The relationship between elastin cross linking and alveolar wall rupture in human pulmonary emphysema.

To better define the role of mechanical forces in pulmonary emphysema, we employed methods recently developed in our laboratory to identify microscopic level relationships between airspace size and elastin-specific desmosine and isodesmosine (DID) cross links in normal and emphysematous human lungs. Free DID in wet tissue (a biomarker for elastin degradation) and total DID in formalin-fixed, paraffin-embedded (FFPE) tissue sections were measured using liquid chromatography-tandem mass spectrometry and correlated with alveolar diameter, as determined by the mean linear intercept (MLI) method. There was a positive correlation between free lung DID and MLI (P < 0.0001) in formalin-fixed lungs, and elastin breakdown was greatly accelerated when airspace diameter exceeded 400 µm. In FFPE tissue, DID density was markedly increased beyond 300 µm (P < 0.0001) and leveled off around 400 µm. Elastic fiber surface area similarly peaked at around 400 µm, but to a much lesser extent than DID density, indicating that elastin cross linking is markedly increased in response to early changes in airspace size. These findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.NEW & NOTEWORTHY The current findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.

Free lung desmosine: a potential biomarker for elastic fiber injury in pulmonary emphysema.

INTRODUCTION: Desmosine and isodesmosine (DID) are biomarkers for elastic fibre damage in pulmonary emphysema. However, current methods for measuring lung DID involve tissue hydrolysis and lack specificity for those fibres undergoing breakdown. To address this limitation, free (nonpeptide-bound) DID content in unhydrolyzed tissues was evaluated as a more accurate biomarker in an animal model of pulmonary emphysema. METHODS: Hamsters were treated with either cigarette smoke and lipopolysaccharide (LPS), room air and LPS, or room air alone (controls). Free DID levels in fresh and formalin-fixed lungs were measured by LC-MS/MS and correlated with the mean linear intercept (MLI) measure of airspace size. RESULTS: There was no significant difference in free DID between fresh and formalin-fixed lungs. Animals treated with smoke and LPS had significantly higher levels of free DID than the LPS only group (359 vs. 93.1 ng/g wet lung, respectively; p = 0.0012) and room air controls (undetectable levels; p = 0.0002). There was a significant positive correlation between free DID and MLI (p < 0.0001). CONCLUSIONS: The results support the hypothesis that free lung DID is a sensitive indicator of alveolar wall injury that may be used to study the development of pulmonary emphysema in both animal models and post-mortem human lung tissue.

Evaluation of Acetylfentanyl Following Suspected Heroin Overdose When Complicated by the Presence of Toxic Fentanyl and Alprazolam Concentrations.

A 34-year-old male was reported to be snorting a white powder that was believed to contain heroin. Toxicological analysis revealed free morphine (356 µg/L), fentanyl (34.7 µg/L), alprazolam (64.9 µg/L), and acetylfentanyl (32.9 µg/L) in femoral blood and 6-monoacetylmorphine (6-MAM, <10.0 µg/L) in vitreous fluid. Norfentanyl was only detected in stomach contents (<1.00 µg/total). Heroin, fentanyl, and acetylfentanyl were also detected in solid dose evidence submitted by law enforcement. The fentanyl and alprazolam concentrations might normally be associated with a fatal outcome and are supported with the distribution of fentanyl and alprazolam being consistent with an acute intoxication. In addition, the presence of 6-MAM and a free versus total morphine ratio of 67.9% provide supporting evidence of a rapid death following intranasal (IN) administration. However, the presence of illicit acetylfentanyl complicates toxicologic interpretation due to overlapping recreational and fatal concentrations of this compound reported in the literature as well as a potential for postmortem redistribution (PMR). Reported acetylfentanyl concentrations have also varied when presented with significant fentanyl concentrations and underscore the need to consider a wide range of illicit opioid compounds when investigating drug-related deaths. Based on our comprehensive toxicologic analysis, the results suggest an acute intoxication primarily by IN administration of acetylfentanyl and fentanyl. In addition, we suggest the presence of alprazolam, 6-MAM, and a percentage free morphine is also consistent with rapid death. The cause of death was officially attributed to an acute combined intoxication of acetylfentanyl, fentanyl, alprazolam, and heroin, with the manner of death as accidental.

Five Postmortem Case Reports with Qualitative Analysis of Cyclopropylfentanyl by LC-MS-MS.

