Everolimus depletes plaque macrophages, abolishes intraplaque neovascularization and improves survival in mice with advanced atherosclerosis.

BACKGROUND AND AIMS: Inhibition of the mechanistic target of rapamycin (mTOR) is a promising approach to halt atherogenesis in different animal models. This study evaluated whether the mTOR inhibitor everolimus can stabilize pre-existing plaques, prevent cardiovascular complications and improve survival in a mouse model of advanced atherosclerosis. METHODS: ApoE-/-Fbn1C1039G+/- mice (n = 24) were fed a Western diet (WD) for 12 weeks. Subsequently, mice were treated with everolimus (1.5 mg/kg daily) or vehicle for another 12 weeks while the WD continued. RESULTS: Despite hypercholesterolemia, everolimus treatment was associated with a reduction in circulating Ly6Chigh monocytes (15 vs. 28% of total leukocytes, p = 0.046), a depletion of plaque macrophages (2.1 vs. 4.1%, p = 0.040) and an abolishment of intraplaque neovascularization, which are all indicative of a more stable plaque phenotype. Moreover, everolimus reduced hypoxic brain damage and improved cardiac function, which led to increased survival (100 vs. 67% of animals, p = 0.038). CONCLUSIONS: Everolimus enhances features of plaque stability and counters cardiovascular complications in ApoE-/-Fbn1C1039G+/- mice, even when administered at a later stage of the disease.

Keratinous matrices for the assessment of drugs of abuse consumption: A correlation study between hair and nails.

Keratinous matrices - hair and nails - accumulate substances over time and allow retrospective investigation of past consumption. Analysis of these matrices can provide information complementary to blood and urine analysis or can be used as standalone. So far, research has primarily focused on the detection of substances in hair, while studies in nails are scarce. In this study, we assessed concentrations of drugs of abuse and their metabolites in hair, fingernails, and toenails collected from the same individuals to evaluate differences and correlations between matrices. A total of 26 hair, 24 fingernail, and 18 toenail samples were collected. Samples were analysed by a validated liquid chromatography-tandem mass spectrometry method able to simultaneously detect the following compounds: amphetamine (AMP), methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine, morphine (MOR), codeine (COD), 6-monoacetylmorphine (6-MAM), methadone (MTD), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine (COC), benzoylecgonine (BE), and ecgonine methyl ester (EME). Strong positive correlations between hair, fingernails, and toenails were present for COC, BE, EME, AMP and MDMA. MOR, COD, 6-MAM, MTD and EDDP showed positive trends. Concentrations were generally higher in nails compared to hair. Ratios between parent compounds and their metabolites were assessed for 6-MAM/MOR, EDDP/MTD, BE/COC and EME/COC. Preliminary cut-off concentrations for COC, BE, EME and AMP in fingernails and toenails were proposed. In light of these results, nails can be considered as a useful alternative to hair for monitoring of long-term drug consumption. However, care should be taken regarding the variability in the accumulation of compounds between the matrices.

A straightforward, validated liquid chromatography coupled to tandem mass spectrometry method for the simultaneous detection of nine drugs of abuse and their metabolites in hair and nails.

Hair and nails allow for a stable accumulation of compounds over time and retrospective investigation of past exposure and/or consumption. Owing to their long window of detection (weeks to months), analysis of these matrices can provide information complementary to blood and urine analysis or can be used in standalone when e.g. elimination from the body has already occurred. Drugs of abuse are often used together and, therefore, multi-analyte methods capable of detecting several substances and their metabolites in a single run are of importance. This paper presents the development and validation of a method based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for the simultaneous detection of nine drugs of abuse and their metabolites in hair and nails. We focused on a simple and straightforward sample preparation to reduce costs, and allow application in routine laboratory practice. Chromatographic and mass spectrometric parameters, such as column type, mobile phase, and multiple reaction monitoring transitions were optimized. The method was validated according to the European Medicine Agency guidelines with an assessment of specificity, limit of quantification (LOQ), linearity, accuracy, precision, carry-over, matrix effects, recovery, and process efficiency. Linearity ranged from 25 to 20 000 pg mg-1 hair and from 50 to 20 000 pg mg-1 nails, and the lowest calibration point achieved the requirements for the LOQ (25 pg mg-1 for hair and 50 pg mg-1 for nails). Although it was not the main focus of the article, the reliability of the method was proven through successful participation in a proficiency test, and by investigation of authentic hair and nail samples from self-reported drug users. In the future, the method should allow comparison between the two matrices to acquire an in-depth knowledge of nail analysis and to define cutoff levels for nail analysis, as they exist for hair.

