Absolute Quantification of Human Serum Albumin Isoforms by Internal Calibration Based on a Top-Down LC-MS Approach.

Well-characterized biomarkers using reliable quantitative methods are essential for the management of various pathologies such as diabetes, kidney, and liver diseases. Human serum albumin (HSA) isoforms are gaining interest as biomarkers of advanced liver pathologies. In view of the structural alterations observed for HSA, insights into its isoforms are required to establish them as reliable biomarkers. Therefore, a robust absolute quantification method seems necessary. In this study, we developed and validated a far more advanced top-down liquid chromatography-mass spectrometry (LC-MS) method for the absolute quantification of HSA isoforms, using myoglobin (Mb) as an internal standard for quantification and for mass recalibration. Two different quantification approaches were investigated based on peak integration from the deconvoluted spectrum and extracted ion chromatogram (XIC). The protein mixture human serum albumin/myoglobin eluted in well-shaped separated peaks. Mb allowed a systematic mass recalibration for every sample, resulting in extremely low mass deviations compared to conventional deconvolution-based methods. In total, eight HSA isoforms of interest were quantified. Specific-isoform calibration curves showing good linearity were obtained by using the deconvoluted peaks. Noticeably, the HSA ionization behavior appeared to be isoform-dependent, suggesting that the use of an enriched isoform solution as a calibration standard for absolute quantification studies of HSA isoforms is necessary. Good repeatability, reproducibility, and accuracy were observed, with better sensitivity for samples with low albumin concentrations compared to routine biochemical assays. With a relatively simple workflow, the application of this method for absolute quantification shows great potential, especially for HSA isoform studies in a clinical context, where a high-throughput method and sensitivity are needed.

Probe Electrospray Ionization Tandem Mass Spectrometry for the Detection and Quantification of Benzodiazepines.

BACKGROUND: Legally prescribed benzodiazepines (BZDs) and designer BZDs are widely misused and must be determined in multiple contexts (eg, overdose, drug-facilitated sexual assaults, or driving under the influence of drugs). This study aimed to develop a method for measuring serum BZD levels using probe electrospray ionization (PESI) mass spectrometry and an isotope dilution approach. METHODS: A tandem mass spectrometer equipped with a probe electrospray ionization source in multiple reaction monitoring mode was used. Isotope dilution was applied for quantification using a deuterated internal standard at a fixed concentration for alprazolam, bromazepam, diazepam, nordiazepam, oxazepam, temazepam, zolpidem, and zopiclone. This method included designer BZDs: clonazolam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, meclonazepam, nifoxipam, and pyrazolam. Sample preparation was done by mixing 10 µL of serum with 500 µL of an ethanol/ammonium formate 0.01 mol/L buffer. Complete validation was performed, and the method was compared with liquid chromatography coupled with mass spectrometry (LC-MS/MS) and immunoassays (IC) by analyzing 40 real samples. RESULTS: The analysis time for identification and quantification of the 18 molecules was 2.5 minutes. This method was fully validated, and the limits of quantification varied from 5 to 50 mcg/L depending on the molecule. In the 40 real samples, 100% of molecules (n = 89) were detected by both LC-MS/MS and PESI-MS/MS, and regression analysis showed excellent agreement between the 2 methods (r 2 = 0.98). On IC, bromazepam and zolpidem were not detected in 2 and 1 cases, respectively. CONCLUSIONS: PESI-MS/MS allows serum BZD detection and measurement. Given the isotope dilution approach, a calibration curve was not required, and its performance was similar to that of LC-MS/MS, and its specificity was higher than that of IC.

Glyphosate presence in human sperm: First report and positive correlation with oxidative stress in an infertile French population.

