Development and validation of a method for quantification of human insulin and its synthetic analogues in plasma and post-mortem sera by LC-MS/HRMS.

Analysis of human insulin and its synthetic analogues is increasingly requested for clinical monitoring, for anti-doping purposes, but also for forensic cases. Indeed, insulin analogues may be abused for suicide or homicide - whence their forensic interest. Collection and storage conditions, as well as the phenomenon of degradation make post-mortem serum samples analytically challenging and consequently, the rate of exogenous insulin administration as cause of death is undoubtedly underestimated. However, with recent technological advances and the development of new extraction techniques particularly for anti-doping analyses, detection of insulins in post-mortem samples seems to be achievable. This study describes the first validated quantitative method for analysis human insulin and its six analogues (lispro, aspart, glulisine, glargine, detemir and degludec) in plasma and post-mortem sera. Various extraction processes, namely precipitation + solid phase extraction (SPE), filtration + SPE, precipitation + SPE + immunopurification, and filtration + immunopurification, were assessed to evaluate the lowest limit of detection for all target analogues. The selected sample preparation consists of filtration step followed by immunopurification extraction with an anti-body precoated ELISA plate for plasma. For post-mortem sera, the first step of precipitation was added to remove matrix interferences. The extracts were analyzed by ultra-high-performance liquid chromatography-high resolution mass spectrometry (LC-HRMS), interfaced by electrospray (ESI). The method was validated with respect linearity, precision, accuracy, recovery, matrix effect, dilution and carryover. The limit of quantification (LOQ) in plasma was 0.5 ng/mL for human insulin and rapid-acting insulins, 1.0 ng/mL for glargine, 2.5 ng/mL for degludec and 10 ng/mL for detemir. Two types of post-mortem sera were studied based on the post-mortem interval (PMI): inferior or superior to 48 h. The obtained LOQ were the same for each analogue, independent from the PMI: 1.0 ng/mL for human insulin and rapid-acting insulins, 1.0 ng/mL for glargine, 2.5 ng/mL for degludec and 10 ng/mL for detemir. At the LOQ level, for all insulins and all samples, accuracy was between 70 and 130% and precision inferior to 30%. The validated method was applied to five subjects participating in therapeutic monitoring of insulin and to seven post-mortem cases.

Hubless keypoint-based 3D deformable groupwise registration.

We present a novel algorithm for Fast Registration Of image Groups (FROG), applied to large 3D image groups. Our approach extracts 3D SURF keypoints from images, computes matched pairs of keypoints and registers the group by minimizing pair distances in a hubless way i.e. without computing any central mean image. Using keypoints significantly reduces the problem complexity compared to voxel-based approaches, and enables us to provide an in-core global optimization, similar to the Bundle Adjustment for 3D reconstruction. As we aim to register images of different patients, the matching step yields many outliers. Then we propose a new EM-weighting algorithm which efficiently discards outliers. Global optimization is carried out with a fast gradient descent algorithm. This allows our approach to robustly register large datasets. The result is a set of diffeomorphic half transforms which link the volumes together and can be subsequently exploited for computational anatomy and landmark detection. We show experimental results on whole-body CT scans, with groups of up to 103 volumes. On a benchmark based on anatomical landmarks, our algorithm compares favorably with the star-groupwise voxel-based ANTs and NiftyReg approaches while being much faster. We also discuss the limitations of our approach for lower resolution images such as brain MRI.

Activational action of testosterone on androgen receptors protects males preventing temporomandibular joint pain.

BACKGROUND: Testosterone protects male rats from Temporomandibular Joint (TMJ) pain. This study investigated whether this protective effect is mediated by an organizational action of testosterone during nervous system development, by central estrogen and androgen receptors and by the 5α-reduced metabolite of testosterone, dihydrotestosterone. METHODS: A pharmacological approach was used to assess the ability of the androgen receptor antagonist flutamide, the estrogen receptor antagonist ICI 182 780 and the 5-α reductase inhibitor dutasteride to block the protective effect of testosterone, evaluated through the behavioral response induced by a TMJ injection of 0.5% formalin. Flutamide and ICI 182 780 were injected into the medullary subarachnoid space, and dutasteride and testosterone were systemically administered. RESULTS: The TMJ injection of 0.5% formalin induced a significant nociceptive behavioral response in gonadectomized male and naïve female, but not in sham gonadectomized male rats, confirming that endogenous testosterone prevents TMJ nociception in males. Testosterone administration prevented formalin-induced TMJ nociception in males gonadectomized either in the neonatal (at the day of birth) or adult period and in naïve female rats, suggesting that the protective effect of testosterone on TMJ nociception does not depend on its organizational actions during critical periods of development. The administration of flutamide and dutasteride but not of ICI 182 780 blocked the protective effect of testosterone. CONCLUSIONS: We conclude that the protective effect of testosterone on TMJ nociception depends on activational actions of dihydrotestosterone on androgen receptors rather than on organizational androgenic actions during central nervous system development or estrogenic actions.

