Metabolic ratios and SNPs implicated in tramadol-related deaths.

Tramadol (TR) metabolism is performed by polymorphic enzymes that are influenced by genetic polymorphisms. Within this scope, the study presented here aimed to describe 41 genetic variants within CYP2D6, CYP2B6, and CYP3A4 genes in 48 cases of TR-related death that may be involved in the response to TR and to assess whether there is a correlation between these genetic variants and metabolic ratios (MRs). Blood samples from 48 victims of a TR-related death were analyzed to determine the concentrations of TR and its metabolites [O-desmethyltramadol (M1) & N-desmethyltramadol (M2)] using a LC-MS/MS method. All the samples were also genotyped for 41 common CYP2D6, CYP2B6, and CYP3A4 single nucleotide polymorphisms (SNPs) using the HaloPlex Target Enrichment system. Cases with the T/- genotype (rs35742686 in CYP2D6) had significantly higher M2/M1 ratio than cases with T/T genotype and cases with the G/A genotype (rs35599367 in CYP3A4) had significantly higher MR2 (TR/M2) ratio than cases with G/G genotype. The frequency of tested SNPs which belong to CYP2D6, CYP2B6, and CYP3A4 revealed the over-presentation of 2 SNPs (rs1058172 in CYP2D6 and rs4803419 in CYP2B6) in TR overdose group, which could have toxicological implications. These results indicate these polymorphisms in CYP2D6, CYP2B6, and CYP3A4 might influence the function and could increase the risk of toxicity. However, these findings should be supported in future studies with larger groups of cases.

Putatively lethal ingestion of isopropyl alcohol-related case: interpretation of post mortem isopropyl alcohol and acetone concentrations remains challenging.

Isopropyl alcohol, or propan-2-ol (IPA), is found in numerous chemicals including alcohol-based hand rubs whose use has been recently widely extended to the general population since the onset of the COVID-19 pandemic. This widespread of IPA use could potentially, but not necessarily, be responsible for an increase in IPA poisoning cases (e.g., in alcoholics and/or for suicide attempt, even more in a lockdown situation). Forensic identification of IPA-related fatalities remains challenging as IPA post mortem detection can also result from antemortem or post mortem production, or post mortem contamination. In order to illustrate this issue, we report the case of a 33-year-old man found dead with a bottle of pure IPA liquid close to him. Toxicological positive results only consisted in IPA (464, 260, 465 and 991 mg/L) and acetone (1560, 2340, 3040 and 1360 mg/L) in blood, vitreous humour, urine and bile, respectively (determinations using headspace gas chromatography with flame ionization detection). These IPA absolute concentrations and IPA-to-acetone ratios appear inferior to those usually reported in the literature (higher than 1000 mg/L and 1.1, respectively) in IPA poisoning cases. In conclusion, this death can be cautiously regarded as an IPA ingestion-related fatality in the hypothesis of a survival time which have promoted IPA metabolism to acetone: this hypothesis is supported by the putative limited IPA-ingested dose. This report emphasizes the fact that post mortem IPA and acetone concentration interpretation involves to take account of (i) results in multiple biological specimens, (ii) complete case history, and (iii) a search of possible IPA presence at the scene of death.

Involuntary 5F-ADB-related intoxication following e-cigarette use.

The detection of synthetic cannabinoid (SC) intoxication cases is challenging, even more when the involved SC identification is requested in a forensic context. This situation can be complicated by new modes of SC consumption, non-specific symptomatology, and analytical pitfalls. To illustrate these issues, we report the case of a 16-year-old man who presented symptoms evocating of a seizure disorder in the minutes following the use of a friend's e-cigarette. At admission in the emergency department, his electroencephalogram was interpreted as coherent with a recent seizure episode. 5F-ADB, a third generation SC, was detected in the e-liquid and in an early collected (H2 after the e-cigarette use) serum sample (0.50 µg/L), but not in urine samples (H18 and H38). One 5F-ADB metabolite, O-desmethyl-5F-ADB (M5), was detectable in urine up to at least 38 h after intoxication. Neither 5F-ADB nor its metabolites could be detected in victim's hair sampled 3 months after the intoxication. Although leading to a non-specific symptomatology, acute SC intoxication should be considered when the case history is related to e-cigarette or e-liquid use. Early biological samples are recommended, even if analytical screening can be positive for SC metabolites in urine sampled until 2 days after exposure. Accordingly, data from the literature and the present case underscore the relevance of adding both main 5F-ADB metabolites (M5 and 5-OH-pentyl-ADB) to mass spectrum databases used for toxicological screening in order to reduce the risk of false-negative results in intoxication cases involving 5F-ADB.