Environmental exposure to endocrine disruptors, such as pesticides, could contribute to a decline of human fertility. Glyphosate (GLY) is the main component of Glyphosate Based Herbicides (GBHs), which are the most commonly herbicides used in the world. Various animal model studies demonstrated its reprotoxicity. In Europe, GLY authorization in agriculture has been extended until 2034. Meanwhile the toxicity of GLY in humans is still in debate. The aims of our study were firstly to analyse the concentration of GLY and its main metabolite, amino-methyl-phosphonic acid (AMPA) by LC/MS-MS in the seminal and blood plasma in an infertile French men population (n=128). We secondly determined Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) using commercial colorimetric kits and some oxidative stress biomarkers including malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) by ELISA assays. We next analysed potential correlations between GLY and oxidative stress biomarkers concentration and sperm parameters (sperm concentration, progressive speed, anormal forms). Here, we detected for the first time GLY in the human seminal plasma in significant proportions and we showed that its concentration was four times higher than those observed in blood plasma. At the opposite, AMPA was undetectable. We also observed a strong positive correlation between plasma blood GLY concentrations and plasma seminal GLY and 8-OHdG concentrations, the latter reflecting DNA impact. In addition, TOS, Oxidative Stress Index (OSI) (TOS/TAS), MDA blood and seminal plasma concentrations were significantly higher in men with glyphosate in blood and seminal plasma, respectively. Taken together, our results suggest a negative impact of GLY on the human reproductive health and possibly on his progeny. A precaution principle should be applied at the time of the actual discussion of GLY and GBHs formulants uses in Europe by the authorities.

Comparison of homocitrulline and carbamylated albumin as biomarkers of carbamylation reactions in hemodialyzed patients.

To describe the association between levels of homocitrulline (HCit) and the degree of albumin carbamylation in a cohort of hemodialyzed patients. Plasma total and protein-bound HCit concentrations in samples from hemodialyzed patients included in NICOREN trial were determined by LC-MS/MS at baseline and after 24 weeks of treatment with either sevelamer or nicotinamide. HCit concentrations at all timepoints and in both groups were positively and significantly correlated with the degree of albumin carbamylation. Plasma concentrations of total HCit, protein-bound HCit and carbamylated albumin did not decrease after 24 weeks of treatment with either sevelamer or nicotinamide. The present results demonstrate that plasma total and protein-bound HCit concentrations were closely associated with albumin carbamylation in hemodialyzed patients. Therefore, total and protein-bound HCit concentrations might be valuable biomarkers of the overall intensity of protein carbamylation in this context. Given the less complex and time-consuming analytical methods required, these markers should be favored in future clinical studies of carbamylation reaction.

Designer Benzodiazepines: Effects, Toxicity, and Interactions.

PURPOSE: Although designer benzodiazepines (DBZDs) constitute a minor part of new psychoactive substances, they deserve the greatest attention because of their popularity among drug users and increasing number and availability. This review covers the effects of different DBZDs, available pharmacological evaluation tools, and their reported toxicity and potential pharmacodynamic and pharmacokinetic interactions with other drugs commonly co-abused with DBZDs. METHODS: For this narrative review, a nonsystematic search was performed on PubMed, EMBASE, Google Scholar, and PubMed Central databases between June and July 2021. RESULTS: The current consensus hypothesis suggests that DBZDs mediate their effects through interactions with the GABA A receptor, producing similar effects to benzodiazepines used in therapy, including sedation, hypnosis, anxiolysis, muscle relaxation, euphoria, amnesia, and addiction. Owing to the complexity of their action mechanism and the numerous GABA A subtype receptors, the pharmacodynamic metrics of DBZDs are very difficult to establish. The pharmacological effects of DBZD are related to their structure, influencing their binding to GABA A receptor subunits. Quantitative structure-activity relationship studies successfully predicted the biological activity and relative potency of DBZD but could not predict the main pharmacological effect of a given DBZD. Exploring the effects by netnographic studies is one of the available alternatives, despite its limitations. DBZDs are usually identified in the context of polysubstance use. Pharmacodynamic interactions between DBZDS and other CNS depressants, such as opioids, have been extensively reported. However, pharmacokinetic interactions between DBZDs and opioids are considered less important, and contradictory conclusions about their clinical significance have been reported. CONCLUSIONS: Understanding the mechanism of action and other pharmacological metrics is highly important in the clinical management of DBZDs.

Impacts of dietary exposure to pesticides on faecal microbiome metabolism in adult twins.

