Proarrhythmic drugs, drug levels, and polypharmacy in victims of sudden arrhythmic death syndrome: An autopsy-based study from Denmark.

BACKGROUND: Sudden arrhythmic death syndrome (SADS), characterized by an unknown or inconclusive cause of death at autopsy, together with a negative or non-lethal toxicology screening, is the most common cause of sudden cardiac death (SCD) in victims under the age of 35. The complete causality of SADS remains unclear, with drugs being a potential risk factor. OBJECTIVE: To describe the toxicological profiles of SADS victims, focusing on proarrhythmic drugs, drug levels, and polypharmacy. METHODS: All deaths in Denmark from 2000-2019 aged 1-35 years and 2007-2019 aged 36-49 years were examined through death certificates, national registries, and autopsy reports with toxicology screenings. We investigated all sudden unexpected death victims with an autopsy performed, and where cause of death was unknown or inconclusive, including negative or non-lethal drug findings (SADS). RESULTS: We identified 477 SADS victims; 313 (66%) had a positive toxicology screening (adjudicated non-lethal), with an average of 2.8 drugs/case. More than half of the SADS victims with a positive toxicology screening had QT-prolonging or brugadogenic drugs present. Polypharmacy was present in 66%, psychotropic polypharmacy in 37%, and QT-prolonging polypharmacy in 22%, with the most frequent overall and QT-prolonging drug combination being an antipsychotic and a psychoanaleptic drug. QT-prolonging drugs were more often present at suprapharmacological levels than non-QT-prolonging drugs. CONCLUSION: A majority of the SADS population had a positive toxicology, with a notably large proportion having proarrhythmic drugs and polypharmacy. This highlights the need for future focus on drugs as a risk factor for SADS.

Toxicological profile using mass spectrometry in sudden cardiac arrest survivors admitted to a tertiary centre.

BACKGROUND: There has been no previous thorough toxicological examination of a cohort of patients with resuscitated sudden cardiac arrest. We aimed to determine the qualitative and quantitative drug composition in a resuscitated sudden cardiac arrest population, using forensic toxicology, with focus on prescribed, non-prescribed, and commonly abused drugs. METHODS: Individuals aged 18-90 years with resuscitated sudden cardiac arrest of presumed cardiac causes were prospectively included from a single tertiary center. Data from the sudden cardiac arrest hospitalization was collected from medical reports. Drugs used during resuscitation or before the blood sampling were identified and excluded in each patient. Mass spectrometry-based toxicology was performed to determine the absence or presence of most drugs and to quantify the findings. RESULTS: Among 186 consecutively enrolled resuscitated sudden cardiac arrest patients (median age 62 years, 83% male), 90% had a shockable rhythm, and were primarily caused by ischemic heart disease (66%). In total, 90 different drugs (excluding metabolites) were identified, and 82% of patients had at least one drug detected (median of 2 detected drugs (IQR:1-4)) (polypharmacy). Commonly abused drugs were present in 16%, and QT-prolonging drugs were present in 12%. Polypharmacy (≥5drugs) were found in 19% of patients. Importantly, none had potentially lethal concentrations of any drugs. CONCLUSION: In resuscitated sudden cardiac arrest patients with cardiac arrest of presumed cardiac cause, routine toxicological screening provides limited extra information. However, the role of polypharmacy in sudden cardiac arrest requires further investigation. No occult overdose-related cardiac arrests were identified.

Analysis of seized peptide and protein-based doping agents using four complimentary methods: Liquid chromatography coupled with time of flight mass spectrometry, liquid chromatography-ultraviolet, Bradford, and immunoassays.

