Quantification of Flualprazolam in Blood by LC-MS-MS: A Case Series of Nine Deaths.

The emergence of novel designer benzodiazepines continues to be a public health concern. Flualprazolam is one of these drugs. It was initially identified in 2017. User forums suggest it is slightly more potent than alprazolam and has longer-lasting central nervous system depressant effects. Here we report a simple, sensitive liquid chromatography-tandem mass spectrometry method for flualprazolam and report a series of nine cases in which flualprazolam was quantified. As is typical of forensic toxicology in the twenty-first century, all the cases had more than one drug present. None of the deaths could be directly attributed to flualprazolam alone, but all were likely due to a combination of sedative drugs. However, this paper still adds to the data available to allow interpretation of postmortem flualprazolam concentrations.

Femoral blood concentrations of the designer benzodiazepine etizolam in post-mortem cases.

Etizolam is a thienodiazepine that although licensed for clinical usage in Japan, India and South Korea is commonly abused and detected in post-mortem cases around the world. To date, there are limited data in the literature to allow for the interpretation of blood concentrations of etizolam in post-mortem cases. A liquid chromatography with tandem mass spectrometry method was used to quantitate etizolam concentrations in 28 post-mortem cases where etizolam was detected. The median concentration of etizolam in femoral blood was 8.5 ng/mL (range 1.0-172.0 ng/mL; n = 24); in antemortem plasma, the etizolam concentration range was 4-44 ng/mL (n = 4). The mean age of the individuals abusing etizolam was 38.5 ± 8.4 years (median 39 years), with the majority being male (86%). In all of the cases, multiple drugs were detected, with the most common being pregabalin (61%) followed by morphine/heroin (54%), diazepam (54%) and benzoylecgonine (21%), illustrating the increasing problem of poly-substance use in drug abusers. The cause of death in the cases in which etizolam was detected was multi-drug toxicity in 87.5% of the cases, with 12.5% unrelated to drug use (hangings and blunt-force trauma). These data will further help forensic practitioners with the interpretation of post-mortem etizolam concentrations.

Post-mortem diagnosis of kidney impairment: An experimental study.

The determination of the role that drugs may have played in a death is an important part of the investigation into unexplained deaths. Renal impairment may lead to a reduction in drug excretion rate and therefore an accumulation of drugs or metabolites, leading to possible toxic or lethal effects. Creatinine levels are known to be stable in the post mortem period and in life can give an indication of kidney function. There are however widely reported limitations when using creatinine in isolation and so we investigated the usefulness of using estimated glomerular filtration rate (eGFR) for scoring an individual as having renal impairment using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. We analysed unpreserved vitreous for creatinine in 812 individuals using an isotope dilution mass spectrometry (ID-MS) traceable enzymatic. We found that the biochemical analysis of post mortem vitreous creatinine and subsequent calculation of eGFR is a useful adjunct to the standard testing that takes place during a post-mortem examination and can assist in death investigation. Using an eGFR of <60 mL/min/1.73 m2 gave a sensitivity of 94.3% and specificity of 97.3% when scoring an individual as having renal impairment. We therefore recommend the calculation of eGFR for the determination of possible renal impairment in post mortem investigations. It is, of course, always pertinent to interpret any results using a wealth of case information. Extreme caution should be exercised in cases where insufficient clinical information/history is available, particularly in cases in which there is suspected diabetic ketoacidosis, dehydration or hospitalisation prior to death.

Primary hyperparathyroidism: findings from the retrospective evaluation of cases over a 6-year period from a regional UK centre.