BACKGROUND: Dietary habits have a profound influence on the metabolic activity of gut microorganisms and their influence on health. Concerns have been raised as to whether the consumption of foodstuffs contaminated with pesticides can contribute to the development of chronic disease by affecting the gut microbiome. We performed the first pesticide biomonitoring survey of the British population, and subsequently used the results to perform the first pesticide association study on gut microbiome composition and function from the TwinsUK registry. METHODS: Dietary exposure of 186 common insecticide, herbicide, or fungicide residues and the faecal microbiome in 65 twin pairs in the UK was investigated. We evaluated if dietary habits, geographic location, or the rural/urban environment, are associated with the excretion of pesticide residues. The composition and metabolic activity of faecal microbiota was evaluated using shotgun metagenomics and metabolomics respectively. We performed a targeted urine metabolomics analysis in order to evaluate whether pesticide urinary excretion was also associated with physiological changes. RESULTS: Pyrethroid and/or organophosphorus insecticide residues were found in all urine samples, while the herbicide glyphosate was found in 53% of individuals. Food frequency questionnaires showed that residues from organophosphates were higher with increased consumption of fruit and vegetables. A total of 34 associations between pesticide residue concentrations and faecal metabolite concentrations were detected. Glyphosate excretion was positively associated with an overall increased bacterial species richness, as well as to fatty acid metabolites and phosphate levels. The insecticide metabolite Br2CA, reflecting deltamethrin exposure, was positively associated with the phytoestrogens enterodiol and enterolactone, and negatively associated with some N-methyl amino acids. Urine metabolomics performed on a subset of samples did not reveal associations with the excretion of pesticide residues. CONCLUSIONS: The consumption of conventionally grown fruit and vegetables leads to higher ingestion of pesticides with unknown long-term health consequences. Our results highlight the need for future dietary intervention studies to understand effects of pesticide exposure on the gut microbiome and possible health consequences.

Self-poisoning with 60 tablets of Apixaban, a pharmacokinetics case report.

A 67-year-old man was admitted to the emergency department about 5 h after deliberate self-poisoning with 300 mg of Apixaban. The clinical examination did not show any organ dysfunctions or haemorrhagic signs, and the patient's life was not in danger. The first analysis, upon admission, showed a concentration of 2655 µg l-1 of Apixaban. The Cmax was observed 17 h after the intake (3654 µg l-1 ), about four times the classical Tmax value (median [range]: 4 h [2-4]). The Apixaban was then eliminated following a first order elimination with a calculated half-life of 10.8 h. The anti-Xa activity seems to be linearly related to concentration up to 4000 µg l-1 . This report suggests that the use of activated charcoal should be effective up to 17 h after a massive intake.

Fully automated sample preparation procedure to measure drugs of abuse in plasma by liquid chromatography tandem mass spectrometry.

For the analysis of drugs and pharmaceutical compounds in biological matrices, extraction procedures are typically used for LC-MS/MS analysis often requiring manual steps in sample preparation. In this study, we report a fully automated extraction method carried out by a programable liquid handler directly coupled to an LC-MS/MS system for the determination of 42 components (illicit drugs and/or metabolites) (plus 20 deuterated internal standards). The acquisition was performed in positive ionization mode with up to 15 MRM transitions per compound, each with optimized collision energy (MRM spectrum mode) to enable qualitative library searching in addition to quantitation. After placing the sample tube into the system, no further intervention was necessary: automated preparation used 50 µL of blood or plasma with 3 µL of extracted sample injected for analysis. The method was validated according to the requirements of ISO 15189. The limit of detection and quantification was 1-5 ng/mL depending on the compound. Stability experiments found that historic calibration curve data files could accurately quantify for up to 1 month with less than 20% uncertainty. Comparison to a QuEChERS method was made using patient samples providing a regression correlation R2 = 0.98 between the two methods. This approach was successfully designed to support parallel sample preparation and analysis therefore significantly increasing sample throughput and reduced cycle times. Graphical abstract ᅟ.

Characterization and identification of eight designer benzodiazepine metabolites by incubation with human liver microsomes and analysis by a triple quadrupole mass spectrometer.

Designer benzodiazepines (DBZDs) have become of particular importance in the past few years. The metabolite monitoring of DBZD in biological fluids could be of great interest in clinical and forensic toxicology. However, DBZD metabolites are not known or not commercially available. The identification of some DBZD metabolites has been mostly explored by self-administration studies or by in vitro studies followed by high-resolution mass spectrometry. The question arose whether a unit resolution instrument could be efficient enough to allow the identification of DBZD metabolites. In this study, we used an in vitro experiment where eight DBZDs (diclazepam, flubromazepam, etizolam, deschloroetizolam, flubromazolam, nifoxipam, meclonazepam and clonazolam) were incubated with human liver microsomes (HLMs) and metabolite identification was carried out by using a UHPLC coupled to a QTRAP triple quadrupole linear iontrap tandem mass spectrometer system. Post-mortem samples obtained from a real poisoning case, involving deschloroetizolam and diclazepam, were also analysed and discussed. Our study using HLM allowed the identification of 26 metabolites of the 8 DBZDs. These were denitro-, mono- or di-hydroxylated and desmethyl metabolites. In the forensic case, diclazepam was not detected whereas its metabolites (lormetazepam and lorazepam) were present at high concentrations in urine. We also identified hydroxy-deschloroetizolam in urine, while the parent compound was not detected in this matrix. This supports the approach that LC coupled to a simple QTRAP could be used by laboratories to identify other not-known/not-commercialized new psychoactive substance (NPS) metabolites.