Influence of Body Mass Index on Hair Ethyl Glucuronide Concentrations.

AIM: Analysis of ethyl glucuronide (EtG) concentrations in hair is increasingly used to estimate the consumption of alcohol of the prior months. Linear correlations between the amount of alcohol consumed and the concentration of EtG in hair have been reported, and several variables that may influence this correlation have been investigated: e.g. cosmetic hair treatments, gender influences or hair color. Here, we investigate the influence of body mass index (BMI) on this correlation. METHODS: A post hoc analysis on the influence of BMI on the relation between amounts of alcohol consumed and the measured EtG concentrations in hair in 199 participants. RESULTS: Our data show higher EtG concentrations in participants with high BMI (≥25) compared to participants with low BMI (<25) (P = 0.001) across a wide range of amounts of alcohol consumed. CONCLUSIONS: We conclude that BMI should be taken into account when interpreting hair EtG concentrations. SHORT SUMMARY: Ethyl glucuronide concentrations in hair (hEtG) can be used to estimate the consumption of alcohol of the prior months. Body mass index (BMI) influences this relation and BMI should be taken into account when interpreting hEtG concentrations in participants with high BMI (≥25) compared to participants with low BMI (<25).

Hair ethyl glucuronide concentrations in teetotalers: Should we re-evaluate the lower cut-off?

AIMS: Ethyl glucuronide in hair (hEtG) can be used to assess the retrospective consumption of alcohol. A lower cut-off of 7pg/mg hair in the 0-3cm proximal scalp hair segment has been used for repeated alcohol consumption in the previous three months. While a concentration below this cut-off is stated not to contradict self reported abstinence, it is often used to assess whether an individual has remained abstinent in the period prior to hair sampling. Here, we address hEtG concentrations in alcohol consuming individuals and critically evaluate this cut-off value. METHODS: Ten individuals remained abstinent from alcohol for 12 weeks. A lock of hair was cut before the start of the study, and the regrown hairs were cut after twelve weeks of abstinence. Hair EtG concentrations were measured both at baseline and after 12 weeks of abstinence. Study compliance was assessed by urine analysis every 2-3 days by liquid chromatography-tandem mass spectrometry with a lower limit of quantification (LLOQ) of 0.1µg/mL. HEtG concentrations were assessed in the first 3cm hair using gas chromatography-tandem mass spectrometry with an LLOQ of 0.2pg/mg. RESULTS: At the beginning of the study, participants had hEtG concentrations ranging between

Continuous administration of the mTORC1 inhibitor everolimus induces tolerance and decreases autophagy in mice.

BACKGROUND AND PURPOSE: Everolimus is an allosteric inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1) widely known for its potent autophagy stimulating properties. Because everolimus shows poor solubility and stability in aqueous solutions, long-term in vivo administration in preclinical models is challenging. The aim of the present study was to evaluate the effects of short-term and long-term everolimus administration on mTORC1 inhibition and autophagy induction in mice. EXPERIMENTAL APPROACH: We developed a vehicle in which everolimus was solubilized and stable at 37°C for at least 1 month. Using osmotic minipumps, GFP microtubule-associated protein light chain 3 transgenic mice were treated continuously either with vehicle or everolimus (1.5 mg·kg-1 per day) for 3 or 28 days. Alternatively, a regimen consisting of intermittent everolimus administration (every other day) for 56 days by oral gavage was used. Autophagy markers and mTORC1 activation status were investigated in the liver. KEY RESULTS: As expected, everolimus inhibited mTORC1 and stimulated autophagy in the liver after 3 days of treatment. However, continuous administration for 28 days resulted in hyperactivation of the Akt1-mTORC1 pathway accompanied by a remarkable decrease in autophagy markers. Everolimus given intermittently for 56 days partially rescued mTORC1 sensitivity to the drug but without inducing autophagy. The failure to induce autophagy following long-term everolimus administration was due to uncoupling of the mTORC1 substrate unc-51 like autophagy activating kinase 1. CONCLUSIONS AND IMPLICATIONS: Our data encourage the use of intermittent everolimus regimens to prevent tolerance and to extend its activity.