In January 2018, the Drug Enforcement Agency temporarily designated cyclopropylfentanyl as a Schedule I drug. Over the course of 5 months (December 2017-May 2018), the Nassau County Medical Examiner Toxicology Laboratory qualitatively identified and confirmed cyclopropylfentanyl in specimens obtained from five postmortem cases. We describe the five cases and include pertinent autopsy findings and decedent histories, along with results for cyclopropylfentanyl determined in postmortem cardiac blood. Samples were prepared by an alkaline liquid-liquid extraction, with sample pH adjusted to >9 and utilizing an extraction solvent consisting of 90:10 hexane:ethyl acetate. Instrumental analysis was achieved via liquid chromatography tandem mass spectrometry with a dual jetstream electrospray source operating in positive ion mode. Two ion transitions were monitored for each analyte of interest and the internal standard. The estimated concentration range of cyclopropylfentanyl in the reported cases was 5.6 to 82 ng/mL for five postmortem cardiac blood specimens. All five cases included cyclopropylfentanyl in the established cause of death.

Screening of Novel Psychoactive Substances in Postmortem Matrices by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS-MS).

Novel psychoactive substances (NPS) are newly emerging compounds, natural and synthetic, that are often sold as "legal" alternatives to controlled substances. These substances can include phenethylamines, tryptamines, synthetic cathinones, piperazines and others. Most novel psychoactive substances and similar designer drugs are often based on the chemical backbone of classic drugs of abuse but are not regularly encountered in postmortem casework. These compounds may go undetected in forensic toxicology labs where suitable identification methods for NPS may not exist. In order to keep up with this ever-expanding list of pharmacologically active and toxicologically hazardous substances, there is an increased need for a screening panel suitable for postmortem human samples such as whole blood and urine. In order to address this increased prevalence of NPS, a method was developed and validated to identify 50 NPS in postmortem samples using liquid chromatography triple quadrupole mass spectrometry. The data presented here represents a validated liquid chromatography triple quadrupole mass spectrometry method to qualitatively identify NPS in postmortem samples. The limit of detection for all compounds was set at 2.5 ng/mL. To evaluate the prevalence of NPS in the region, blood and urine specimens from 110 postmortem cases were submitted for analysis based on initial screening results and/or case history. Of those cases, 28% were positive for several NPS, though only a few were included in the cause of death. The robustness of this method proves that it is suitable for the continual addition of newer substances, ensuring up-to-date simultaneous drug detection.

Uptake and distribution of the abused inhalant 1,1-difluoroethane in the rat.

1,1-Difluoroethane (DFE) is a halogenated hydrocarbon used as a propellant in products designed for dusting electronic equipment and air brush painting. When abused, inhaled DFE produces intoxication and loss of muscular coordination. To investigate DFE toxicokinetics, groups (n = 3) of Sprague-Dawley rats were exposed to 30 s of 20 L/min DFE. The experimental model was designed to mimic exposure during abuse, a protocol which has not been conducted. Tissue collection (blood, brain, heart, liver, and kidney) occurred at 0, 10, 20, 30, 45, 60, 120, 240, 480, and 900 s. Average peak DFE levels were blood 352, brain 519, heart 338, liver 187, and kidney 364 mg/L or mg/kg. The total percent uptake of the administered dose was 4.0%. Uptake into individual compartments was 2.72, 0.38, 0.15, 0.41, and 0.32% for blood, brain, heart, liver, and kidney, respectively. All animals showed signs of intoxication within 20 s manifested as lethargy, prostration and loss of righting reflex. Marked intoxication continued for about 4 min when DFE averaged 21 mg/L in blood and 17 mg/kg in brain. Between 4 and 8 min, animals continued to show signs of sedation as evidenced by reduced aggression and excitement during handling. No discernable intoxication was evident after 8 min and blood and brain levels had fallen to 10 and 6 mg/L or kg, respectively. Plots of concentration (log) versus time were consistent with a two compartment model. Initial distribution was rapid with average half life (t((1/2))) during the alpha phase of 9 s for blood, 18 s for brain and 27 s in cardiac tissue. During beta slope elimination average t((1/2)) was 86 s in blood, 110 s in brain and 168 s in heart. Late elimination half lives were longer with blood gamma = 240 s, brain gamma = 340 s, and heart gamma = 231 s. Following acute exposure the Vd = 0.06 L, beta = 0.48 min(-1), AUC = 409.8 mg.min L(-1), and CL from blood was 0.03 L min(-1). The calculated toxicokinetic data may underestimate these parameters if DFE is abused chronically due to continued uptake into lowly perfused tissues with repeated dosing.

A validated method for the quantitation of 1,1-difluoroethane using a gas in equilibrium method of calibration.