Is it worth carrying out determination of N-butane in postmortem samples? A case report and a comprehensive review of the literature.

The aim of this article is to illustrate the importance of N-butane determination in postmortem samples through a case report and to propose actions and precautions to be taken into consideration when butane is suspected to be involved in cases of death. The case concerns a 15-year-old boy found dead after sniffing a cigarette lighter refill. Toxicological investigation revealed the presence of butane in the heart and femoral blood (1280 and 1170 µg/L, respectively), in the gastric contents (326 µg/L), and in the liver (1010 µg/kg) and lung tissues (210 µg/kg). Propane was present only in the blood samples at concentrations tenfolds lower.Butane can be involved in three kinds of fatalities: deliberate inhalations including volatile substance abuse (VSA), involuntary exposure, and homicides. A fatal outcome of butane inhalation can be caused by asphyxia and/or cardiac arrhythmia. In the context where butane exposure is evidenced by non-toxicological investigations, the usefulness of the determination of butane in postmortem samples is often questionable. However, it is admitted that butane-related deaths are generally underreported. Several difficulties including sample handling and storage, substantial variation in tissue concentrations, and lack of a lethal threshold make the interpretation of butane results challenging. In our opinion, systematic toxicological methods should be developed in order to analyze butane, at least when it concerns a typical VSA victim, even when butane is not actually suspected to be the cause of death.

QuEChERS sample preparation prior to LC-MS/MS determination of opiates, amphetamines, and cocaine metabolites in whole blood.

Modern LC-MS/MS instruments have sensitivity and scanning velocity high enough to analyze many different compounds in single runs. Consequently, the sample preparation procedure has become the bottleneck for developing efficient, rapid, and cheap multi-compound methods. Here, we examined one-step sample preparation based on quick, easy, cheap, effective, rugged, and safe (QuEChERS) salts to set up and validate a LC-MS/MS method for the simultaneous determination of 35 drugs of abuse and their metabolites in whole blood. Despite large differences in physicochemical properties, this simplified QuEChERS extraction method yielded satisfactory recoveries (until 96%) for the 35 molecules. The amounts of QuEChERS salts had no influence on extraction yield. Chromatographic separation was obtained in less than 6 min. LLOD and LLOQ were 3 and 5 ng/mL, respectively. The procedure was successfully validated and then applied to 253 cases of driving under the influence of drugs (DUID), collected over a 6-month period.

Relative exchangeable copper: a promising tool for family screening in Wilson disease.

BACKGROUND: Family screening is a main step for the diagnosis in Wilson disease. This study was undertaken to evaluate the value of relative exchangeable copper for family screening. METHODS: Data from family screening were collected from the French National Center of Reference for Wilson disease. Subjects who were first- or second-degree relatives of the index case underwent clinical examination and biological parameters. RESULTS: Of 127 subjects examined, copper abnormalities or low ceruloplasminemia were detected in 21 subjects, corresponding to 5 patients with Wilson disease, 14 heterozygous ATP7B carriers and 2 subjects with no ATP7B mutations. Relative exchangeable copper determination significantly discriminates heterozygous ATP7B carriers and subjects with no ATP7B mutations from WD patients with a cutoff of 15%. CONCLUSIONS: Exchangeable copper appears to be a promising tool for family screening in Wilson disease.

Hexahydrocannabinol poisoning reported to French poison centres.