Hair ethyl glucuronide and serum carbohydrate deficient transferrin for the assessment of relapse in alcohol-dependent patients.

OBJECTIVES: Ethyl glucuronide in hair (hEtG) and serum carbohydrate deficient transferrin (%CDT) are valuable markers for alcohol abuse, but their diagnostic accuracy to monitor abstinence and relapse is unclear. Here, we investigate to what extent repeated measurements of hEtG and %CDT can be used to monitor relapse in alcohol-dependent patients during abstinence treatment. DESIGN AND METHODS: HEtG and %CDT were measured in individuals starting treatment for alcohol dependence both at treatment entry and 3months later. Alcohol consumption and relapse episodes were recorded using the Time Line Follow Back and by alcohol breath and urine tests, and correlated with hEtG and %CDT measurements. RESULTS: Fifteen patients completed the study, of which nine had one or more relapses. Hair EtG and serum %CDT identified whether a relapse occurred in 78% and 57% of cases, respectively. Only hEtG correlated with the amount of alcohol consumed before treatment entry (Pearson r=0.92; p<0.001). The specificity of %CDT to assess abstinence during treatment was 100%. HEtG had a specificity of only 17%; however, in all patients who remained abstinent, hEtG decreased with >85% from initial values. Mean hEtG, but not %CDT, differed significantly between patients who relapsed and patients who remained abstinent (p=0.034). CONCLUSIONS: HEtG was more sensitive than serum %CDT to assess relapse in alcohol-dependent patients and was positively correlated with the amounts of alcohol consumed. In contrast, serum %CDT was more specific for assessing abstinence. We highlight the benefit of repeated measurements of hEtG and serum %CDT for monitoring abstinence during treatment.

Ethyl glucuronide concentrations in hair: a controlled alcohol-dosing study in healthy volunteers.

Ethyl glucuronide (EtG) is a minor phase II metabolite of alcohol that accumulates in hair. It has been established as a sensitive marker to assess the retrospective consumption of alcohol over recent months using a cut-off of ≥7 pg/mg hair to assess repeated alcohol consumption. The primary aim was to assess whether amounts of alcohol consumed correlated with EtG concentrations in hair. Additionally, we investigated whether the current applied cut-off value of 7 pg/mg hair was adequate to assess the regular consumption of low-to-moderate amounts of alcohol. A prospective controlled alcohol-dosing study in 30 healthy individuals matched on age and gender. Individuals were instructed to drink no alcohol (N = 10), 100 g alcohol per week (N = 10) or 150 g alcohol per week (N = 10) for 12 consecutive weeks, before and after which hair was collected. Throughout the study, compliance to daily alcohol consumption was assessed by analyzing urine EtG three times weekly. Participants in the non-drinking group had median EtG concentrations of 0.5 pg/mg hair (interquartile range (IQR) 1.7 pg/mg; range < 0.21-4.5 pg/mg). Participants consuming 100 and 150 g alcohol per week showed median EtG concentrations of 5.6 pg/mg hair (IQR 4.7 pg/mg; range 2.0-9.8 pg/mg) and 11.3 pg/mg hair (IQR 5.0 pg/mg; range 7.7-38.9 pg/mg), respectively. Hair EtG concentrations between the three study groups differed significantly from one another (p < 0.001). Hair EtG concentrations can be used to differentiate between repeated (low-to-moderate) amounts of alcohol consumed over a long time period. For the assessment of repeated alcohol use, we propose that the current cut-off of 7 pg/mg could be re-evaluated.

Influence of repeated permanent coloring and bleaching on ethyl glucuronide concentrations in hair from alcohol-dependent patients.