1,1-Difluoroethane (DFE), also known as Freon 152A, is a member of a class of compounds known as halogenated hydrocarbons. A number of these compounds have gained notoriety because of their ability to induce rapid onset of intoxication after inhalation exposure. Abuse of DFE has necessitated development of methods for its detection and quantitation in postmortem and human performance specimens. Furthermore, methodologies applicable to research studies are required as there have been limited toxicokinetic and toxicodynamic reports published on DFE. This paper describes a method for the quantitation of DFE using a gas chromatography-flame-ionization headspace technique that employs solventless standards for calibration. Two calibration curves using 0.5 mL whole blood calibrators which ranged from A: 0.225-1.350 to B: 9.0-180.0 mg/L were developed. These were evaluated for linearity (0.9992 and 0.9995), limit of detection of 0.018 mg/L, limit of quantitation of 0.099 mg/L (recovery 111.9%, CV 9.92%), and upper limit of linearity of 27,000.0 mg/L. Combined curve recovery results of a 98.0 mg/L DFE control that was prepared using an alternate technique was 102.2% with CV of 3.09%. No matrix interference was observed in DFE enriched blood, urine or brain specimens nor did analysis of variance detect any significant differences (alpha = 0.01) in the area under the curve of blood, urine or brain specimens at three identical DFE concentrations. The method is suitable for use in forensic laboratories because validation was performed on instrumentation routinely used in forensic labs and due to the ease with which the calibration range can be adjusted. Perhaps more importantly it is also useful for research oriented studies because the removal of solvent from standard preparation eliminates the possibility for solvent induced changes to the gas/liquid partitioning of DFE or chromatographic interference due to the presence of solvent in specimens.

Assessing free and total morphine following heroin overdose when complicated by the presence of toxic amitriptyline levels.

A 43-year-old female was reported to inject heroin, which led to her rapid death. Because of the potential for criminal charges, laboratory results that could verify "hotshot" heroin overdose were valuable. Initial toxicological analysis detected morphine (0.78 mg/L), amitriptyline (2.91 mg/L), and nortriptyline (2.80 mg/L) in femoral blood. Because these tricyclic antidepressant levels alone might normally be associated with a fatal outcome, the ratio of free versus total morphine (88.6%) and presence of 6-monoacetylmorphine in vitreous fluid were used support a history of rapid death following intravenous (IV) administration. The distribution of amitriptyline and nortriptyline was consistent with accumulation of drug after chronic dosing. Our other results suggest that the low morphine level in vitreous humor fluid (0.16 mg/L) relative to free morphine in femoral blood (0.78 mg/L) may also be an indicator of limited survival time following exposure to morphine. Based upon comprehensive toxicologic analysis, we determined overdose due to IV abuse of heroin was likely to have precipitated the fatal outcome. This case underscores the need for complete toxicologic workup and to consider individual variation in the dose response during toxicologic interpretation of postmortem results.

A motor vehicle accident fatality involving the inhalation of 1,1-difluoroethane.

A 24-year-old female driver with a history of substance abuse was pronounced dead following a single car motor vehicle accident. A surviving front seat passenger witnessed the decedent inhaling "Dust Off" cleaner just prior to losing control of the vehicle. The propellant compound used in this product is the halogenated hydrocarbon 1,1-difluoroethane (DFE). Sealed autopsy specimens were examined for the presence and subsequent quantitation of DFE utilizing an Agilent 6850 gas chromatograph (GC)-flame-ionization detector. The levels of DFE obtained were as follows: 29.8 mg/L in femoral blood, 40.3 mg/L in pulmonary arterial blood, 85.6 mg/L in aortic blood, 79.9 mg/L in chest cavity blood, 21.2 mg/L in vitreous, 11.7 mg/kg in brain, 27.9 mg/kg in liver, 71.0 mg/L in urine, and 51.8 mg/total gastric contents. The presence of DFE was confirmed in the decedent's urine by injection on an Agilent 6890/5973 GC-mass spectrometer in full scan mode. This case presents a uniquely witnessed observation of the apparent impairing effects and consequences of the acute inhalation of halogenated hydrocarbons such as DFE and the operation of a motor vehicle. The proximity of time of death to inhalant use may also provide insight to postmortem distribution patterns of DFE in relation to normal physiologic blood flow. With further investigations, estimating the time of final use of an inhalant prior to death may be deciphered from such patterns, although a degree of caution should be applied in deaths resulting from severe trauma in which normal tissue structure is compromised because postmortem redistribution may result.

Fatal cardiac arrhythmia after repeated exposure to 1,1-difluoroethane (DFE).

A 42-year-old man was found dead after repeated exposure to 1,1-difluoroethane (DFE, Freon 152a), a propellant found in CRC Duster, a product intended for the removal of dust and lint. Toxicologic analysis detected DFE in femoral blood 136.3 mg/L, brain 117.5 mg/kg, liver 87.6 mg/kg, lung 60.3 mg/kg, adipose 235.7 mg/kg, and vitreous fluid 25.1 mg/L. The cause of death was determined to be a fatal cardiac arrhythmia due to intoxication with 1,1-difluoroethane. After comparison to previously published cases involving DFE, we suggest that analysis of adipose tissue for DFE and similar compounds, along with blood and other tissues, may be useful in distinguishing between acute versus chronic exposure. Adipose may also be a valuable alternate specimen for detection in cases where loss or elimination from blood is likely to have occurred.

