A two-sample approach to retrograde extrapolation of blood THC concentrations - Is it feasible?

BACKGROUND: Retrograde extrapolation of drug concentrations in blood can be relevant in cases of drug-impaired driving and is regularly used in forensic toxicology in Norway. Δ9-tetrahydrocannabinol (THC) has complex, multi-compartmental pharmacokinetics, which makes retrograde extrapolation of blood THC concentrations problematic. In the present study, we evaluated an approach to retrograde extrapolation in which momentary rates of decrease of THC were estimated from two consecutive blood samples in apprehended drivers. MATERIAL AND METHODS: Data were collected from apprehended drivers in Norway 2000-2020. We included 548 cases in which THC was detected in two consecutive blood samples collected ≥ 20 min apart. THC concentrations were measured by GC-MS and UHPLC-MS/MS. In each case, THC concentrations and the time between the two sampling points (Δt) were used to estimate the rate constant k. The relationship between THC concentration and k was modelled by linear regression. RESULTS: The median Δt was 31 min (interquartile range, IQR = 9). The median blood THC concentration was 2.4 µg/L (IQR = 3.4) at the first sampling point and 2.3 µg/L (IQR =3.1) at the second. The concentration decreased in 62% and increased in 38% of all cases. However, considering measurement uncertainty, the changes were not statistically significant in 87% of cases. The mean of k was 0.12 h-1, corresponding to an apparent t1/2 of 6.0 h. The t1/2 predicted from linear regression of k against THC concentration ranged from 0.93 to 13 h for the highest and lowest concentrations observed (36 and 0.63 µg/L, respectively). The time from driving to blood collection had a median of 1.7 h (IQR = 1.5), and did not correlate with k. CONCLUSIONS: The apparent t1/2 of THC calculated from the mean of k was 6.0 h, which is shorter than the terminal elimination t1/2 suggested in previous population studies. This indicates that blood samples were often taken during the late distribution phase of THC. Because Δt was short relative to the rates of decrease expected in the late distribution and elimination phases, the underlying true concentration changes related to in vivo pharmacokinetics were small and masked by the relatively larger "false" changes introduced by random analytical and pre-analytical error. Therefore, individual values of k calculated from only two blood samples taken a short time apart are unreliable, and a two-sample approach to retrograde extrapolation of THC cannot be recommended.

Comparative Evaluation of Carboxyhemoglobin Quantification in Postmortem Whole Blood by CO-Oximetry and Headspace Gas Chromatography with Flame Ionization Detection and Atom Absorption Spectrophotometry.

A comparative evaluation of two methods used for carboxyhemoglobin (COHb) determination in postmortem whole blood was performed: carbon monoxide (CO)-oximetry measuring at 128 wavelengths and headspace gas chromatography with flame ionization detection (HS-GC--FID) where CO was determined after catalytic reduction of CO to CH4 and Fe was determined by atom absorption spectrophotometry (AAS, 248.3 nm). An aliquot of 100 µL whole blood was loaded into the CO-oximetry module. In the HS-GC--FID analysis, to 1.0 mL of whole blood, 3.0 mL of saponin solution was added, mixed and centrifuged. To 20 mL HS vials, 400 µL of the supernatant was added and the vials were immediately sealed. One milliliter of potassium hexacyanoferrat (III) solution was added through the HS septum and mixed. The samples were incubated at 70°C for 5 min. CO was separated using He as carrier gas and a CP-Molsieve 5 Å PLOT capillary column. Fe was determined using 400 µL of the saponin supernatant diluted to 10 mL by water. During a period of ∼3 years, 124 postmortem whole blood samples were analyzed. Bland-Altman method comparison showed satisfactory agreement and no significant bias between the methods for the whole saturation range (5 to 85% COHb). Five samples, all with %COHb >40, showed deviations of more than 10% COHb in absolute terms. One sample, in the lower COHb range <10%, was false negative on the CO-oximetry method. The between-assay accuracy, reported as bias, at 60% COHb was -0.8% and -9.0%, and precision, reported as relative standard deviation, was 1.6% and 7.7%, for the CO-oximetry and HS-GC--FID-AAS methods, respectively. Both methods obtained satisfactory results in proficiency testing rounds, with z-scores <±2 (n = 11). This study showed that the CO-oximetry method based on the 128-wavelength principle and the HS-GC--FID-AAS method are comparable and satisfactory for %COHb determination in postmortem whole blood.

Deaths associated with MDMA in the period 2000-2019. / Dødsfall assosiert med MDMA i perioden 2000­19.