Long-Term Analgesia following a Single Application of Fentanyl Transdermal Solution in Pigs.

INTRODUCTION: In animal research, obtaining efficient and constant pain control is regulatory but challenging. The gold standard pain management consists of opioid analgesic administration, such as buprenorphine or fentanyl extended-release patches. However, as in all drugs with a short half-life time, repeated buprenorphine administrations are needed, leading to multiple injections that affect the research protocol. On the other hand, fentanyl patch efficacy is discussed in some species. These elements highlight the need of an optimal formulation of analgesic drugs for laboratory animals. In this study, we investigated how Recuvyra®, a fentanyl transdermal solution (FTS), validated in dog perioperative pain management, could provide sustained analgesia after a single topical administration in pigs in a surgical context. METHODS: A total of 11 minipigs were used in this study. As a preliminary experiment, two different doses were tested as a single application on five pigs: two pigs at full dose (2.6 mg/kg) and three pigs at half dose (1.3 mg/kg). Plasma fentanyl dosages were performed during 4 consecutive days, using liquid chromatography with tandem mass spectrometry detection. The efficacy of FTS was then evaluated in a perioperative period. Six minipigs benefited from a surgical intervention comprising a laparotomy. The FTS was blotted on the skin in a single application 20 min before the surgical incision and plasma fentanyl dosages, clinical examination (body weight, food intake, heart rate, and body temperature) and pain assessment were performed for 7 consecutive days. RESULTS: During the preliminary experiment, all fentanyl concentrations reached the minimum effective concentration (MEC) extrapolated in pigs (fentanylemia ≥0.2 ng/mL) throughout the 4 days. The half dose was chosen for the next step of the study. After the surgical intervention, all plasma fentanyl concentrations remained above the MEC up to 7 days post administration. Pig clinical examinations and pain evaluations showed efficient and constant pain control at the half dose, and few adverse events were observed. DISCUSSION AND CONCLUSION: This study confirms the pharmacological and clinical efficacy of FTS at 1.3 mg/kg in pigs throughout at least 7 postoperative days following laparotomy. The clinical analgesic effect of FTS appears more efficient and well-tolerated than the one observed with repeated injections of buprenorphine. This analgesic drug formulation could be universally used in animal research to provide optimal perioperative pain management and long-term analgesia.

Pitfalls of toxicological investigations in hair, bones, and nails in extensively decomposed bodies: illustration with two cases.

It is difficult to carry out toxicological investigations in biological samples collected from extensively decomposed bodies and to interpret obtained results as several pitfalls should be considered: redistribution phenomena, degradation of xenobiotics during the postmortem period, contamination by putrefaction fluids, and external contamination. This work aims to present two cases in order to illustrate and discuss these difficulties in this tricky situation. Case#1: the body of a 30-year-old woman was found in a wooded area (1 month after she has been reported missing by her family): hair and a femur section were sampled. Case#2: the decomposed corpse of a 52-year-old man was found in a ditch: hair and nails were sampled. After decontamination steps, toxicological investigations were performed using liquid chromatography with high-resolution mass spectrometry and tandem mass spectrometry detection methods. In case#1, the same drugs or metabolites (benzodiazepines, propranolol, tramadol, acetaminophen, paroxetine, and oxetorone) were detected in hair and in bone specimens. This result combination strongly suggests intakes close to the time of death for three of them (oxazepam, lormetazepam, and propranolol). In case#2, results of toxicological investigations in hair and nails [(hair/nail concentration in ng/mg) nordiazepam (1.12/1.06), oxazepam (0.113/0.042), zolpidem (0.211/< 0.01), hydroxyzine (0.362/< 0.01), and cetirizine (0.872/1.110)] were both consistent with several drug intakes but were not contributory to cause of death determination. In case of positive toxicological results in biological samples collected from extensively decomposed bodies (such as hair, bones, or nails), it is challenging to determine the time, and even more, the level of the dose of exposure(s).