BACKGROUND: Ethyl glucuronide (EtG), a minor metabolite of alcohol, is used as a sensitive marker in hair to detect the retrospective consumption of alcohol. The proximal 0-3 cm hair segment is often used for analysis, providing information on alcohol consumption over the past 3 months. Using more distal segments would allow the detection of alcohol consumption over longer time periods, thereby addressing the chronicity of the consumption. In view of this, permanent coloring and bleaching were shown in vitro to alter EtG concentrations in hair, but no in vivo studies are available to prove or disprove this. AIMS: To investigate the influence of repeated bleaching and permanent coloring on EtG concentrations in vivo and to assess the stability of EtG concentrations in distal compared to proximal hair segments. METHODS: Hair samples from alcohol-dependent patients with uncolored/unbleached (N=4), permanent coloration (N=5) and bleached hair (N=5) were analyzed in two to six 3 cm long segments for EtG concentrations, and alcohol consumption and hair cosmetic treatments were assessed. RESULTS: We observed that hair bleaching and permanent coloring reduces EtG concentrations by 82±11% and 65±24%, respectively, with correlations between the number of cosmetic treatments and the decrease in EtG concentrations. EtG remained stable in untreated hair samples up to 18 cm. CONCLUSIONS: EtG is a sensitive marker to assess chronic alcohol consumption up to 18 months in alcohol-dependent patients with no cosmetic hair treatments. However, in alcohol-dependent patients who color or bleach their hair, care should be taken when interpreting EtG measurements.

Retrospective evaluation of therapeutic drug monitoring of clozapine and norclozapine in Belgium using a multidrug UHPLC-MS/MS method.

OBJECTIVE: Clozapine is an atypical antipsychotic with a narrow therapeutic range and serious toxic side effects. According to AGNP-TDM consensus guidelines, therapeutic drug monitoring (TDM) of clozapine and its metabolite norclozapine is strongly recommended. 330 serum samples, sent to the toxicological laboratory of Ziekenhuis Netwerk Antwerpen for monitoring of clozapine, were tested with a new ultra-high performance liquid chromatography-tandem mass spectrometric method (UHPLC-MS/MS). The aim of this research was to evaluate this method for TDM of clozapine and norclozapine, but also to determine other antipsychotics present in these serum samples. DESIGN AND METHODS: Serum samples were taken just prior to the morning dose of the antipsychotic (trough concentration). All samples were, after a simple liquid-liquid extraction with methyl t-butylether, analyzed using a fully validated UHPLC-MS/MS method which is able to quantitate 16 different antipsychotics and 8 of their major metabolites. Serum concentrations were compared with the therapeutic ranges as defined by the AGNP-TDM guidelines. RESULTS: For clozapine, only 22.3% of the serum concentrations were within the therapeutic range of 350-600 ng/mL, while 67.9% of the concentrations were below 350 ng/mL. For norclozapine, 68.2% of the serum samples were within the therapeutic range of 100-600 ng/mL. The mean clozapine:norclozapine ratio was 1.7 (SD 0.8). 218 of the 330 serum samples contained other antipsychotics than clozapine. Only 52.5% of these concentrations were within the proposed range. CONCLUSION: This retrospective study highlights the importance of TDM for clozapine and other APs, since many patients show suboptimal serum concentrations.

Hair ethyl glucuronide levels as a marker for alcohol use and abuse: a review of the current state of the art.

BACKGROUND: Ethyl glucuronide (EtG) is a minor alcohol metabolite that has been proposed as a stable marker in hair to detect and quantify alcohol consumption over long time periods. METHODS: We provide an outline of currently available techniques for EtG hair sample analysis and highlight the pitfalls related to data interpretation. The literature of EtG analysis has been reviewed from January 1980 up to August 2013. In addition, we present an overview of the clinical and forensic studies which have used EtG quantification in hair as a marker for alcohol consumption/abstinence and we provide suggestions for future research. RESULTS: EtG is a stable marker in hair that can be used to detect and quantify alcohol consumption over long time periods. This alcohol metabolite remains in hair after complete elimination of alcohol. Currently, there are three main analytical techniques used to quantify EtG in hair: gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS/MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). No standardized protocols are yet available for the analysis of EtG levels in hair samples, and the current protocols vary in sample preparation and extraction procedures. Variables such as hair length, cosmetic treatment, gender, and pathophysiological conditions influence the final results and should be taken into account. CONCLUSIONS: EtG quantification in hair is a useful tool for the objective detection of alcohol consumption over extended time periods, but care should be taken when interpreting the results.