INTRODUCTION: Hexahydrocannabinol is a hexahydro derivative of cannabinol. Poisoning with hexahydrocannabinol was first observed in Europe in May 2022. METHOD: This is a retrospective observational study of cases of self-reported hexahydrocannabinol exposure reported to French poison centres between 1 January 2022 and 31 May 2023. RESULTS: There were 37 cases, including 19 in May 2023. The median age of the patients was 36 (interquartile range 28-43) years, and most were men. Eight patients had a history of substance use disorder. The route of exposure was ingestion in 24, inhalation (smoking or vaping) in 10, inhalation and ingestion in two and sublingual in one. Clinical features were neurological (85 per cent), cardiovascular (61 per cent), gastrointestinal (33 per cent), psychiatric (27 per cent) and ocular (21 per cent). Fifty-nine per cent of the patients were hospitalized. In four patients, the Poisoning Severity Score was 0 (asymptomatic); in 15 patients, the Score was 1 (minor); in 16, the Score was 2 (moderate); and in two cases, the Score was 3 (severe). In 70 per cent of patients, the outcome was known, and all recovered. Testing of biological samples was only undertaken in six cases. Five patients had positive blood or urine tests for hexahydrocannabinol; in two patients, tetrahydrocannabinol and metabolites were also detected. In addition, there was an additional patient in whom Δ8- and Δ9-tetrahydrocannabinol was detected in the substances used. DISCUSSION: Clinical effects reported in this series included neuropsychiatric and somatic effects. Whilst these cases related to self-reported hexahydrocannabinol use, it is likely that tetrahydrocannabinol use also contributed to the effects in a substantial proportion of cases. This study has some limitations, such as the lack of available information due to the retrospective nature of the study. As a result, it probably overestimates the number of moderate and severe cases due to under-reporting of cases of little or no severity. Analysis of the patient's blood and urine was performed only in six patients, so we cannot be certain that the products consumed by the other patients were hexahydrocannabinol. CONCLUSION: The clinical effects attributed to hexahydrocannabinol were neurological, cardiovascular, gastrointestinal, psychiatric and ocular predominantly and were sometimes serious.

Early detection of liver injuries by the Serum enhanced binding test sensitive to albumin post-transcriptional modifications.

Early and sensitive biomarkers of liver dysfunction and drug-induced liver injury (DILI) are still needed, both for patient care and drug development. We developed the Serum Enhanced Binding (SEB) test to reveal post-transcriptional modifications (PTMs) of human serum albumin resulting from hepatocyte dysfunctions and further evaluated its performance in an animal model. The SEB test consists in spiking serum ex-vivo with ligands having specific binding sites related to the most relevant albumin PTMs and measuring their unbound fraction. To explore the hypothesis that albumin PTMs occur early during liver injury and can also be detected by the SEB test, we induced hepatotoxicity in male albino Wistar rats by administering high daily doses of ethanol and CCl4 over several days. Blood was collected for characterization and quantification of albumin isoforms by high-resolution mass spectrometry, for classical biochemical analyses as well as to apply the SEB test. In the exposed rats, the appearance of albumin isoforms paralleled the positivity of the SEB test ligands and histological injuries. These were observed as early as D3 in the Ethanol and CCl4 groups, whereas the classical liver tests (ALT, AST, PAL) significantly increased only at D7. The behavior of several ligands was supported by structural and molecular simulation analysis. The SEB test and albumin isoforms revealed hepatocyte damage early, before the current biochemical biomarkers. The SEB test should be easier to implement in the clinics than albumin isoform profiling.

Chlordecone determination in serum by LC-MS/MS and the importance of low limit of detection.

Chlordecone is an organochlorine insecticide that has been used intensively from 1973 to 1993 in the French West Indies banana fields to control root borers. This use has resulted in persistent pollution of soils and waters, and people have been and are still exposed mainly through food. Epidemiological studies showed that this exposure is associated with health disorders, including prostate cancer, prematurity, cognitive or motor development. The measurement of chlordecone in serum is considered as the best surrogate, though no clear and definitive cut-off value has been established. This renders necessary the development of analytical methods with the lowest limit of detection as possible. While most published methods have utilized GC-MS or GC-MS/MS, in the present study we report an LC-MS/MS method based on a simple QuEChERS salts extraction. The whole procedure was validated according to ISO 15189 requirements and reached LOD and LOQ values of 0.007 and 0.02 µg/L, respectively. It was applied to more 10 000 serum samples of French Indies inhabitants. More than a half had a concentration below 0.1 µg/L and more than one third of them exhibiting a concentration below 0.05 µg/L. The capability of this LC-MS/MS method to detect very low concentrations highlights its utility in exploring the health impact of chlordecone.