Lead: a hidden "untested" risk in neonatal blood transfusion.

BACKGROUND: Neonates may be exposed to lead (Pb) through blood transfusions from donors. Pb exposure has neurological, cardiovascular, renal, and other adverse effects. The study aimed to (i) determine the blood lead levels (BLLs) in different blood product units (whole blood, packed red blood cells (pRBCs), platelets, and plasma transfused to neonates) and (ii) estimate the proportion of units with high BLLs. METHODS: Residual blood from blood bank bags that were used for neonatal transfusion were collected: 25 samples from each type of blood product except for whole blood (10 samples). The Pb analysis was performed using the atomic absorption method. The study was conducted at the Suez Canal University Hospital, Egypt. RESULTS: The mean of BLL in pRBCs, platelets, plasma, and whole blood were 136, 199, 108, and 130 µg/L, respectively; 60% contained Pb above 50 µg/L. The highest BLLs were in platelet units. CONCLUSIONS: The present study showed for the first time that platelets and plasma in addition to whole blood and pRBCs used for neonatal transfusions are sources of Pb. Re-evaluation of the guidelines is mandatory for the safety of the neonates. Long-term neurodevelopment assessment of neonates exposed to high Pb is warranted.

Case report: relevance of metabolite identification to detect new synthetic opioid intoxications illustrated by U-47700.

Today, new psychoactive substances (NPS) producers increasingly appear to be targeting new synthetic opioids (NSOs), and the recent emergence of NSOs is causing considerable concern in North America and in Europe. For toxicologists, NSO detection in a forensic context presents three additional difficulties to the general NPS analytical detection challenge: (i) high frequency of new products, (ii) low concentrations (in µg/L range and under) in biological samples related to their high opioid potency, and (iii) extensive metabolism. In this context, the present work aims to highlight the relevance of NSO metabolite detection in potential intoxication cases. Illustration is given with U-47700, an emerging NSO, (i) that was identified in a powder recently collected in France and in a fatality case, (ii) whose metabolites were in vitro produced using human liver microsomes and their mass spectra (MS) added in our MS/MS and HRMS libraries, and (iii) for which metabolism data were compared to those of the literature: U-47700 was identified in the powder and at 3040 µg/L in peripheral blood in the fatality case. In addition, high amounts of several U-47700 metabolites, especially N-desmethyl-U-47700, were observed in urine. Even if metabolite formation may largely depend on the enzymatic activity as well as on the length of the survival time, confrontation of these results to data found in the literature strongly suggests that this metabolite is regularly a better blood and (mainly) urine biomarker of U-47700 intake than U-47700 itself. Indeed, in this fatality and in other previous reports, N-desmethyl-U-47700 produced the main observed chromatographic signal (i) systematically in vitro and (ii) commonly in vivo, especially in urines. N,N-Didesmethyl-U-47700 is also sometimes a better biomarker of U-47700 intake than U-47700 itself. Accordingly, we suggest adding N-desmethyl-U-47700 (and N,N-didesmethyl-U-47700) in mass spectrum databases used for toxicological screening in order to reduce the risk of false-negative results in intoxication cases involving U-47700.

[Congenital hyperinsulinism revealed by sudden infant death]. / Hyperinsulinisme congénital révélé par une mort inattendue de l'enfant.

We report the case of a girl of 5 and a half months admitted for discomfort and consciousness loss at home and supported on sudden infant death protocol. Workup was negative. Autopsy showed only signs of asphyxia. Microscopic examination of the pancreas showed hypertrophic beta cells of Langerhans islets, explaining death linked to severe hypoglycemia by inappropriate insulin hypersecretion. This observation highlights the importance of the management of sudden infant unexpected death according to the protocol of the National Health Authority, which includes an autopsy with complete sampling, which in this case resulted in a diagnosis of unknown disease the lifetime of the child.

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.

Testing for Drugs in Exhaled Breath Collected With ExaBreath in a Drug Dependence Population: Comparison With Data Obtained in Urine After Liquid Chromatographic-Tandem Mass Spectrometric Analyses.

