Bidirectional Effect of Long-Term Δ9-Tetrahydrocannabinol Treatment on mTOR Activity and Metabolome.

Brain aging is associated with cognitive decline, reduced synaptic plasticity, and altered metabolism. The activity of mechanistic target of rapamycin (mTOR) has a major impact on aging by regulating cellular metabolism. Although reduced mTOR signaling has a general antiaging effect, it can negatively affect the aging brain by reducing synaptogenesis and thus cognitive functions. Increased mTOR activity facilitates aging and is responsible for the amnestic effect of the cannabinoid receptor 1 agonist Δ9-tetrahydrocannabinol (THC) in higher doses. Long-term low-dose Δ9-THC had an antiaging effect on the brain by restoring cognitive abilities and synapse densities in old mice. Whether changes in mTOR signaling and metabolome are associated with its positive effects on the aging brain is an open question. Here, we show that Δ9-THC treatment has a tissue-dependent and dual effect on mTOR signaling and the metabolome. In the brain, Δ9-THC treatment induced a transient increase in mTOR activity and in the levels of amino acids and metabolites involved in energy production, followed by an increased synthesis of synaptic proteins. Unexpectedly, we found a similar reduction in the mTOR activity in adipose tissue and in the level of amino acids and carbohydrate metabolites in blood plasma as in animals on a low-calorie diet. Thus, long-term Δ9-THC treatment first increases the level of energy and synaptic protein production in the brain, followed by a reduction in mTOR activity and metabolic processes in the periphery. Our study suggests that a dual effect on mTOR activity and the metabolome could be the basis for an effective antiaging and pro-cognitive medication.

Fatty acid esters as novel metabolites of γ-hydroxybutyric acid: A preliminary investigation.

γ-Hydroxybutyric acid (GHB) is a substance frequently abused as a knockout agent. Because of possible amnesia experienced by victims of GHB exposure and the short detection time of GHB in biological samples, the proof of GHB uptake is often challenging for forensic toxicologists. For this reason, various approaches have been evaluated to prolong the detection of GHB intake. In the present study, a fatty acid ester of GHB (4-palmitoyloxy butyrate [GHB-Pal; 3-carboxypropyl hexadecanoate]) was synthesized with the intent of examining whether such esters could be detected as metabolites of GHB in blood samples. Using the structurally elucidated synthesis product (structural elucidation by means of high performance liquid chromatography quadrupole time of flight mass spectrometry [LC-QToF-MS]), an LC triple quadrupole mass spectrometric (LC-MS/MS) method was established for the detection of GHB-Pal. Blood (plasma) samples from four cases in which GHB was previously detected at relevant concentrations (56.1-96.5 µg/ml) were analyzed with respect to GHB-Pal. Signals for GHB-Pal, as well as possible signals for other fatty acid esters of GHB, were detectable in these specimens. (Negative) control samples (20 plasma samples and 20 red blood cell/blood clot samples; from cases in which an intake of GHB or its precursors was not assumed) were all negative for GHB-Pal. To evaluate a possible forensic benefit of GHB fatty acid esters (prolongation of the detection window of a GHB uptake), the analysis of additional plasma samples collected after GHB uptake (or controlled GHB administration) and quantification of GHB fatty acid esters are needed.

Follow up: palmitic acid ester of tetrahydrocannabinol (THC) and palmitic acid diester of 11-hydroxy-THC - unsuccessful search for additional THC metabolites.

OBJECTIVES: In a previous investigation we searched for the occurrence of palmitic acid ester compounds of delta9-tetrahydrocannabinol (THC) and its primary metabolite 11-hydroxy-delta9-THC (11-OH-THC) in human body fluids and tissues (THC palmitic acid monoester [THC-Pal] and 11-OH-THC palmitic acid diester [11-OH-THC-DiPal]). As those esters could not be detected in various human body fluids (e.g. blood) or tissues (e.g. adipose tissue) we extended the investigation analyzing adipose tissue samples of mice previously given synthetic THC or a cannabis extract. METHODS: In total, 48 adipose tissue samples previously tested positive for THC by means of a liquid chromatographic triple quadrupole mass spectrometric (LC-QQQ-MS) method were analyzed for the presence of THC-Pal and 11-OH-THC-DiPal by means of LC-QQQ-MS. RESULTS: THC-Pal and 11-OH-THC-DiPal were not detected among the adipose tissue samples analyzed despite the presence of high THC concentrations within the adipose tissue. THC concentrations in adipose tissue were in a range of approximately 7-2,595 ng/g (median: 468 ng/g, average: 704 ng/g). CONCLUSIONS: A (site-specific) synthesis of 11-OH-THC palmitic acid monoesters (11-hydroxy-delta9-THC-1-palmitate and 11-palmitoyloxy-delta9-THC) still remains to be done. After synthesis of these monoesters, their presence in the body fluids and tissues after THC administration should be investigated.