BACKGROUND: The use of MDMA (3,4-methylenedioxymethamphetamine), also known as ecstasy, has increased in Norway in recent years. Since MDMA has the potential to be toxic and cause death, we studied whether increased availability and use correlates with the increase in MDMA-associated deaths. MATERIAL AND METHOD: The study includes post-mortems with findings of MDMA in blood, linked to information about cause of death from the Norwegian Cause of Death Registry. These data were compared with the number of arrested drug drivers with MDMA detected in their blood as well as annual seizure statistics from Kripos (The National Criminal Investigation Service) in the period 2000-2019. RESULTS: In the period 2000-2019, MDMA was detected in 142 fatalities, and the cause of death was known for 132 of these. The number of annual MDMA-associated deaths varied from 1 to 18. The median MDMA concentration among the fatalities increased from 1.9 µmol/L (interquartile range (IQR) 0.9 to 5.0) in 2000-2004 to 3.8 µmol/L (1.4 to 12.0) in 2015-2019. In 47/132 (36 %) of cases, MDMA and other central nervous system (CNS) stimulant drugs contributed to the death. Among arrested drug drivers with detected MDMA, the annual number of detected cases was 7-262 in this period, but the median concentration remained stable. INTERPRETATION: MDMA may have contributed to numerous deaths in Norway. Increased availability, increased use and increased strength of contents seem to be significant.

Relationship between betahydroxybutyrate (BHB) and acetone concentrations in postmortem blood and cause of death.

Unexpected death caused by diabetic or alcoholic ketoacidosis is easily overlooked due to the non-specific symptoms. Although the acid betahydroxybutyrate (BHB) is the most abundant ketone body formed in conditions with ketoacidosis, routine analysis in postmortem investigations often only includes the neutral ketone body acetone. This study aims to evaluate the usefulness of implementing routine BHB analysis in postmortem cases, by investigating the relationship between BHB and acetone concentrations in postmortem blood and the main cause of death. From our database of forensic autopsy cases examined from 2012 to 2015, there were 376 cases with BHB and/or acetone detected in postmortem blood that could be paired with data from the Norwegian Cause of Death Registry. Cases were categorized into three groups based on cause of death: "Diabetes-related" (n = 38), "Alcohol-related" (n = 35) and "Other" (n = 303). Analysis of BHB in blood was performed using UHPLC-MS/MS (limit of quantification (LOQ) 52 mg/L) and of acetone using HS-GC-FID (LOQ 87 mg/L). For the purpose of the study, the acetone method was also validated for a LOQ of 23 mg/L. The median BHB concentration was significantly higher in the group of diabetes-related deaths (671 mg/L, range 68-1311 mg/L) compared to the group of alcohol-related (304 mg/L, range 65-1555 mg/L, p <0.001) and other causes of deaths (113 mg/L, range 0-1402 mg/L, p <0.001). In seven deaths (1.9%), the BHB blood concentration was above the suggested pathological threshold of 250 mg/L, without detection of acetone in blood above 23 mg/L. In 15% of deaths by other causes than diabetes or alcohol, a pathologically significant BHB blood concentration was detected. Our results indicate that BHB is a more reliable marker of pathologically significant ketoacidosis than acetone, and we suggest that BHB should be routinely analyzed in postmortem investigations.

Studies on Para-Methoxymethamphetamine (PMMA) Metabolite Pattern and Influence of CYP2D6 Genetics in Human Liver Microsomes and Authentic Samples from Fatal PMMA Intoxications.

Para-methoxymethamphetamine (PMMA) has caused numerous fatal poisonings worldwide and appears to be more toxic than other ring-substituted amphetamines. Systemic metabolism is suggested to be important for PMMA neurotoxicity, possibly through activation of minor catechol metabolites to neurotoxic conjugates. The aim of this study was to examine the metabolism of PMMA in humans; for this purpose, we used human liver microsomes (HLMs) and blood samples from three cases of fatal PMMA intoxication. We also examined the impact of CYP2D6 genetics on PMMA metabolism by using genotyped HLMs isolated from CYP2D6 poor, population-average, and ultrarapid metabolizers. In HLMs, PMMA was metabolized mainly to 4-hydroxymethamphetamine (OH-MA), whereas low concentrations of para-methoxyamphetamine (PMA), 4-hydroxyamphetamine (OH-A), dihydroxymethamphetamine (di-OH-MA), and oxilofrine were formed. The metabolite profile in the fatal PMMA intoxications were in accordance with the HLM study, with OH-MA and PMA being the major metabolites, whereas OH-A, oxilofrine, HM-MA and HM-A were detected in low concentrations. A significant influence of CYP2D6 genetics on PMMA metabolism in HLMs was found. The catechol metabolite di-OH-MA has previously been suggested to be involved in PMMA toxicity. Our studies show that the formation of di-OH-MA from PMMA was two to seven times lower than from an equimolar dose of the less toxic drug MDMA, and do not support the hypothesis of catechol metabolites as major determinants of fatal PMMA toxicity. The present study revealed the metabolite pattern of PMMA in humans and demonstrated a great impact of CYP2D6 genetics on human PMMA metabolism.