Analysis and identification of seized doping-related products are important tasks for customs or forensic laboratories in order to prevent potentially dangerous and illegal compounds to go into circulation. At the Section of Forensic Chemistry in Copenhagen, we have a workflow consisting of four complimentary validated methods to identify common doping-related substances: liquid chromatography-ultraviolet (LC-UV), LC coupled with time of flight mass spectrometry (LC-TOF-MS), the colorimetric Bradford assay, and an immunoassay. The Bradford assay screens for peptide or proteins in the sample, and the immunoassay confirmed human chorionic gonadotropin (hCG). LC-UV was carried out with a C4 protein column for identification of peptides and proteins from a standard reference library, based on retention times and ratios between peak areas at 220, 254, and 280 nm. LC-TOF-MS was performed using a C18 column, and identification was based on comparison of the retention time and the accurate mass with those of reference standards. In 2019, we received 36 samples for peptide/protein analysis, all of which were tested using the LC-UV, LC-TOF-MS, and colorimetric method, and samples suspected of containing hCG were confirmed with an immunoassay. We found a total of 15 samples containing an illegal doping substance, 12 samples containing substances not prohibited by the Danish Doping List, and nine samples containing no peptides or proteins. In conclusion, the four complimentary methods constitute a suitable approach for identifying common peptide/protein doping substances in the day-to-day routine of a forensic laboratory, with limited sample preparation and interpretation of data.

Increased risk of fatal intoxication and polypharmacy among psychiatric patients at death.

Patients suffering from psychiatric disorders have an excess mortality and a shorter life span expectancy compared to the general population. Furthermore, they are treated with multiple drugs and are known to have an increased risk of drug abuse. In this study, we aimed at investigating the pharmaceutical drug and drug of abuse profiles of the deceased included in the Danish prospective autopsy-based forensic study on psychiatric patients, SURVIVE. Using the postmortem systematic toxicological analysis results, we identified 129 different consumed compounds in our population (n = 443). Polypharmacy (≥5 compounds) was detected in 39.5% of the deceased. Deceased with a psychiatric diagnosis or who died from a fatal intoxication had significantly more compounds at the time of their death compared to having either no psychiatric diagnosis or another cause of death, respectively. Evidence of drug abuse was present, as 29.8% of our total population had consumed either methadone or illicit drugs of abuse, excluding tetrahydrocannabinol. Of those deceased with a psychiatric diagnosis, 33.6% had either consumed methadone or illicit drugs of abuse, a greater number than those without a psychiatric diagnosis. Fatal intoxication was the most frequent cause of death (40.6%) with methadone as the major intoxicant. Here, we found that those without a psychiatric diagnosis had fewer fatal pharmaceutical drug intoxications compared to the psychiatric diagnosis groups. Our findings add further context to understanding the excess mortality of psychiatric patents, since there is an increased occurrence of fatal intoxication, polypharmacy, and drug abuse in this population.

Concentrations of aripiprazole and dehydroaripiprazole in hair segments from deceased individuals with mental disorders.

Segmental hair analysis provides information regarding previous long-term drug exposure, which is useful in the evaluation of cause of death for individuals with mental disorders. The aim was to analyze postmortem concentrations of the antipsychotic drug aripiprazole and its active metabolite dehydroaripiprazole in hair segments from individuals with known aripiprazole intake. Hair samples were collected during autopsy. Each sample was segmented into one to six 1cm segments, depending on the length of the hair shaft. Pulverized hair was extracted and analyzed using a previously published ultra-high-performance liquid chromatography-tandem mass spectrometric method. The 10th-90th percentile of aripiprazole concentrations in all hair segments (n=78) from 17 individuals were 0.024ng/mg-11ng/mg with a median of 2.3ng/mg, and the 10th-90th percentile concentrations of dehydroaripiprazole were 0.020ng/mg-11ng/mg, with a median of 2.6ng/mg, in all hair segments (n=71). The metabolite-to-parent drug ratios ranged from 0.21 to 1.5, with a median of 0.72. The administered doses were calculated for each individual based on aripiprazole prescription data and pharmacy pickups, giving dose estimates of 1mg-32mg daily. A positive significant correlation was observed between concentrations in hair and blood, whereas no trends were observed between the concentrations in hair and the estimated doses. Besides aripiprazole, other antipsychotic drugs were found in several hair segments, indicating a high degree of polypharmacy among all subjects. The present study establishes concentrations of aripiprazole and dehydroaripiprazole in hair segments from 17 deceased individuals with long-term aripiprazole use. In addition, hair analysis demonstrates the possibility of evaluating polypharmacy.

In Vitro Metabolism and Hepatic Intrinsic Clearance of the Synthetic Cannabinoid Receptor Agonist JWH-122 and Its Four ω-Halogenated Analogues.