Follow up: palmitic acid ester of tetrahydrocannabinol (THC) and palmitic acid diester of 11-hydroxy-THC - unsuccessful search for additional THC metabolites.

OBJECTIVES: In a previous investigation we searched for the occurrence of palmitic acid ester compounds of delta9-tetrahydrocannabinol (THC) and its primary metabolite 11-hydroxy-delta9-THC (11-OH-THC) in human body fluids and tissues (THC palmitic acid monoester [THC-Pal] and 11-OH-THC palmitic acid diester [11-OH-THC-DiPal]). As those esters could not be detected in various human body fluids (e.g. blood) or tissues (e.g. adipose tissue) we extended the investigation analyzing adipose tissue samples of mice previously given synthetic THC or a cannabis extract. METHODS: In total, 48 adipose tissue samples previously tested positive for THC by means of a liquid chromatographic triple quadrupole mass spectrometric (LC-QQQ-MS) method were analyzed for the presence of THC-Pal and 11-OH-THC-DiPal by means of LC-QQQ-MS. RESULTS: THC-Pal and 11-OH-THC-DiPal were not detected among the adipose tissue samples analyzed despite the presence of high THC concentrations within the adipose tissue. THC concentrations in adipose tissue were in a range of approximately 7-2,595 ng/g (median: 468 ng/g, average: 704 ng/g). CONCLUSIONS: A (site-specific) synthesis of 11-OH-THC palmitic acid monoesters (11-hydroxy-delta9-THC-1-palmitate and 11-palmitoyloxy-delta9-THC) still remains to be done. After synthesis of these monoesters, their presence in the body fluids and tissues after THC administration should be investigated.

Enantioselective Quantification of Amphetamine and Metabolites in Serum Samples: Forensic Evaluation and Estimation of Consumption Time.

In forensic toxicology, amphetamine intoxications represent one of the most common case groups and present difficult questions for toxicologists. Estimating the time of consumption and the current influence of the stimulant is particularly difficult when only total amphetamine concentrations are considered. Stereoselective analysis and the consideration of metabolites can provide valuable information to facilitate interpretation. An enantioselective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of amphetamine, norephedrine and 4-hydroxyamphetamine was developed. Validation showed satisfactory selectivity, sensitivity, linearity (0.5-250 ng/mL), precision and accuracy for all enantiomers. The method was applied to a collective of 425 forensic serum samples and 30 serum samples from psychiatric inpatients stating their last time of amphetamine consumption. Norephedrine and 4-hydroxyamphetamine were detected more frequently at higher amphetamine concentrations and at lower amphetamine (R)/(S) concentration ratios, possibly indicating recent consumption. Mean (R)/(S) ratio of amphetamine was 1.14, whereas higher ratios (mean 1.36) were found for amphetamine concentrations below 100 ng/mL. The (R)/(S) ratios of psychiatric inpatients significantly correlated with the reported time intervals to last consumption. The use of amphetamine (R)/(S) ratios and the simultaneous detection of metabolites are promising factors that can facilitate estimation of consumption time and current impairment.

Detectability of various cannabinoids in plasma samples of cannabis users: Indicators of recent cannabis use?

To assess the informative value of determining (minor) cannabinoids in plasma of cannabis users, detection rates of 14 cannabinoids (next to Δ9 -THC and THC-COOH) were determined. 11-OH-THC, THCA, CBC, CBN, and CBD were the most frequent detectable cannabinoids. The dependency of cannabinoid detectability on the plasma Δ9 -THC concentration was examined.

Decarbonylation: A metabolic pathway of cannabidiol in humans.

Cannabidiol (CBD) is a non-psychoactive cannabinoid, which is of growing medical interest. Previous studies on the metabolism of CBD showed mainly the formation of hydroxylated or oxidized derivatives, the formation of carboxylic acids or modifications of the aliphatic side chain. Using incubation of CBD with hepatic microsomes of mice, the formation of carbon monoxide was reported. We investigated the phase I metabolism of CBD and cannabidivarin (CBDV) using in vitro experiments with human liver microsomes in order to discover so far not considered metabolites. Identification of metabolites was done by liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC-QToF-MS). Within these experiments, we came across decarbonylation of CBD and CBDV. Further investigations were focused on observed decarbonylated CBD (DCBD). To confirm this metabolite in humans in vivo, plasma samples containing large amounts of cannabinoids as well as serum and urine samples, collected after a voluntary intake of a CBD-containing food supplement, were analyzed by LC coupled to triple quadrupole mass spectrometry (LC-QQQ-MS). DCBD was detected in in vitro incubation mixtures, serum samples, and urine samples (after alkaline or enzymatic hydrolysis) collected after the voluntary intake, as well as in plasma samples of cannabis users. DCBD appears to be an important supplementary human metabolite that might be helpful for the analytical confirmation of a CBD uptake and might improve the interpretation of the consumption of CBD-containing products. Results of this study indicate a prolonged detectability of DCBD (in serum) in comparison to CBD after oral CBD ingestion.

