbV range ( R 2 = 0.98-0.99), and coefficients of variation were within 20% of mean values. The usability of the platform was evaluated in the analysis of a set of breath samples of clinical origin, allowing for a throughput of nearly 100 TD tubes for 24 h of continuous operation. All of these characteristics enhance the implementation of TD-PTR-ToF-MS for large-scale clinical studies.

Objective: N-acylethanolamines play different roles in energy balance; anandamide (AEA) stimulates energy intake and storage, N-palmitoylethanolamide (PEA) counters inflammation, and N-oleoylethanolamide (OEA) mediates anorectic signals and lipid oxidation. Inconsistencies in the association of plasma N-acylethanolamines with human obesity and cardiometabolic risk have emerged among previous studies, possibly caused by heterogeneous cohorts and designs, and by unstandardized N-acylethanolamine measurements. We aimed to characterize changes in the plasma profile, including N-acylethanolamine levels and ratios associated with obesity, menopause in women, and ageing in men, and to define the significance of such a profile as a biomarker for metabolic imbalance., Methods: Adult, drug-free women (n = 103 premenopausal and n = 81 menopausal) and men (n = 144) were stratified according to the body mass index (BMI) into normal weight (NW; BMI: 18.5-24.9 kg/m 2 ), overweight (OW; BMI: 25.0-29.9 kg/m 2 ), and obese (OB; BMI ≥30.0 kg/m 2 ). Anthropometric and metabolic parameters were determined. Validated blood processing and analytical procedures for N-acylethanolamine measurements were used. We investigated the effect of BMI and menopause in women, and BMI and age in men, as well as the BMI-independent influence of metabolic parameters on the N-acylethanolamine profile., Results: BMI and waist circumference directly associated with AEA in women and men, and with PEA in premenopausal women and in men, while BMI directly associated with OEA in premenopausal women and in men. BMI, in both genders, and waist circumference, in women only, inversely associated with PEA/AEA and OEA/AEA. Menopause increased N-acylethanolamine levels, whereas ageing resulted in increasing OEA relative abundance in men. AEA and OEA abundances in premenopausal, and PEA and OEA abundances in lean menopausal women, were directly associated with hypertension. Conversely, PEA and OEA abundances lowered with hypertension in elderly men. Insulin resistance was associated with changes in N-acylethanolamine ratios specific for premenopausal (reduced PEA/AEA and OEA/AEA), menopausal (reduced OEA/AEA) women and men (reduced OEA/AEA and OEA/PEA). PEA and OEA levels increased with total cholesterol, and OEA abundance specifically increased with HDL-cholesterol. Elevated triglyceride levels were associated with increased N-acylethanolamine levels only in menopausal women., Conclusions: Obesity-related N-acylethanolamine hypertone is characterized by imbalanced N-acylethanolamine ratios. The profile given by a combination of N-acylethanolamine absolute levels and ratios enables imbalances to be identified in relationship with different metabolic parameters, with specific relevance according to gender, menopause and age, representing a useful means for monitoring metabolic health. Finally, N-acylethanolamine system appears a promising target for intervention strategies., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Bidirectional communication between neurons and astrocytes shapes synaptic plasticity and behavior. D-serine is a necessary co-agonist of synaptic N-methyl-D-aspartate receptors (NMDARs), but the physiological factors regulating its impact on memory processes are scantly known. We show that astroglial CB 1 receptors are key determinants of object recognition memory by determining the availability of D-serine at hippocampal synapses. Mutant mice lacking CB 1 receptors from astroglial cells (GFAP-CB 1 -KO) displayed impaired object recognition memory and decreased in vivo and in vitro long-term potentiation (LTP) at CA3-CA1 hippocampal synapses. Activation of CB 1 receptors increased intracellular astroglial Ca 2+ levels and extracellular levels of D-serine in hippocampal slices. Accordingly, GFAP-CB 1 -KO displayed lower occupancy of the co-agonist binding site of synaptic hippocampal NMDARs. Finally, elevation of D-serine levels fully rescued LTP and memory impairments of GFAP-CB 1 -KO mice. These data reveal a novel mechanism of in vivo astroglial control of memory and synaptic plasticity via the D-serine-dependent control of NMDARs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: The 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., Methods: Adult, 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/m 2 ), overweight (OW; BMI: 25.0-29.9 kg/m 2 ), and obese (OB; BMI ≥ 30.0 kg/m 2 ) classes. Anthropometric and metabolic parameters were determined. Plasma 2-arachidonoylglycerol was measured by a validated liquid chromatography-mass spectrometry assay., Results: 2-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)., Conclusions: Plasma 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.

This study examined the respective influences of cannabinoid type-1 (CB1) receptors expressed either in forebrain GABAergic neurons, in cortical glutamatergic neurons, or in astrocytes on the turnover rates of the endocannabinoids N-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), and the non-cannabinoid N-acylethanolamides, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA), in mouse forebrain regions. To this end, conditional mutant mice lacking CB1 receptors from either of these cell types were pre-treated systemically with JZL195, a dual inhibitor of fatty acid amide hydrolase, the enzyme degrading AEA, PEA, and OEA, and of monoacylglycerol lipase, the main 2-AG-degrading enzyme. The analyses of frontocortical, hippocampal, and striatal AEA, 2-AG, PEA, and OEA concentrations revealed that their respective baseline concentrations were not influenced by the mouse genotype. On the other hand, the accumulation of frontocortical and/or hippocampal 2-AG levels in JZL195-pre-treated mice was dependent on the mouse genotype. Thus, JZL195-induced 2-AG accumulation rates were diminished in the frontal cortex of mice lacking CB1 receptors in glutamatergic neurons while their respective values were increased in the frontal cortex and hippocampus of mice lacking these receptors in astrocytes. These genotypic differences occurred with parallel and proportionate changes in the fractional rate constants for degradation of 2-AG, thus providing a mechanism whereby the baseline levels of 2-AG remained constant between genotypes. Besides suggesting a cell-type-specific control of frontocortical and/or hippocampal 2-AG synthesis and degradation rates by CB1 receptors, this study highlights the interest of assessing endocannabinoid turnover rates when questioning the status of the endocannabinoid system., (© 2015 International Society for Neurochemistry.)