BACKGROUND: Although there are international guidelines on diagnosis and management of primary hyperparathyroidism (PHPT), clinical practice varies in different centres. Periodic review of diagnostic work-up, surgical treatment by parathyroidectomy (PTX) and clinical surveillance in nonsurgical treatment group among patients with PHPT is expected to improve the quality of care. We report a retrospective study of cases with PHPT managed at a regional centre in the United Kingdom. METHODS: Clinical data of cases with calcium ≥2.6 mmol/L and parathyroid hormone (PTH) ≥9.0 pmol/L was procured from biochemistry database from January 2011 to December 2016. Laboratory parameters, imaging studies for renal stones, osteoporosis and localisation of parathyroid adenomas, type of treatment received (PTX or nonsurgical), complications of treatment, other medical co-morbidities and mortality during follow-up was recorded in each case to examine the outcomes of care of patients with PHPT. RESULTS: The study included 160 patients: 127 (79%) females and 33 (21%) males. Median age was 70 years in females and 74 in males. Thirty cases (19% of 159) had renal stones and 47 (37.3% of 126) had osteoporosis. Eighty-one cases (51%) received PTX. Logistic regression analysis showed that higher calcium levels (odds ratio (OR) = 73.991; p < 0.001), peak PTH (OR = 1.023; p = 0.025), peak alkaline phosphatase (OR = 0.985, p < 0.001), lower age (OR = 0.985, p < 0.001) and male gender (OR = 0.209, p < 0.002) as statistically significant predictors for patients receiving PTX. Higher age at diagnosis of PHPT was associated with increased risk of co-existent hypertension (OR = 10.904, p = 0.001) and fractures (OR = 1.067, p = 0.004). Higher peak calcium concentration was an independent predictor of acute kidney injury (OR = 9.631, p = 0.011). PTX cured 76 cases (94%) with only 7 (9%) postoperative complications. Twenty-four cases (15%) died from the entire cohort (only one from PTX group) during a median follow-up period of 3.6 years (interquartile range = 1.5). CONCLUSIONS: PTX treatment is associated with cure of disease in patients with PHPT with acceptable risk of complications. Improvements in diagnostic work-up and follow-up care should improve the morbidity from PHPT.

Multiple fatalities in the North of England associated with synthetic fentanyl analogue exposure: Detection and quantitation a case series from early 2017.

BACKGROUND: Synthetic fentanyl analogues are highly potent opioid drugs which have no pharmaceutical use in humans. We detected the synthetic fentanyl analogues; carfentanil, butyryl fentanyl, fluorobutyrylfentanyl, furanylfentanyl, and alfentanil as well as fentanyl itself in 25 cases in early 2017. There have been no previous reports of synthetic fentanyl deaths in the United Kingdom (UK). METHODS: Cases in which the history clearly stated drug use but where a post mortem blood morphine concentration was lower than would be expected to explain the sudden death, were referred for further analysis by high resolution accurate mass (HRAM) mass spectrometry. RESULTS: 25 post mortem cases in which synthetic fentanyl analogues were implicated in the cause of death were reported from January to May 2017. No cases were seen in June 2017. The age range was 21-54 years and 22 were male. There was a history of heroin use, or markers of heroin use on toxicology screening in 21/25 cases. Carfentanil and fentanyl were detected in 7 cases. Multiple synthetic fentanyl analogues were present in 13 cases, with the remaining 5 cases having only carfentanil present. Synthetic fentanyl analogues were detected in combination with other drugs in all cases. Significant concentrations of ethanol were detected in only 2 cases. The concentration range of carfentanil in blood was 90-4004pg/mL. Of note, the 3 cases in which ante mortem carfentanil was quantified ranged from 21 to 98pg/mL. In all cases, death was attributed to combined central nervous system depression. CONCLUSIONS: This paper highlights a new and rapid emergence of these drugs into the UK illicit drug arena. Synthetic fentanyl analogues represent a significant challenge both analytically and clinically within the groups who misuse drugs. It is worthwhile considering the possibility of the presence of these drugs in cases in which a toxicological cause of death is not apparent analytically but there is a history of drug use and circumstantial evidence exists to support a drug-related death as the most likely cause. It may be that synthetic fentanyl analogues should be screened for routinely to avoid reporting any false negative results, but the cost implications and viability of this have not been fully evaluated.

Death from Ingestion of E-Liquid.

BACKGROUND: The use of e-liquids is becoming more prevalent. There is a risk that such liquids may be ingested by mouth rather than being vaped/ inhaled. Due to the high concentration of drugs such as nicotine in these liquids, there may be toxic, and possibly fatal consequences. CASE REPORT: We report the death of a 32-year-old male who ingested nicotine-containing e-liquid while under the influence of alcohol. A serum sample taken 24 h after collapse contained nicotine at a concentration of 1600 ng/mL of nicotine. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Death secondary to e-liquid ingestion is still very rare, but has the potential for causing deaths due to the easy access of such liquids to the general public. Such toxicity should be considered in individuals who present in the early phases with symptoms of stimulant toxicity, but also in the latter phase where there may be autonomic depressive effects.