State of the art in bile analysis in forensic toxicology.

In forensic toxicology, alternative matrices to blood are useful in case of limited, unavailable or unusable blood sample, suspected postmortem redistribution or long drug intake-to-sampling interval. The present article provides an update on the state of knowledge for the use of bile in forensic toxicology, through a review of the Medline literature from 1970 to May 2015. Bile physiology and technical aspects of analysis (sampling, storage, sample preparation and analytical methods) are reported, to highlight specificities and consequences from an analytical and interpretative point of view. A table summarizes cause of death and quantification in bile and blood of 133 compounds from more than 200 case reports, providing a useful tool for forensic physicians and toxicologists involved in interpreting bile analysis. Qualitative and quantitative interpretation is discussed. As bile/blood concentration ratios are high for numerous molecules or metabolites, bile is a matrix of choice for screening when blood concentrations are low or non-detectable: e.g., cases of weak exposure or long intake-to-death interval. Quantitative applications have been little investigated, but small molecules with low bile/blood concentration ratios seem to be good candidates for quantitative bile-based interpretation. Further experimental data on the mechanism and properties of biliary extraction of xenobiotics of forensic interest are required to improve quantitative interpretation.

Using bone marrow matrix to analyze meprobamate for forensic toxicological purposes.

Bone marrow (BM) analysis is of forensic interest for postmortem toxicological investigations where blood samples are unavailable or unusable. Due to the lack of studies, it remains difficult to interpret concentrations of xenobiotics measured in this matrix. Based on a statistical approach published previously to interpret meprobamate concentrations in bile and vitreous humor, we propose here a diagnostic test for interpretation of BM meprobamate concentrations from analysis of 99 sets of autopsy data. The mean age was 48 years (range 18-80 years, one unknown) for males and 50 years (range 19-80 years, one unknown) for females, with a male/female ratio at 0.768. A BM concentration threshold of 11.3 µg/g was found to be statistically equivalent to that of a blood meprobamate concentration threshold of 50 µg/ml in distinguishing overdose from therapeutic use. The intrinsic qualities of this diagnostic test were good with sensitivity of 0.82 and specificity of 0.92. Compared to previous tests published with the same objective on vitreous humor and bile, this study shows that BM is a useful alternative matrix to reveal meprobamate overdose when blood, vitreous humor, and bile are not available or unusable.

Postmortem measurement of caffeine in bone marrow: influence of sample location and correlation with blood concentration.

Bone marrow (BM) analysis is of forensic interest in postmortem toxicological investigation in case of limited, unavailable or unusable blood samples. However, it remains difficult to determine whether a drug BM concentration is therapeutic or represents overdose, due to the lack of studies on this alternative matrix. Given the variations in BM composition in the body, sample location was suggested to be a relevant factor in assessing BM concentration. The aim of the present study was to compare postmortem caffeine concentrations in various BM sample locations and secondly to consider the correlation between BM and blood concentrations. Six BM samples (right and left side: proximal and medial femur and 5th rib) and a blood sample were collected from 21 forensic autopsies. Gas chromatography coupled to tandem mass spectrometry was performed. Blood caffeine concentrations ranged from 60 to 7591ng/mL. Femoral and rib BM concentrations ranged from 51 to 6171ng/g and 66 to 7280ng/g, respectively. Blood concentrations were always higher than BM concentrations. As a good correlation was demonstrated between blood and rib BM and between blood and the average of the four femoral BM concentrations, blood caffeine concentrations could be correctly extrapolated from BM concentrations. BM caffeine concentration was found to depend on sample location. Rib BM caffeine concentrations appeared to be systematically greater than averaged femur values and concentrations were much more variable between the 4 femur BM samples than between the 2 ribs. From a practical point of view, for caffeine analysis, rib BM appeared more relevant than femoral BM, which requires multisampling to overcome the concentration variability problem.

Interpretation of drug concentrations in an alternative matrix: the case of meprobamate in bile.

Investigating toxicological causes of death may require alternative matrices when the usual ones are lacking. Whereas forensic toxicology uses bile almost only for xenobiotic screening, a diagnostic test interpreting postmortem bile concentrations of meprobamate is reported. Based on 128 sets of autopsy data, its intrinsic qualities were good, with 0.95 sensitivity and 0.93 specificity. In a French forensic population, the positive and negative predictive factors were 0.90 and 0.97, respectively. It is a useful means of revealing overdoses where blood samples are not available or of confirming blood tests when postmortem redistribution is suspected.

Toxicologic aspects of deaths due to falls from height.

A prospective study of 161 victims of falls from height is reported. The aim was to determine the interest of systematic qualitative and quantitative toxicological analysis in such fatalities. The primary cause of death was suicide (84.5%), followed by accidents (7%) and homicide (1%). In the remaining 7.5%, cause of death was undetermined. In the suicides, there was evidence of psychotropic medicines in 57% of the observations, with a much higher proportion of benzodiazepines and antidepressants in women than in men. Quantitative toxicologic analysis showed overdosing on medication in 16 suicide victims, with toxic levels in 11 of these. Systematic qualitative and quantitative toxicologic analysis made a significant contribution to the diagnosis of suicide by revealing either an unknown psychiatric treatment or a toxic level.

