Prevalence of carboxy-Δ8 -tetrahydrocannabiniol in antidoping samples.

Δ9 -Tetrahydrocannabinol (Δ9 -THC) is usually the primary psychoactive agent in cannabis preparations. Recently, products containing another isomer, Δ8 -tetrahydrocannabinol (Δ8 -THC), have become available for sale. Δ8 -THC exists naturally in the cannabis plant at very low concentrations; hence, the Δ8 -THC present in most of the above-mentioned products is likely to be manufactured synthetically. A surge in popularity of these products, coupled with little oversight to ensure purity and potency, has led to reports of adverse events. Workplace drug testing programs as well as many sporting organizations prohibit the use of cannabinoids. Carboxy-Δ9 -THC (Δ9 -THC-COOH) is the targeted urinary metabolite for detection of cannabis use. The proliferation of products containing Δ8 -THC, which metabolizes to Δ8 -THC-COOH, presents analytical complexity with respect to separation and quantification of the individual isomers as well as legal complexity with respect to lack of clarity around the legal status of Δ8 -THC. This study aims to estimate the prevalence of Δ8 -THC use in the athlete community by monitoring for Δ8 -THC-COOH in samples collected for antidoping. A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method was utilized to resolve Δ8 and Δ9 -THC-COOH. One thousand samples with a presumptive Δ9 -THC-COOH finding in routine screening were analyzed by the above LC-MS/MS method. Approximately 12% of samples contained Δ8 -THC-COOH at relative abundances between 5% and 100% of total carboxy-THC content.

δ13 C values of urinary 19-norandrosterone in antidoping samples and potential for adverse findings from boar offal consumption.

19-Norandrosterone (19NA) is the preferred urinary target compound to identify doping with nandrolone or related 19-norsteroids. At concentrations between 2.5 and 15 ng/mL, isotope ratio mass spectrometry (IRMS) is required to establish exogenous origin of urinary 19NA. An absolute difference of 3‰ between urinary 19NA and an endogenous reference compound (ERC) constitutes a finding for exogenous origin of 19NA. Over the last 3 years, 77 samples containing urinary 19NA between 2.5 and 15 ng/mL were analyzed at our laboratory. The measured δ13 C values for 19NA ranged from -29.5‰ to -16.8‰. In comparison, the δ13 C values for the corresponding urinary ERCs ranged from -22.4‰ to -16.2‰. Due to the considerable overlap in values between the target compound and the natural range of urinary ERCs, it can be challenging to distinguish between endogenous and exogenous origins of urinary 19NA. In addition, it is well known that consumption of offal from non-castrated pigs can produce 19NA in urine. To determine whether this could cause a positive IRMS finding under the current IRMS positivity criteria, meat from non-castrated boars fed a mixture of corn and soy was consumed by 13 volunteers. Two volunteers produced 19NA findings above 2.5 ng/mL, and the measured isotope values, while inconsistent with documented 19-norsteroid preparations, did meet IRMS positivity criteria. However, these increases in 19NA urinary concentrations were short-lived due to rapid elimination. Timely follow-up collections may help support a claim for dietary exposure when low urinary concentrations of 19NA with pseudo-endogenous isotope values are observed.

Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.

Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.

Activated neutrophil fluorescent imaging technique for human lungs.

Neutrophil activation is an integral process to acute inflammation and is associated with adverse clinical sequelae. Identification of neutrophil activation in real time in the lungs of patients may permit biological stratification of patients in otherwise heterogenous cohorts typically defined by clinical criteria. No methods for identifying neutrophil activation in real time in the lungs of patients currently exist. We developed a bespoke molecular imaging probe targeting three characteristic signatures of neutrophil activation: pinocytosis, phagosomal alkalinisation, and human neutrophil elastase (HNE) activity. The probe functioned as designed in vitro and ex vivo. We evaluated optical endomicroscopy imaging of neutrophil activity using the probe in real-time at the bedside of healthy volunteers, patients with bronchiectasis, and critically unwell mechanically ventilated patients. We detected a range of imaging responses in vivo reflecting heterogeneity of condition and severity. We corroborated optical signal was due to probe function and neutrophil activation.

Optical Molecular Imaging of Inflammatory Cells in Interventional Medicine-An Emerging Strategy.

The optical molecular imaging of inflammation is an emerging strategy for interventional medicine and diagnostics. The host's inflammatory response and adaptation to acute and chronic diseases provides unique signatures that have the potential to guide interventions. Thus, there are emerging a suite of molecular imaging and sensing approaches for a variety of targets in this area. This review will focus on two key cellular orchestrators that dominate this area, neutrophils and macrophages, with recent developments in molecular probes and approaches discussed.

Metabolism of methylstenbolone studied with human liver microsomes and the uPA⁺/⁺-SCID chimeric mouse model.

Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method.