Death cases involving certain new psychoactive substances: A review of the literature.

In the last decades, more and more new psychoactive substances (NPS) were introduced on the drug market which were sold as "legal" alternatives for classic drugs and misused medications. Due to an increased number of available substances and a growing utilization by users of common drugs but also by inexperienced users because of the supposed "legal" status, also undesired adverse effects of these NPS, at worst leading to death, became apparent. This review summarizes fatalities previously described in scientific literature which were attributed to the use of NPS or such cases, in which intake of NPS was proven or even assumed to contribute to death. This summary includes an overview of substances involved (particularly synthetic cannabinoids ("spice"), novel opioids and synthetic cathinones ("bath salts")) as well as of postmortem concentrations determined in various biological matrices. The compiled data assist forensic toxicologists with the interpretation of death cases involving NPS.

Palmitic acid ester of tetrahydrocannabinol (THC) and palmitic acid diester of 11-hydroxy-THC - Unsuccessful search for additional THC metabolites in human body fluids and tissues.

Fatty acid conjugates of hydroxy-metabolites of tetrahydrocannabinol (THC) or cannabinol have already been reported as metabolites in rats. In the herein presented investigation, palmitic acid esters of THC and its primary metabolite 11-hydroxy-delta9-tetrahydrocannabinol (11-OH-THC) were synthesized using esterification with palmitic acid chloride. Structural elucidation of the products was conducted using nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography coupled to quadrupole time of flight mass spectrometry (LC-QToF-MS). For the confirmation of a previous cannabis use, body fluids (femoral blood, heart blood, urine, bile) of 27 death cases (all with known cannabis use), including adipose tissue homogenates of six of these cases as well as eleven plasma samples (probably all with regular cannabis use, confirmed by a high 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THC-COOH) concentration (except one sample, >200ng/mL), were tested for THC and its main metabolites 11-OH-THC and THC-COOH using gas chromatography coupled to mass spectrometry (GC-MS). These samples as well as further tissue homogenates of autopsy cases (liver, kidney, brain) were additionally tested for the presence of THC palmitic acid ester or 11-OH-THC palmitic acid diester by means of a liquid chromatographic triple quadrupole mass spectrometric (LC-QQQ-MS) method, in order to evaluate a possible presence of these conjugates in humans. In none of the analyzed samples (in total 196 specimens; plasma (N=11), femoral blood (N=23), heart blood (N=25), urine (N=23), bile (N=27), liver (N=27), kidney (N=27), brain (N=27), adipose tissue (N=6)), palmitic acid esters of THC or 11-OH-THC could be proven. Even if the existence of these esters in human samples cannot be ruled out definitely, suitability as cannabis consumption markers does not seem likely based on our findings.

Mono-/polyintoxication with 5F-ADB: A case series.

5F-ADB is an indazole-based synthetic cannabinoid. In recent years, it has been detected in legal high products as well as in biological samples and is associated with serious adverse health, behavioral effects and even death. Due to the fast pace of the market of synthetic cannabinoids, data on such newly appearing substances are scarce. As pharmacological properties are often investigated in vitro or by using animal experiments, reports on synthetic cannabinoid findings in human samples along with corresponding case history descriptions are valuable for the interpretation of upcoming routine cases. Herein we report five cases with verified 5F-ADB consumption, including three fatalities, a case of driving under the influence of drugs as well as a case of grievous bodily harm. In four cases, 5F-ADB could be detected in blood or plasma. Concentrations were in the range of 0.11-0.57 µg/L. In one instance 5F-ADB consumption was verified by the detection of 5F-ADB metabolites in postmortem body fluids. The described cases illustrate various adverse effects including confusion (possibly even psychosis), collapse, loss of consciousness, unsafe driving style or changing moods that might be attributed to 5F-ADB.

Considerations regarding the validation of chromatographic mass spectrometric methods for the quantification of endogenous substances in forensics.

The requirement for correct evaluation of forensic toxicological results in daily routine work and scientific studies is reliable analytical data based on validated methods. Validation of a method gives the analyst tools to estimate the efficacy and reliability of the analytical method. Without validation, data might be contested in court and lead to unjustified legal consequences for a defendant. Therefore, new analytical methods to be used in forensic toxicology require careful method development and validation of the final method. Until now, there are no publications on the validation of chromatographic mass spectrometric methods for the detection of endogenous substances although endogenous analytes can be important in Forensic Toxicology (alcohol consumption marker, congener alcohols, gamma hydroxy butyric acid, human insulin and C-peptide, creatinine, postmortal clinical parameters). For these analytes, conventional validation instructions cannot be followed completely. In this paper, important practical considerations in analytical method validation for endogenous substances will be discussed which may be used as guidance for scientists wishing to develop and validate analytical methods for analytes produced naturally in the human body. Especially the validation parameters calibration model, analytical limits, accuracy (bias and precision) and matrix effects and recovery have to be approached differently. Highest attention should be paid to selectivity experiments.