High throughput identification and quantification of 16 antipsychotics and 8 major metabolites in serum using ultra-high performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Therapeutic drug monitoring of antipsychotics is important for optimizing therapy, explaining adverse effects, non-response or poor compliance. We developed a UHPLC-MS/MS method for quantification of 16 commonly used and recently marketed antipsychotics and 8 metabolites in serum. METHODS: After liquid-liquid extraction using methyl tert-butyl ether, analysis was performed on an Agilent Technologies 1290 Infinity LC system coupled with an Agilent Technologies 6460 Triple Quadrupole MS. Separation with a C18 column and gradient elution at 0.5 mL/min resulted in a 6-min run-time. Detection was performed in dynamic MRM, monitoring 3 ion transitions per compound. Isotope labeled internal standards were used for every compound, except for bromperidol and levosulpiride. RESULTS: Mean recovery was 86.8%. Matrix effects were -18.4 to +9.1%. Accuracy ranged between 91.3 and 107.0% at low, medium and high concentrations and between 76.2 and 113.9% at LLOQ. Within-run precision was <15% (CV), except for asenapine and hydroxy-iloperidone. Between-run precision was aberrant only for 7-hydroxy-N-desalkylquetiapine, asenapine and reduced haloperidol. No interferences were found. No problems of instability were observed, even for olanzapine. The method was successfully applied on patient samples. CONCLUSIONS: The liquid-liquid extraction and UHPLC-MS/MS technique allows robust target screening and quantification of 23 antipsychotics and metabolites.

Ethyl glucuronide determination in meconium and hair by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Ethyl glucuronide (EtG) detection in non-conventional matrices, such as hair and meconium, can provide useful information on alcohol abuse over a long time frame, for example during pregnancy or after a withdrawal treatment. This study reports on the development, validation and application of a new hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for the analysis of EtG in meconium and hair. For each matrix, the sample preparation and the chromatographic separation were thoroughly optimised. Additionally, experiments with reversed-phase liquid chromatography were also performed in the development stages. Analyses were carried out using a Phenomenex Luna HILIC column (150 mm x 3 mm, 5 microm) and a mobile phase composed by ammonium acetate 2mM and acetonitrile, in gradient. Different SPE cartridges (Oasis MAX, Oasis WAX, aminopropyl silica) and solvents were tested in order to obtain the highest recoveries and cleanest extracts. Optimal results were obtained for meconium with aminopropyl cartridges, while for hair an incubation of 16 h with 2 mL of water and acetonitrile (50/50, v/v) provided good results. The analytical method was validated for both matrices (meconium and hair) by assessing linearity, precision, accuracy, recovery and limit of quantification. The calibration curve concentrations ranged from 50 to 1200 pg/mg for meconium and from 20 to 1000 pg/mg for hair. Real meconium and hair samples were analyzed and results were consistent with literature.

Analysis of gamma-hydroxybutyric acid, DL-lactic acid, glycolic acid, ethylene glycol and other glycols in body fluids by a direct injection gas chromatography-mass spectrometry assay for wide use.

Analysis of blood of severely intoxicated patients always requires prompt investigation. Diagnosis of intoxication with ethylene glycol, gamma-hydroxybutyric acid or D-lactic acid takes hours, since several different procedures are required. Rapid derivatization of the common hydroxyl function may resolve this analytical problem. Here we describe a fast method for the simultaneous measurement of ethylene glycol, glycolic acid, gamma-hydroxybutyric acid and racemic lactic acid. Only 20 microl of serum, plasma or urine are required for immediate derivatization at 70 degrees C with 750 microl of bis-N,O-trimethylsilyl trifluoroacetamide after adding 20 microl of internal standard solution (1,3-propylene glycol) and 20 microl of the catalyst dimethylformamide. After centrifugation an aliquot is transferred to a gas chromatographic system and analyzed with electron-impact mass spectrometry in selective ion monitoring mode. The derivatized acids and ethylene glycol are well separated and detected with a limit of detection ranging from 0.12 mg/l for ethylene glycol to 0.95 mg/l for gamma-hydroxybutyric acid, while the limit of quantification ranged from 0.4 mg/l for ethylene glycol to 3.15 mg/l for gamma-hydroxybutyric acid. The method is linear from 0.5 to 1800 mg/l blood for ethylene glycol, from 0.7 to 1200 mg/l for lactic acid, from 1.2 to 1800 mg/l for glycolic acid, and from 3.2 to 200 mg/l for gamma-hydroxybutyric acid, with analytical recoveries, accuracy, day-to-day and within-day precision well within the required limits. Total analysis time with one calibrator was 30 min, derivatization time included. This method is very suitable for emergency toxicology, since several toxic substances can be quantified simultaneously in a fast and sensitive manner.