Objective: High-protein diets favor weight loss and its maintenance. Whether these effects might be recapitulated by certain amino acids is unknown. Therefore, the impact of leucine supplementation on energy balance and associated metabolic changes in diet-induced obese (DIO) mice during and after weight loss was investigated., Methods: DIO C57BL/6J mice were fed a normocaloric diet to induce weight loss while receiving or not the amino acid leucine in drinking water. Body weight, food intake, body composition, energy expenditure, glucose tolerance, insulin, and leptin sensitivity were evaluated. Q-PCR analysis was performed on muscle, brown and white adipose tissues., Results: DIO mice decreased body weight and fat mass in response to chow, but supplementation with leucine did not affect these parameters. During weight maintenance, mice supplemented with leucine had improved glucose tolerance, increased leptin sensitivity, and lower respiratory quotient. The latter was associated with changes in the expression of several genes modulating fatty acid metabolism and mitochondrial activity in the epididymal white and the brown adipose tissues, but not muscle., Conclusions: Leucine supplementation might represent an adjuvant beneficial nutritional therapy during weight loss and maintenance, because it improves lipid and glucose metabolism and restores leptin sensitivity in previously obese animals., (Copyright © 2013 The Obesity Society.)

Background: Leucine supplementation might have therapeutic potential in preventing diet-induced obesity and improving insulin sensitivity. However, the underlying mechanisms are at present unclear. Additionally, it is unclear whether leucine supplementation might be equally efficacious once obesity has developed., Methodology/principal Findings: Male C57BL/6J mice were fed chow or a high-fat diet (HFD), supplemented or not with leucine for 17 weeks. Another group of HFD-fed mice (HFD-pairfat group) was food restricted in order to reach an adiposity level comparable to that of HFD-Leu mice. Finally, a third group of mice was exposed to HFD for 12 weeks before being chronically supplemented with leucine. Leucine supplementation in HFD-fed mice decreased body weight and fat mass by increasing energy expenditure, fatty acid oxidation and locomotor activity in vivo. The decreased adiposity in HFD-Leu mice was associated with increased expression of uncoupling protein 3 (UCP-3) in the brown adipose tissue, better insulin sensitivity, increased intestinal gluconeogenesis and preservation of islets of Langerhans histomorphology and function. HFD-pairfat mice had a comparable improvement in insulin sensitivity, without changes in islets physiology or intestinal gluconeogenesis. Remarkably, both HFD-Leu and HFD-pairfat mice had decreased hepatic lipid content, which likely helped improve insulin sensitivity. In contrast, when leucine was supplemented to already obese animals, no changes in body weight, body composition or glucose metabolism were observed., Conclusions/significance: These findings suggest that leucine improves insulin sensitivity in HFD-fed mice by primarily decreasing adiposity, rather than directly acting on peripheral target organs. However, beneficial effects of leucine on intestinal gluconeogenesis and islets of Langerhans's physiology might help prevent type 2 diabetes development. Differently, metabolic benefit of leucine supplementation is lacking in already obese animals, a phenomenon possibly related to the extent of the obesity before starting the supplementation.

Context: Physiological transient imbalance typical of adolescence needs to be distinguished from hyperandrogenism-related dysfunction. The accurate determination of circulating androgens is the best indicator of hyperandrogenism. However, reliable reference intervals for adolescent and young women are not available., Objective: The aim of the study was to define androgen reference intervals in young women and to analyze the impact of the menstrual phase and ovulation efficiency over the androgen profile as assessed by reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique., Participants: Female high school students aged 16-19 years were included in the study., Main Outcome Measures: The study was performed on reference subjects properly selected among an unbiased population. Normal-weight, drug and disease free, eumenorrheic females with no signs of hyperandrogenism were included. The steroid hormone profile was determined by a validated in-house LC-MS/MS method. A statistical estimation of overall and menstrual phase-specific reference intervals was performed. A subgroup of anovulatory females was identified based on progesterone circulating levels. The impact of ovulation efficiency over hormonal profile was analyzed., Results: A total of 159 females satisfied healthy criteria. Androgen levels did not vary according to menstrual phase, but a significantly higher upper reference limit was found for T in the luteal phase compared to the follicular phase. Higher T and androstenedione levels were observed in anovulatory compared to ovulatory females, paralleled by higher LH and FSH and lower 17-hydroxyprogesterone and 17β-estradiol levels., Conclusions: 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.

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 indefiniteness 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 oleoyl-ethanolamide, 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 on-line purification was chosen, allowing high-throughput and reliability. We estimated gender-specific reference intervals on 121 healthy normal weight subjects fulfilling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas significantly higher MAG levels were found in males compared with females. MAGs also significantly 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 definitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research field into the clinical field.

Background: The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects., Methods: 0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis., Results: Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone>1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone<1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established., Conclusion: 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., (Copyright © 2010 Elsevier Inc. All rights reserved.)