The number of new psychoactive substances (NPS) emerging on the illicit drug market has increased over the last decade. Halogenation of existing illicit drugs is a particular trend, with the purpose of both circumventing the law and altering the toxicodynamic and toxicokinetic profiles of the compounds. This study investigates the in vitro impact of JWH-122 ω-halogenation (fluoro, chloro, bromo and iodo) on the metabolism, apparent intrinsic hepatic clearance and analytical targets for detecting drug consumption. Metabolite profiling was conducted with pooled human liver microsomes, suspended rat hepatocytes and pooled human hepatocytes. The in vitro half-life was also determined in pooled human hepatocytes. All samples were analysed by liquid chromatography/high-resolution mass spectrometry. All compounds, except for JWH-122, showed high formation rates of phase I metabolites, predominantly ω-COOH and methylnaphthyl hydroxylation metabolites. Phase II metabolites were ω-O-glucuronides, methylnaphthyl O-glucuronides and ω-glutathione conjugates. The relative ion intensity of the glutathione conjugates increased with the ω-halogen size, with I-JWH-122 having the highest intensity. Stability studies gave a low half-life and a high intrinsic hepatic clearance for JWH-122 (1305 mL/min/kg) and MAM-2201 (1408 mL/min/kg). Cl-, Br- and I-JWH-122 showed increasing half-life with increasing ω-halogen size, with intrinsic clearance values of 235-502 mL/min/kg. The recommended analytical targets for consumption of JWH-122 or ω-halogenated JWH-122 analogues are the ω-COOH metabolites for unspecific profiling and the methylnaphthyl hydroxylated metabolites to distinguish the compounds. Furthermore, ω-halogenation with larger halogens appears to increase the intrinsic hepatic stability, thereby prolonging exposure and possibly the duration of action.

Segmental Hair Analysis-Interpretation of the Time of Drug Intake in Two Patients Undergoing Drug Treatment.

The present study involved segmental testing of hair in two clinical cases with known dosage histories. Hair analysis confirmed the first patient's exposure to the prescribed sertraline and citalopram for several months. Citalopram was generally distributed along the hair shaft in accordance with the drug ingestion period. By contrast, "false" positive results were observed for sertraline in distal hair segments, corresponding to a period of no sertraline exposure, which may indicate incorporation from sweat or sebum, which transport the drugs along the hair surface. The second patient received various drugs during her treatment for brain cancer. Metoclopramide, morphine, oxazepam, paracetamol, sumatriptan, tramadol, and zopiclone, which had been part of the therapy, were all detected in the proximal hair segment. The results of these two cases indicated that results-especially concerning the time of drug intake-must be interpreted with caution and allow for the possibility of incorporation from sweat or sebum.

Post-mortem toxicology in young sudden cardiac death victims: a nationwide cohort study.

Aims: Several drugs increase the risk of ventricular fibrillation and sudden cardiac death (SCD). We aimed to investigate in detail the toxicological findings of all young SCD throughout Denmark. Methods and results: Deaths in persons aged 1-49 years were included over a 10-year period. Death certificates and autopsy reports were retrieved and read to identify cases of sudden death and establish cause of death. All medico-legal autopsied SCD were included and toxicological reports collected. Positive toxicology was defined as the presence of any substance (licit and/or illicit). All toxicological findings had previously been evaluated not to have caused the death (i.e. lethal concentrations were excluded). We identified 620 medico-legal autopsied cases of SCD, of which 77% (n = 477) were toxicologically investigated post-mortem, and 57% (n = 270) had a positive toxicology profile. Sudden cardiac death with positive toxicology had higher rates of sudden arrhythmic death syndrome (SADS), compared with SCD with negative toxicology (56% vs. 42%, P < 0.01). In total, 752 agents were detected, and polypharmacy (defined as the presence of more than one drug) was present in 61% (n = 164), all substances combined. Psychotropic drugs were the most frequent (62%, n = 467), and 82% (n = 385) were in pharmacological or subpharmacological levels. Conclusion: We found that more than half of all toxicologically investigated SCD victims have positive post-mortem toxicological findings, and polypharmacy is displayed in a considerable proportion. SCD with positive toxicology had higher rate of SADS, suggesting that the compounds may play a proarrhythmic role in these cases.