Asphyxiation death caused by oxygen-depleting cargo on a ship.

The extreme danger associated with entering enclosed spaces loaded with oxygen-depleting organic cargo in ships and tanks is obviously underestimated, both among crew and management. We present a case report to highlight this occupational hazard and to increase the knowledge about the imperative precautions, in order to prevent future accidents. An experienced customs officer was found lifeless at the bottom of the unattended cargo hold on a ship loaded with woodchips. The oxygen content in the cargo atmosphere was below 2%, which is incompatible with life. Forensic autopsy revealed injuries related to the fall, and there were no positive toxicological findings in blood, lung or urine. Management and workers must be taught about the extreme rapidity of developing unconsciousness and asphyxiant death when entering enclosed spaces loaded with oxygen-depleting cargo. Even a single inhalation can result in unconsciousness and death. Dozens of annual deaths and severe injuries can easily be prevented if simple precautions are followed.

Novel psychoactive substances. / Nye psykoaktive stoffer.

There has been a significant increase in the number of new intoxicants on the illegal drugs market globally, also in Norway. The substances are given the name NPS: Novel Psychoactive Substances, and are mainly sold over the Internet. Uncertain dosage of potent substances entails a risk of accidental overdose, and therefore serious intoxication and death. In this article we provide an overview of current knowledge with regard to these substances.

Is toxicity of PMMA (paramethoxymethamphetamine) associated with cytochrome P450 pharmacogenetics?

In 2010-2013, 29 fatal intoxications related to the designer drug paramethoxymethamphetamine (PMMA, 4-methoxymethamphetamine) occurred in Norway. The current knowledge about metabolism and toxicity of PMMA in humans is limited. Metabolism by the polymorphic cytochrome P450 (CYP) 2D6 enzyme to the psychoactive metabolite 4-hydroxymethamphetamine (OH-MA), and possibly by additional enzymes, is suggested to be involved in its toxicity. The aim of this work was to study the association between CYP genetics, PMMA metabolism and risk of fatal PMMA toxicity in humans. The frequency distribution of clinically relevant gene variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A5, and the phenotypic blood CYP2D6 metabolic ratio (OH-MA/PMMA) in particular, were compared in fatal PMMA intoxications (n=17) and nonfatal PMMA abuse controls (n=30), using non-abusers (n=305) as references for the expected genotype frequencies in the Norwegian population. Our study demonstrated that the CYP2D6 enzyme and genotype are important in the metabolism of PMMA to OH-MA in humans, but that other enzymes are also involved in this biotransformation. In the fatal PMMA intoxications, the blood concentrations of PMMA were higher and the CYP2D6 metabolic ratios were lower, than in the nonfatal PMMA abuse controls (median (range) 2.1 (0.03-5.0) vs 0.3 (0.1-0.9) mg/L, and ratio 0.6 (0.0-4.6) vs 2.1 (0.2-7.4) p=0.021, respectively). Overall, our findings indicated that, in most cases, PMMA death occurred rapidly and at an early stage of PMMA metabolism, following the ingestion of large and toxic PMMA doses. We could not identify any genetic CYP2D6, CYP2C9, CYP2C19 or CYP3A5 predictive marker on fatal toxicity of PMMA in humans. The overrepresentation of the CYP2D6 poor metabolizer (PM) genotype found in the nonfatal PMMA abuse controls warrants further investigations.

Codeine-spiked beer in a date rape case?

A case of suspected drug-facilitated sexual assault, involving codeine and acetaminophen, possibly mixed in beer, was recently addressed at the Norwegian Institute of Public Health. To examine the case, a small study was performed, spiking beer with preparations containing codeine and acetaminophen and observing the concentrations, appearance, and taste of the solutions. The study revealed the majority of the preparations to be quickly soluble in beer, achieving high concentrations, but at the expense of strong taste and drastic visible changes in the beer.

Codeine to morphine concentration ratios in samples from living subjects and autopsy cases after incubation.

The codeine to morphine concentration ratio is used in forensic toxicology to assess if codeine has been ingested alone or if morphine and/or heroin have been ingested in addition. In our experience, this interpretation is more difficult in autopsy cases compared with samples from living persons, since high morphine concentrations are observed in cases where only codeine is assumed to have been ingested. We have investigated if codeine and morphine glucuronides are subject to cleavage to the same extent in living and autopsy cases in vitro. We included whole blood samples from eight living subjects and nine forensic autopsy cases, where only codeine ingestion was suspected. All samples were incubated for 2 weeks at 37°C and analyzed for codeine and six codeine metabolites using liquid chromatography tandem mass spectrometry. A reduction in the codeine to morphine concentration ratio was found, both in samples from living subjects (mean 33%, range 22-50%) and autopsy cases (mean 37%, range 13-54%). The increase in the morphine concentrations was greater in the autopsy cases (mean 85%, max 200%) compared with that of the living cases (mean 51%, max 87%). No changes were seen for codeine or codeine-6-glucuronide concentrations. The altered ratios might mislead the forensic toxicologist to suspect morphine or heroin consumption in cases where only codeine has been ingested.