Biochemical Screening for Nonadherence Is Associated With Blood Pressure Reduction and Improvement in Adherence.

We hypothesized that screening for nonadherence to antihypertensive treatment using liquid chromatography-tandem mass spectrometry-based biochemical analysis of urine/serum has therapeutic applications in nonadherent hypertensive patients. A retrospective analysis of hypertensive patients attending specialist tertiary care centers was conducted in 2 European countries (United Kingdom and Czech Republic). Nonadherence to antihypertensive treatment was diagnosed using biochemical analysis of urine (United Kingdom) or serum (Czech Republic). These results were subsequently discussed with each patient, and data on follow-up clinic blood pressure (BP) measurements were collected from clinical files. Of 238 UK patients who underwent biochemical urine analysis, 73 were nonadherent to antihypertensive treatment. Their initial urinary adherence ratio (the ratio of detected to prescribed antihypertensive medications) increased from 0.33 (0-0.67) to 1 (0.67-1) between the first and the last clinic appointments. The observed increase in the urinary adherence ratio in initially nonadherent UK patients was associated with the improved BP control; by the last clinic appointment, systolic and diastolic BPs were ≈19.5 and 7.5 mm Hg lower than at baseline (P=0.001 and 0.009, respectively). These findings were further corroborated in 93 nonadherent hypertensive patients from Czech Republic-their average systolic and diastolic BPs dropped by ≈32.6 and 17.4 mm Hg, respectively (P<0.001), on appointments after the biochemical analysis. Our data show that nonadherent hypertensive patients respond to liquid chromatography-tandem mass spectrometry-based biochemical analysis with improved adherence and significant BP drop. Such repeated biochemical analyses should be considered as a therapeutic approach in nonadherent hypertensive patients.

Risk Factors for Nonadherence to Antihypertensive Treatment.

Nonadherence to antihypertensive treatment is a critical contributor to suboptimal blood pressure control. There are limited and heterogeneous data on the risk factors for nonadherence because few studies used objective-direct diagnostic methods. We used high-performance liquid chromatography-tandem mass spectrometry of urine and serum to detect nonadherence and explored its association with the main demographic- and therapy-related factors in 1348 patients with hypertension from 2 European countries. The rates of nonadherence to antihypertensive treatment were 41.6% and 31.5% in the UK and Czech populations, respectively. Nonadherence was inversely related to age and male sex. Each increase in the number of antihypertensive medications led to 85% and 77% increase in nonadherence (P<0.001) in the UK and Czech populations, respectively. The odds of nonadherence to diuretics were the highest among 5 classes of antihypertensive medications (P≤0.005 in both populations). The predictive model for nonadherence, including age, sex, diuretics, and the number of prescribed antihypertensives, showed area under the curves of 0.758 and 0.710 in the UK and Czech populations, respectively. The area under the curves for the UK model tested on the Czech data and for the Czech model tested on UK data were calculated at 0.708 and 0.756, respectively. We demonstrate that the number and class of prescribed antihypertensives are modifiable risk factors for biochemically confirmed nonadherence to blood pressure-lowering therapy. Further development of discriminatory models incorporating these parameters might prove clinically useful in assessment of nonadherence in countries where biochemical analysis is unavailable.

Estimation of postmortem interval using vitreous potassium levels in cases of fatal road traffic collision

AIM OF THE STUDY: To produce a formula that can accurately predict postmortem interval (PMI) based on vitreous potassium levels using road traffic collision fatalities. MATERIAL AND METHODS: Vitreous humour samples were taken from 78 individuals who had died following road traffic collisions between 2010 and 2015. Samples were obtained from both eyes and were sent for on-site analysis. Measurement of potassium was by an indirect ion-specific electrode Siemens diagnostics ADVIA 2400 chemistry system. Exact time of death was known from police reports, the time of postmortem was recorded and the postmortem interval was calculated. Linear regression was then used to analyse the relationship between the two. The impact of age was also assessed. RESULTS: PMI was between 6 and 162 hours. As vitreous potassium increases, the PMI also increases; exhibiting a linear relationship. This is illustrated by a regression equation of PMI = 6.42[K+] - 40.94, R = 0.67 (p < 0.001). This produced a formula closely comparable with three other studies proposed in previous literature and produces estimates that may exceed one calendar day. When both age and medical intervention are accounted for there is an insignificant improvement in prediction. CONCLUSIONS: Validated methods have been used to produce a formula for prediction of PMI using vitreous potassium. Although this is specific to road traffic collisions, the methods are transferable and can be seen to be comparable with other recently published methods. Nonetheless, if greater levels of accuracy are required it is suggested that biomarkers delivering a higher level of precision should still be sought.