BACKGROUND: Exhaled breath is commonly used in alcohol testing but has been recently demonstrated by scientists from Sweden, Switzerland, and the United States to contain a large number of both volatile and nonvolatile substances that can be measured using dedicated devices. ExaBreath is a sampling device that collects the bio-aerosols particles from the donor. Approximately 1-2 minutes exhaled breath is enough for the test. The device collects the very small bio-aerosols on a filter, which is consecutively incubated into methanol to release the drugs at the laboratory. METHODS: Eighteen drug addicts from a methadone substitution program were recruited for this study. There were 5 women and 13 men, aged 25-50 years. The daily methadone dosage ranged from 10 to 120 mg, mostly as syrup. Urine (in plastic tubes with no preservative) and exhaled breath were simultaneously collected. In both fluids, methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) were tested using a specific liquid chromatographic-tandem mass spectrometric method, whereas all other compounds were screened by liquid chromatographic-tandem mass spectrometric method using a home made library of more than 800 compounds. Linearity, imprecision, and limit of quantitation were established. In each analytical batch, low and high controls were included. RESULTS: All 18 urine specimens tested positive for methadone and EDDP. Several other compounds were also identified, including morphine, THC-COOH, benzoylecgonine, nicotine, some antidepressants, and neuroleptics. Methadone and EDDP were identified in exhaled breath from all 18 patients, with concentrations in the range 11-1470 and 29-818 pg per filter, respectively. In 13 cases, the ratio methadone/EDDP in exhaled breath was >1 (range 0.4-2.8). Except nicotine (n = 7), no other substance was detectable in exhaled breath. CONCLUSION: This study gives further support to the possibility of using exhaled breath as a new matrix to document exposure to drugs.

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.

Identification and quantification of 35 psychotropic drugs and metabolites in hair by LC-MS/MS: application in forensic toxicology.

Despite a non-invasive sampling, hair samples are generally collected in limited amounts for an obvious esthetic reason. In order to reduce the required quantity of samples, a multianalytes method allowing simultaneous identification and quantification of 35 psychoactive drugs was developed. After incubation of 50 mg of hair in a phosphate buffer pH 5 for one night at room temperature, the substances of interest were extracted by a simple liquid-liquid extraction step, with a dichloromethane/ether mixture (70:30, v/v). After evaporation under a gentle stream of nitrogen and reconstitution in formate buffer (2 mM, pH 3)/acetonitrile (90:10, v/v), twenty microliter were injected into the LC-MS/MS system for a chromatographic run of 29 min using an Atlantis T3 column (150 × 2.1 mm, 3 µm) (Waters Corp, Milford, USA) and a gradient mixture of 2 mM, pH 3.0 ammonium formate, and 2 mM, pH 3.0 ammonium formate/acetonitrile. The data acquisition was performed in scheduled MRM mode. Intra- and inter-day precisions, estimated using the coefficient of variation and relative bias, were lower than 20 % for all concentration levels, except for two compounds. The limits of detection and quantification ranged from 0.5 to 10 pg/mg. After complete validation, this method has been successfully used in several forensic cases, three of which are reported.

Improved liquid chromatography-tandem mass spectrometric method for the determination of ethyl glucuronide concentrations in hair: applications to forensic cases.

Ethyl glucuronide (EtG) is a direct marker of ethanol consumption, and its assay in hair is an efficient tool for chronic alcoholism diagnosis. In 2012, the Society of Hair Testing proposed a new consensus for hair concentrations interpretation, strongly advising the use of analytical methods providing a limit of quantification of less than 3 pg/mg. The present work describes the optimization and validation of a previously developed liquid chromatography-tandem mass spectrometric method in order to comply with this recommendation. The concentration range of this improved method is from 3 to 1,000 pg/mg. Some cases are then described to illustrate the usefulness of hair EtG: a forensic post-mortem case and two cases of suspension of driving licences. Finally, hair samples of some teetotallers (n = 10) have been analyzed, which allowed neither to quantitate nor to detect any trace of EtG.

Effect of CYP2D6, 2C19, and 3A4 Phenoconversion in Drug-Related Deaths.