Optimization of ICP-MS internal standardization for 26 elements by factorial design experiment.

INTRODUCTION: Internal standardization is a common tool used in inductively coupled plasma - mass spectrometry (ICP-MS) analysis in order to reduce matrix effects, and thus improve the reliability and the robustness of results. However, its efficacy relies on the choice of a proper internal standard (IS) which, ideally, undergoes the same signal variations as the analyte. Thus far, IS selection has mainly relied on the proximity of atomic mass between the analyte and the internal standard. However, while it may be a satisfactory rule of thumb, more recent works suggest that this criterium might not be suitable in several conditions, among which the presence of high amounts of carbon atoms in the sample. This may thus be of particular interest in the case of trace elements determination in biological samples. MATERIALS AND METHODS: In this study, we propose an empirical and global approach to IS selection in ICP-MS through the use of a factorial design of experiments (DoE), with a focus on biological matrices of interest in clinical analysis: human blood and urine. The suitability of 13 potential IS was evaluated for 26 clinically-relevant analytes, including a polyatomic ion obtained through reaction with oxygen, across 324 experimental conditions. RESULTS AND DISCUSSION: The results underline several exceptions to the rule of IS selection based on mass proximity, notably when considering heavy or polyatomic analytes. As a consequence, measurements of said analytes in several extreme experimental conditions using IS selected by mass proximity could yield vastly erroneous results (up to 30 times the theoretical concentrations). By contrast, the use of empirically selected IS yielded much more acceptable results.

Ultrafast Measurement of Metformin in the Clinical Setting Using Probe Electrospray Ionization Mass Spectrometry.

Metformin (MtF) is a treatment used for type 2 diabetes. Lactic acidosis (LA) is a frequent complication that can be either induced by or associated with elevated MtF plasma concentrations. When coupled with a mass spectrometry (MS) system, the probe electrospray ionization (PESI) method allows direct and rapid analysis of different types of matrices without pretreatment. In this study, we developed a PESI-MS method for the determination of MtF in plasma. We used a tandem mass spectrometer equipped with a PESI source in the reaction monitoring mode for the quantitation of MtF. MtF-d6 was chosen as the internal standard (IS), following an isotope dilution (ID) approach. The method was fully validated with six concentration levels (0.5-50 mg/L). The matrix effect was evaluated for each level, and the specificity was tested with a mix of potential co-medications. Using patient samples, the performance was compared with two classical LC-MS-MS and LC-diode array detector (DAD) methods used in external labs. Sample preparation consisted in mixing 10 µL plasma in 1,000 µL ethanol/ammonium formate buffer including MtF-d6 at a fixed concentration of 5 mg/L. The total run time was 0.31 min. ID gave satisfactory results of accuracy and precision (min-max: -12.1 to 15.8% and 1.0-17.1%, respectively). The matrix effect was fully corrected by the internal standard (bias < 1%). The specificity study also reported satisfactory results. Finally, in a representative group of 29 patients (55% with a concentration <5 mg/L, 38% with a concentration >5 mg/L and 7% not detected), we observed almost identical results when comparing LC-DAD and LC-MS-MS to PESI-MS (r2 > 0.99). We propose a specific, sensitive, accurate and ultrafast solution for the measurement of MtF in patient plasma, with no sample preparation or calibration curve building. This could be helpful in a core lab when rapid diagnosis of LA is needed.

Semi-synthetic human albumin isoforms: Production, structure, binding capacities and influence on a routine laboratory test.

Human Serum Albumin (HSA) undergoes Post-Translational-Modifications (PTMs) leading to isoforms affecting its oncotic and non-oncotic properties. HSA is comprised of several isoforms whose abundance may vary with pathologies such as diabetes, kidney and liver diseases. Studying their impact separately may help to understand their sources and potential pathogenicity and further their evaluation as biomarkers. The present study examined semi-synthetic HSA isoforms to investigate independently their structure by means of advanced mass spectrometry techniques (LC-TOF-MS and ICP-MS), influence on the HSA binding/antioxidant activities using a binding capacity test, and potential impact on albumin quantification by a routine immunoturbidimetric assay. Applying different chemical reactions to a commercial HSA solution, we obtained different solutions enriched up to 53 % of native HSA, 78 % of acetylated HSA, 71 % of cysteinylated HSA, 94 % of oxidized HSA, 58 % of nitrosylated HSA and 96 % of glycated HSA, respectively. Moreover, the semi-synthetic isoforms showed differently altered binding capacities for a panel of ligands (Cu, Cd, Au, Ds and L-T4). Furthermore, immunoturbidimetry was found to be insensitive to the presence and abundance of the different isoforms. The fully characterized semi synthetic HSA isoforms obtained should be useful to further investigate their pathogenicity and potential roles as biomarkers.