Enumeration of small and large numerosities: the effect of element visibility.

Precise enumeration is associated with small numerosities within the subitizing range (<4 items), while approximate enumeration is associated with large numerosities (>4 items). To date, there is still debate on whether a single continuous process or dual mutually exclusive processes mediate enumeration of small and large numerosities. Here, we evaluated a compromise between these two notions: that the precise representation of number is limited to small numerosities, but that the approximate representation of numerosity spans across both small and large numerosities. We assessed the independence of precise and approximate enumeration by looking at how luminance contrast affected enumeration of elements that differ by ones (1-8) or by tens (10-80). We found that enumeration functions of ones and tens have different characteristics, which is consistent with the presence of two number systems. Subitizing was preserved for small numerosities. However, simply decreasing element visibility changed the variability signatures of small numerosities to match those of large numerosities. Together, our results suggest that small numerosities are mediated by both precise and approximate representations of numerosity.

The effect of viewing eccentricity on enumeration.

Visual acuity and contrast sensitivity progressively diminish with increasing viewing eccentricity. Here we evaluated how visual enumeration is affected by visual eccentricity, and whether subitizing capacity, the accurate enumeration of a small number (∼3) of items, decreases with more eccentric viewing. Participants enumerated gratings whose (1) stimulus size was constant across eccentricity, and (2) whose stimulus size scaled by a cortical magnification factor across eccentricity. While we found that enumeration accuracy and precision decreased with increasing eccentricity, cortical magnification scaling of size neutralized the deleterious effects of increasing eccentricity. We found that size scaling did not affect subitizing capacities, which were nearly constant across all eccentricities. We also found that size scaling modulated the variation coefficients, a normalized metric of enumeration precision, defined as the standard deviation divided by the mean response. Our results show that the inaccuracy and imprecision associated with increasing viewing eccentricity is due to limitations in spatial resolution. Moreover, our results also support the notion that the precise number system is restricted to small numerosities (represented by the subitizing limit), while the approximate number system extends across both small and large numerosities (indexed by variation coefficients) at large eccentricities.

Loss of reelin expression in breast cancer is epigenetically controlled and associated with poor prognosis.

Reelin is a secreted, signaling protein associated with neuronal cell positioning and migration. Recently, reelin was found to be epigenetically silenced in gastric and pancreatic cancers in which down-regulation was associated with increased migratory ability and reduced survival. Here we analyzed reelin expression by immunohistochemistry in 17 normal breast tissue samples from reduction mammoplasties and in two independent tissue microarrays of 136 and more than 2000 breast cancer biopsy samples, respectively. Results were analyzed with regard to clinical parameters, including BRE (Bloom, Richardson, Elston) grade, nodal status, estrogen receptor and HER2 status, and overall survival. Reelin was expressed in the luminal epithelium and myoepithelium of the normal human breast but not in cancerous breasts. Loss of reelin protein expression correlated significantly with decreased survival (P=0.01) and positive lymph node status (P<0.001). By measuring reelin expression and promoter methylation status in 39 primary breast tumors, as well as in breast cancer-derived cell lines before and after decitabine treatment, we established that reelin expression levels correlated inversely with promoter methylation status, whereas demethylation increased reelin mRNA expression in vitro. Reelin overexpression in MDA-MB231 cells, as well as incubation with recombinant reelin, suppressed cell migration, invadopodia formation, and invasiveness in vitro. We conclude that reelin may play an important role in controlling invasiveness and metastatic potential of breast cancer cells and that its expression is controlled by promoter methylation.

An anatomic and autologous lateral ankle stabilization.

A new technique for stabilization of the lateral ankle ligaments is presented. The procedure uses a split peroneus longus tendon to recreate the calcaneofibular and anterior talofibular ligaments. The new ligaments follow a precise anatomic course that replicates the pathway of the original ligaments. The procedure also capitalizes on interference screw technology so that accurate ligament tension can be obtained. This technique is most useful for severe ligamentous insufficiency involving both the calcaneofibular and anterior talofibular ligaments. Biomechanical rationale for the use of peroneus longus is also discussed.