Molecular autopsy is a very important tool in forensic toxicology. However, many determinants, such as co-medication and physiological parameters, should be considered for optimal results. These determinants could cause phenoconversion (PC), a discrepancy between the real metabolic profile after phenoconversion and the phenotype determined by the genotype. This study's objective was to assess the PC of drug-metabolizing enzymes, namely CYP2D6, 2C19, and 3A4, in 45 post-mortem cases where medications that are substrates, inducers, or inhibitors of these enzymes were detected. It also intended to evaluate how PC affected the drug's metabolic ratio (MR) in four cases. Blood samples from 45 cases of drug-related deaths were analyzed to detect and determine drug and metabolite concentrations. Moreover, all the samples underwent genotyping utilizing the HaloPlex Target Enrichment System for CYP2D6, 2C19, and 3A4. The results of the present study revealed a statistically significant rate of PC for the three investigated enzymes, with a higher frequency of poor metabolizers after PC. A compatibility was seen between the results of the genomic evaluation after PC and the observed MRs of venlafaxine, citalopram, and fentanyl. This leads us to focus on the determinants causing PC that may be mainly induced by drug interactions. This complex phenomenon can have a significant impact on the analysis, interpretation of genotypes, and accurate conclusions in forensic toxicology. Nevertheless, more research with more cases in the future is needed to confirm these results.

Application to several suspected poisoning cases of a validated analytical method for the determination of muscarine in human biological matrices using liquid chromatography with high-resolution mass spectrometry detection.

Despite prevention efforts, many cases of mushroom poisoning are reported around the world every year. Among the different toxins implicated in these poisonings, muscarine may induce parasympathetic neurological damage. Muscarine poisonings are poorly reported in the current literature, implying a lack of available data on muscarine concentrations in human matrices. A validated liquid chromatography with high-resolution mass spectrometry detection (Orbitrap technology) method was developed to determine muscarine concentrations in human urine, plasma, and whole blood samples. Muscarine was determined using 100 µL of biological fluids, and precipitation was used for sample preparation. Liquid chromatography-mass spectrometry was performed using an Accucore Phenyl-X analytical column with the electrospray source in positive ion mode. Muscarine was quantitated in parallel reaction monitoring (PRM) mode with D9-muscarine as the internal standard. The method was validated successfully over the concentration range 0.1-100 µg/L for plasma and whole blood and 1-100 µg/L for urine, with acceptable precision and accuracy (<13.5%), including the lower limit of quantification. Ten real cases of suspected muscarine poisoning were successfully confirmed with this validated method. Muscarine concentrations in these cases ranged from 0.12 to 14 µg/L in whole blood,

Synthetic cathinones in drug-facilitated sexual assault: A case report involving the novel generation substituted cathinone N-ethylpentedrone and a review of the literature.

The use of 3,4-methylenedioxymethamphetamine (MDMA) in drug-facilitated sexual assault (DFSA) is not uncommon. Indeed, the effects associated with the use of this substance may lead to disinhibition. Several synthetic cathinones, such as mephedrone or methylone, also possess marked entactogenic properties. This manuscript aims to (i) report a DFSA case involving a novel cathinone derivative, namely N-ethyl-pentedrone (NEPD) and (ii) review previously reported DFSA cases involving synthetic cathinones. Using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), NEPD was detected in both plasma and urine collected from a 36-year-old male who had been victim of DFSA. Furthermore, an exhaustive, non-period-specific English-language literature search was performed using several different electronic databases to identify DFSA cases involving synthetic cathinones. Overall, five synthetic cathinones have been associated with DFSA:methylenedioxypyrovalerone, 4-methylethcathinone, α -pyrrolidinopentiophenone, mephedrone, α -pyrrolidinohexiophenone, and methylone, which appears to be the most frequently reported. Methylone is the ß-keto analog of MDMA, with which it shares substantial pharmacological similarities. Indeed, the pharmacological effects of methylone are similar to those associated with MDMA. By contrast, little is known regarding NEPD's pharmacological effects in humans. Based on subjective reports, NEPD can produce both positive and negative effects in human. Unlike what is reported in the case of methylone or mephedrone, only a small minority of NEPD users report slightly entactogenics effects. Such properties theoretically make NEPD more suitable for use in a chemsex context than in DFSA context; even though, the boundary between these two specific forms of sexualized drug use can sometimes appear tenuous.

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.