Determination of ketone bodies in blood by headspace gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method for determination of ketone bodies (ß-hydroxybutyrate, acetone, and acetoacetate) in blood is presented. The method is based on enzymatic oxidation of D-ß-hydroxybutyrate to acetoacetate, followed by decarboxylation to acetone, which was quantified by the use of headspace GC-MS using acetone-(13)C(3) as an internal standard. The developed method was found to have intra- and total interday relative standard deviations < 10% for acetone+acetoacetate levels (∼25 to 8300 µM) and D-ß-hydroxybutyrate levels (∼30 to 16500 µM). Recovery values varied from 98 to 107%, demonstrating the suitability of the method for measuring ketone bodies over a wide concentration range. The method has been applied to cases in which ketoacidosis was suspected as the cause of death in diabetics or chronic alcoholics, as well as to cases in which another cause of death was identified.

The influence of P-glycoprotein on cerebral and hepatic concentrations of nortriptyline and its metabolites.

The impact of P-glycoprotein (P-gp) on the distribution of nortriptyline (NT) and its metabolites between brain, liver and serum was studied experimentally. The interaction of NT with P-gp in vitro was confirmed by measurement of P-gp stimulated ATPase activity (Km = 257.6 microM, Vmax = 51.0 nmol phosphate released/mg protein-min). Administration of NT (5 mg/kg, s.c.) to mdrla knockout mice resulted in enhanced brain-serum (1.6-fold, p = 0.012) and liver-serum (1.4-fold, p = 0.019) ratios, as compared to the wild-type mice. For a series of NT doses (2.5, 5, 10, 25, 30 mg/kg, i.p.) inhibition of P-gp with cyclosporine A (CsA, 200 mg/kg, i.p.) in rats led to NT brain- and liver-serum ratios that were on average 1.3- (p = 0.005) and 2.1- (p = 0.001) fold higher than those of the controls, respectively. Verapamil (50 mg/kg) (NT, 5 mg/kg) increased the ratios by a factor of 1.6 (p <0.001) and 10.3 (p <0.001) for brain and liver, respectively. Finally, co-administration of methadone (1 mg/kg) did not alter the brain-serum ratio of NT, but in the liver a slight increase (1.5-fold, p = 0.035) was observed. In conclusion, verapamil yielded complete inhibition of P-gp at the blood-brain barrier and CsA had an effect corresponding to about 50% inhibition. The results show that P-gp influences the penetration of NT into the brain, and that drug-drug interactions may take place.

P-glycoprotein interaction with risperidone and 9-OH-risperidone studied in vitro, in knock-out mice and in drug-drug interaction experiments.

The drug transporter P-glycoprotein (P-gp) influences drug distribution across the blood-brain barrier (BBB) by actively extruding drugs into the neural capillaries. Several psychotropic drugs, including nortriptyline (NT) and risperidone (Risp), are substrates of P-gp. Here we compared the in vitro P-gp interactions of Risp and its major metabolite, 9-OH-Risperidone (OH-Risp), with their distribution over the BBB in P-gp knock-out mice and in rats where P-gp was inhibited. K(m) and V(max) were determined by an in vitro ATPase assay, and V(max)/K(m) ratios of 2.7 and 0.5 were recorded for Risp and OH-Risp, respectively, suggesting that Risp is a better substrate for P-gp than OH-Risp. In Mdr1a (-/-) knock-out mice, the brain-serum ratios of both Risp and OH-Risp were more than ten times those of control mice (14 and 11, respectively). When P-gp was inhibited with cyclosporine A (CsA) in Wistar rats, the effect was an order of magnitude less than that observed for the knock-out mice experiments (1-1.5 times the controls), and co-administration of NT had no effect. In conclusion, both Risp and OH-Risp interact with P-gp in vitro, and P-gp has a profound effect on Risp and OH-Risp distribution over the BBB, as is evident from the knock-out mice experiments. Drug-drug interaction effects in relation to P-gp, however, appear to be more limited.