Epigenetic silencing of argininosuccinate synthetase confers resistance to platinum-induced cell death but collateral sensitivity to arginine auxotrophy in ovarian cancer.

Evidence indicates that acquired resistance of cancers to chemotherapeutic agents can occur via epigenetic mechanisms. Down-regulation of expression of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in the biosynthesis of arginine, has been associated with the development of platinum resistance in ovarian cancer treated with platinum-based chemotherapy. The aim of the present study was to analyse epigenetic regulation of ASS1 in ovarian cancer tissue taken at diagnosis and relapse and determine its significance as a predictor of clinical outcome in patients treated with platinum-based chemotherapy. In addition, expression and epigenetic regulation of ASS1 were analysed in human ovarian cancer cell lines, and ASS1 expression correlated with the ability of the lines to grow in media containing cisplatin, carboplatin or taxol or in arginine-depleted media. Our results show that aberrant methylation in the ASS1 promoter correlated with transcriptional silencing in ovarian cancer cell lines. ASS1 silencing conferred selective resistance to platinum-based drugs and conferred arginine auxotrophy and sensitivity to arginine deprivation. In ovarian cancer, ASS1 methylation at diagnosis was associated with significantly reduced overall survival (p = 0.01) and relapse-free survival (p = 0.01). In patients who relapse, ASS1 methylation was significantly more frequent at relapse (p = 0.008). These data establish epigenetic inactivation of ASS1 as a determinant of response to platinum chemotherapy and imply that transcriptional silencing of ASS1 contributes to treatment failure and clinical relapse in ovarian cancer. The collateral sensitivity of cells lacking endogenous ASS1 to arginine depletion suggests novel therapeutic strategies for the management of relapsed ovarian cancer.

On the importance of the electrostatic environment for the transport properties of freestanding multiwall carbon nanotubes.

Electrical measurements of freestanding multiwall carbon nanotubes using high resistance tunnelling contacts reveal a power law behaviour, I alpha V alpha + 1, with alpha as high as 5.2, followed by a transition to an offset ohmic behaviour. The freestanding electrode geometry allows for a distinction between the predictions from Luttinger liquid and environmental quantum fluctuation (EQF) theories to be made. The high values of exponents found are explained within the EQF formulism, where reflections resulting from the impedance discontinuity caused by the freestanding geometry are included.

Slow recrystallization of tripalmitoylglycerol from MCT oil observed by 2H NMR.

The crystallization and recrystallization of fats have a significant impact on the properties and quality of many food products. While crystallization has been the subject of a number of studies using pure triacylglycerols (TAG), recrystallization in similarly pure systems is rarely studied. In this work, perdeuterated tripalmitoylglycerol ( (2)H-PPP) was dissolved in medium chain triacylglycerol oil (MCT) to yield a saturated solution. The solution was heated to cause partial melting of the solid and dissolution of the molten fraction of (2)H-PPP in MCT and was then cooled to the original temperature to induce recrystallization from the supersaturated solution. (2)H NMR was used to monitor the disappearance of (2)H-PPP from the solution and showed that recrystallization occurred in two steps. The first step was rapid, taking place over a few minutes, and accounted for more than two-thirds of the total recrystallization. The second step was much slower, taking place over a remarkably long timescale of hours to days. It is proposed that dissolution occurs from all parts of the crystals, leaving an etched and pitted surface. The first step of crystallization is the infilling of these pits, while the second step is the continued growth on the smoothed crystal faces.

Updated Epstein-Barr virus (EBV) DNA sequence and analysis of a promoter for the BART (CST, BARF0) RNAs of EBV.

Two sequences required for activity of the Epstein-Barr virus BART RNA promoter in transfection assays have been identified by site-directed mutagenesis. One contains a consensus AP-1 site; the other has some similarity to Ets and Stat consensus binding sites. Candidate sequences were suggested by mapping a region of unmethylated DNA in EBV around the BART promoter followed by in vivo footprinting the promoter in the C666-1 nasopharyngeal carcinoma cell line, which expresses BART RNAs. The data are presented in the context of a revised EBV DNA sequence, known as EBV wt, that is proposed as a future standard sequence for EBV.