Percutaneous absorption and distribution of methanol in a homicide.

A woman was alleged to have committed suicide by consuming a gasoline additive shortly before jumping from a second floor balcony within her home. She was found dead by police with a multitude of injuries, lying nude in a partially evaporated unknown residue that was later determined to be methanol. Samples collected at autopsy were found to contain methanol in the following concentrations: femoral blood 31.2 mg/dL, pulmonary artery blood 111.0 mg/dL, aortic blood 77.8 mg/dL, vitreous fluid 196.4 mg/dL, brain 22.0 mg/100 g, liver 21.2 mg/100 g, and kidney 25.9 mg/100 g using a headspace gas chromatographic method. Significantly, no methanol was detected in samples recovered from the esophagus, stomach, duodenum, small intestine, bile, or urine. These findings are inconsistent with either recent or delayed oral ingestion of methanol. We concluded that absorption of methanol occurred dermally and through the oral mucosa as she lay dying and saturated in the fuel additive. Based upon the toxicological data and a comprehensive forensic investigation (including documentation and analysis of evidence recovered at the scene and the autopsy), the cause of death was determined to be blunt impact trauma and methanol poisoning.

Fatality due to methyl acetylene-propadiene (MAPP) inhalation.

A 33-year-old man died after intentionally inhaling a gaseous mix of methyl acetylene (propyne) and propadiene (allene) commonly known as MAPP, which is used for soldering and welding. He was found with a plastic bag securely placed over his head and a cylinder of MAPP alongside his head. The cylinder had been vented into the bag using a flexible hose. A comprehensive toxicological analysis revealed only a trace of diphenhydramine in the liver and 0.02 mg/L of morphine in the urine. Analysis of blood by headspace gas chromatography (HS-GC) detected two unknown peaks. These were determined to be the components of MAPP gas. MAPP was quantitated in femoral blood (59.6 mg/L) and brain (43.6 mg/kg) using a HS-GC method. The cause of death was attributed to acute MAPP intoxication, and the manner was determined to be suicide. A discussion on the analytical and interpretive considerations commonly encountered when analyzing volatile compounds is also presented.

Two cases involving clomipramine intoxication.

Clomipramine and its active metabolite norclomipramine were identified and quantitated in multiple tissues recovered from two postmortem cases using liquid chromatography-mass spectrometry. In both cases clomipramine toxicity was assessed primarily upon levels determined from brain samples. This communication supplements the database on clomipramine and norclomipramine by providing quantitative determinations of both parent drug and metabolite in multiple tissues. A literature search revealed a paucity of data on clomipramine and norclomipramine levels in general and a total absence of documented brain levels. In patients who have undergone long-term tricyclic antidepressant (TCA) therapy, blood and liver analysis alone may not be sufficient to establish toxicity. Such patients can sequester substantial amounts in liver, a concern because the TCAs are subject to significant postmortem redistribution. When conducting postmortem investigations, the inclusion of brain determination provides valuable information in assessing the magnitude of toxicity in cases involving clomipramine and its active metabolite norclomipramine.

Sudden death caused by 1,1-difluoroethane inhalation.

A 20-year-old man was found dead on the floor next to a computer, with a nearly full can of "CRC Duster" dust remover located next to the deceased on the floor, and an empty can of the same product on the computer desk. Toxicologic evaluation using either gas chromatography/mass spectrometry (GC/MS) or gas chromatography/flame ionization detector (GC/FID) method identified the active ingredient 1,1-difluoroethane (Freon 152a) in all tissues analyzed. Tissue distribution studies revealed highest concentration in central blood, lung, and liver. It is believed that the 1,1-difluoroethane inhalation was the cause of death.

Fatal olanzapine-induced hyperglycemic ketoacidosis.

Olanzapine is an antipsychotic medication linked to the development, or exacerbation of, type 2 diabetes mellitus. This report describes 3 patients being treated with olanzapine who died suddenly and unexpectedly with hyperglycemic ketoacidosis. All had olanzapine concentrations within the therapeutic range. Vitreous glucose concentrations ranged from 640 mg/dL to 833 mg/dL, and blood acetone concentrations from 25.6 mg/dL to 57.6 mg/dL. Beta-hydroxybutyrate concentrations in blood were from 55.2 mg/dL to 110 mg/dL. Low levels of isopropanol were also detected. None had a history or family history of diabetes mellitus. Glycolated (A1C) hemoglobin in 2 cases was 14.3% and 14.7%. No predisposing factors to olanzapine-induced diabetes were identified. It is recommended that chemical testing of patients dying suddenly while being treated with antipsychotic drugs include vitreous glucose and blood acetone determinations to elucidate the cause and mechanism of death in these patients. Warnings concerning this potentially fatal complication of olanzapine therapy should be included in standard pharmaceutical and prescription references.