[Intoxation with paramethoxymethamphetamine]. / Forgiftning med parametoksymetamfetamin.

BACKGROUND: Since the summer of 2010, there has been an epidemic of deaths related to paramethoxymethamphetamine (PMMA) in Norway. We present a review of the pharmacology and toxicology of the substance. MATERIAL AND METHOD: The review is based on a literature search in the databases PubMed, Ovid and MEDLINE. A discretionary selection was made of relevant articles. RESULTS: Paramethoxymethamphetamine and paramethoxyamphetamine (PMA) are two so-called designer amphetamines which appear from time to time on the illegal narcotics market in many countries. They are frequently sold as ecstasy or amphetamine, often mixed with amphetamine or methamphetamine. The substances, known on the street as «Death¼, have potent serotonergic effects and are associated with significant toxicity. Many deaths have been reported worldwide, even after intake of an «ordinary user dose¼. The narcotic effect is not very pronounced and the onset is slow, which may lead to unintentional overdosing. INTERPRETATION: In cases of severe intoxation that are apparently related to intake of amphetamine or ecstasy, PMMA/PMA intoxation should be suspected.

The PMMA epidemic in Norway: comparison of fatal and non-fatal intoxications.

During a 6 month period (July 2010-January 2011) we observed 12 fatal intoxications and 22 non-fatal cases related to the drug paramethoxymethamphetamine (PMMA) in Norway (4.8 mill inhabitants). This toxic designer drug, also known as "Death", is occasionally found in street drugs offered as "ecstasy" or "amphetamine". The present study aimed to evaluate the cause of death, and to compare the PMMA blood concentrations in fatal and non-fatal cases. Methods for identification and quantification of PMMA are presented. The median age of fatalities was 30 years (range 15-50) with 67% males; in non-fatal cases 27 years (20-47) with 86% males. In the 12 fatalities, the median PMMA blood concentration was 1.92 mg/L (range 0.17-3.30), which is in the reported lethal range of 0.6-3.1 mg/L in peripheral blood and 1.2-15.8 mg/L in heart blood. In the 22 non-fatal cases, the median PMMA concentration was 0.07 mg/L (range 0.01-0.65). Poly-drug use was frequent both in fatal and non-fatal cases. The PMA concentrations ranging from 0.00 to 0.26 mg/L in both groups likely represented a PMMA metabolite. Three fatalities were attributed to PMMA only, six to PMMA and other psychostimulant drugs, and three to PMMA and CNS depressant drugs, with median PMMA concentrations of 3.05 mg/L (range 1.58-3.30), 2.56 (1.52-3.23) and 0.52 mg/L (0.17-1.24), respectively. Eight victims were found dead, while death was witnessed in four cases, with symptoms of acute respiratory distress, hyperthermia, cardiac arrest, convulsions, sudden collapse and/or multiple organ failure. In summary, all fatalities attributed to PMMA had high PMMA blood concentrations compared to non-fatal cases. Our sample size was too small to evaluate a possible impact of poly-drug use. A public warning is warranted against use and overdose with illegal "ecstasy" or "speed" drugs.

Lethal methemoglobinemia and automobile exhaust inhalation.

Inhalation of automobile exhaust gas often leads to death by CO intoxication. In some cases the measured carbon monoxide hemoglobin saturation level (COHb) is considerably below what is considered to be lethal. The death in such cases has been attributed to a combination of a high CO2 and a low O2 tension. In a recent case the deceased was found dead in a car equipped with a catalytic converter, with a hose leading exhaust from the engine to the interior of the car. Analysis revealed a moderately elevated COHb and a high methemoglobin saturation level (MetHb) in peripheral blood. No ethanol, narcotics or drugs were detected. Reports mentioning MetHb or methemoglobinemia in post-mortem cases are surprisingly scarce, and very few have related exhaust gas deaths to methemoglobinemia. High-degree methemoglobinemia causes serious tissue hypoxia leading to unconsciousness, arrhythmia and death. The existing literature in this field and the knowledge that exhaust fumes contain nitrogen oxide gases (NOx) that by inhalation and absorption can result in severe methemoglobinemia, led us to postulate that this death could possibly be attributed to a combination of methemoglobinemia and a moderately high COHb concentration.