Metabolic interactions of benzodiazepines with oxycodone ex vivo and toxicity depending on usage patterns in an animal model.

BACKGROUND AND PURPOSE: Opioids and benzodiazepines are frequently combined in medical as well as in non-medical contexts. At high doses, such combinations often result in serious health complications attributed to pharmacodynamics interactions. Here, we investigate the contribution of the metabolic interactions between oxycodone, diazepam and diclazepam (a designer benzodiazepine) in abuse/overdose conditions through ex vivo, in vivo and in silico approaches. EXPERIMENTAL APPROACH: A preparation of pooled human liver microsomes was used to study oxycodone metabolism in the presence or absence of diazepam or diclazepam. In mice, diazepam or diclazepam was concomitantly administered with oxycodone to mimic acute intoxication. Diclazepam was introduced on Day 10 in mice continuously infused with oxycodone for 15 days to mimic chronic intoxication. In silico modelling was used to study the molecular interactions of the three drugs with CYP3A4 and 2D6. KEY RESULTS: In mice, in acute conditions, both diazepam and diclazepam inhibited the metabolism of oxycodone. In chronic conditions and at pharmacologically equivalent doses, diclazepam drastically enhanced the production of oxymorphone. In silico, the affinity of benzodiazepines was higher than oxycodone for CYP3A4, inhibiting oxycodone metabolism through CYP3A4. Oxycodone metabolism is likely to be diverted towards CYP2D6. CONCLUSION AND IMPLICATIONS: Acute doses of diazepam or diclazepam result in the accumulation of oxycodone, whereas chronic administration induces the accumulation of oxymorphone, the toxic metabolite. This suggests that overdoses of opioids in the presence of benzodiazepines are partly due to metabolic interactions, which in turn explain the patterns of toxicity dependent on usage. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Posttranslational-modifications of human-serum-albumin analysis by a top-down approach validated by a comprehensive bottom-up analysis.

The posttranslational modifications (PTM) of human serum albumin (HSA) can result in the development of isoforms that have been identified as potential biomarkers for advanced hepatic diseases. However, previous approaches using top-down (TD) analysis to identify isoforms based on molecular weight may have resulted in misidentifications. The nature of the identified isoforms has never been confirmed in previous works. Here, we aimed to critically evaluate TD for the characterization and determination of HSA isoforms in patients and make an inventory of HSA-PTM. Serum samples from control subjects and patients with liver dysfunctions were analyzed using both top-down (TD) and bottom-up (BU) approaches. TD analysis involved using a LC-TOF-MS system to obtain a multicharged spectrum of HSA, which was deconvoluted to identify isoforms. Spectra were then used for relative quantitation analysis of albumin isoform abundances based on trapezoidal integration. For BU analysis, serums were reduced +/- alkylated, digested with trypsin and analyzed in the Q-TOF, data-dependent acquisition (DDA) mode to generate a SWATH-MS high-resolution mass spectral library of all HSA peptides. Tryptic digests of another set of serum samples were then analyzed using data-independent acquisition (DIA) mode to confirm the presence of HSA isoforms and their modification sites. TD detected 15 isoforms corresponding to various modifications, including glycation, cysteinylation, nitrosylation, and oxidation (di- and tri-). In BU, the spectral library containing 127 peptides allowed for the characterization of the important isoforms with their modified sites, including some modifications that were only characterized in BU (carbamylation, deamidation, and amino-acid substitution). The method used for determining isoforms offered acceptable reproducibility (intra-/inter-assay CVs < 15%) for all isoforms present at relative abundances higher than 2%. Overall, the study found that several isoforms could be missed or misidentified by TD. However, all HSA isoforms identified by TD and reported to be relevant in liver dysfunctions were confirmed by BU. This critical evaluation of TD approach helped design an adequate and reliable method for the characterization of HSA isoforms in patients and offers the possibility to estimate isoform abundances within 3 min. These findings have significant implications for the diagnosis and treatment of liver dysfunctions.