Homicide followed by freezing and burning: usefulness of measuring SCHAD (short-chain 3-hydroxyacyl-CoA dehydrogenase) activity.

An observation of homicide in which the victim was frozen, then burned, was reported. This confirmed the usefulness of measuring SCHAD (short-chain 3-hydroxyacyl-CoA dehydrogenase) activity in the freezing of a human corpse despite unusual circumstances.

Cardiac lesions induced by neuroleptic drugs in the rabbit.

Sudden death seems to be more frequent following treatment with neuroleptic drugs in patients with pre-existing cardiac lesions, especially dilated and hypertrophic myocardiopathy. The present study was undertaken to confirm the hypothesis that myocardial lesions can be induced by neuroleptic drugs. Eight groups of 6 New-Zealand White rabbits were treated for 3 months: group I: controls (saline); group II: 15 mg/kg/day amisulpride; group III: 0.20 mg/kg/day haloperidol; group IV: 3 mg/kg/day levomepromazine; group V: 0.30 mg/kg/day olanzapine; group VI: 1.0 mg/kg risperidone, every 15 days; group VII: levomepromazine+haloperidol, same dose levels as single treatments; group VIII: levomepromazine+risperidone, same dose levels as single treatments. The hearts were immediately weighted and fixed, and paraffin sections were prepared and examined. Ventricular hypertrophy was observed following treatment with olanzapine and was still more marked with the combinations levomepromazine+haloperidol and levomepromazine+risperidone. Amisulpride and haloperidol induced necrotic lesions and levomepromazine, endocardial fibrosis. There was a lack of severe cardiac lesions following treatment with risperidone. The observed cardiac lesions can be compared to those seen in toxic myocarditis. These findings confirm the hypothesis that some neuroleptic drugs induce myocardial lesions. Further studies are warranted to demonstrate the effects of treatments of longer duration and the influence of pre-existing cardiac lesions.

Criminal burning.

We report a study of 40 burnt bodies on which an autopsy was carried out at the Institut de Médecine Légale in Lyon (28 men/12 women, average age = 41 years, minimum age = 3 years, maximum age = 86 years). Criminal deaths (31%) represented the second cause of death after accidents (52%), and before suicide (16%). Criminal burning seemed mainly to be means of covering up homicide, whereas criminal immolation was rarer. The particular characteristics of each of these situations have been highlighted (tying or poisoning in criminal immolation). We deemed it essential to make X-rays, to look for injuries due to trauma and to carry out systematic toxicological analyses in a victim of burning.

Human pentastomiasis discovered postmortem.

The autopsy of an 18-year-old girl who had died suddenly at home revealed generalised pentastomiasis. The location of this disease in the intestines was responsible for death by hemorrhagic enterocolitis. This discovery constituted an atypical case in our daily practice of forensic medicine.

Method for determining if a corpse has been frozen: measuring the activity of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD).

Accidental deaths by exposure to the cold have figured in many publications, whereas the freezing of the corpse is not well known. The study which we have reported was motivated by an observation of homicide in which ignorance of the fact that the body had been frozen was responsible for an underestimation of the time since death. It proved that measuring the activity of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) could reveal that the body had been frozen.

The frog and the scorpion: sado-masochistic injury.

Interpreting the lesions of victims of sexual violence is one of the most difficult and most controversial areas of forensic medicine. The case we report involves the care of a victim of sado-masochism. It identifies the difficulties of both the forensic and legal management of sexual violence in an unusual context.

Arsenic speciation in human organs following fatal arsenic trioxide poisoning--a case report.

The aim of this investigation was to study the distribution of arsenic species in human organs following fatal acute intoxication by arsenic trioxide. The collected autopsy samples of most organs were ground and dried, and the total arsenic was measured by electrothermal atomic absorption spectrometry (ETAAS). The arsenic species--inorganic arsenic, in the form of arsenite [As(III)] and arsenate [As(V)], and its metabolites [monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA)]--were quantified by ETAAS after extraction with methanol/water (1:1, by volume) and separation by HPLC. The results indicate that after acute intoxication, the liver and kidneys show the highest concentrations of total arsenic and that the total concentration in blood is 7- to 350-fold less concentrated than in organs. In all organs, As(III) is the predominant species, and MMA is more concentrated than DMA. MMA and DMA are more prevalent in lipidic organs (49% of total arsenic) compared with other organs (25% of total arsenic). As(V) was found in small quantities in the liver, kidneys, and blood.

False rape: a case report.

A 16-year-old girl was admitted to the emergency department for sexual assault. The forensic examination revealed genital lesions of an age that were incompatible with her statements. She also presented extragenital lesions that resembled self-inflicted lesions. The reports of false rape allegations in the literature have all dealt with the motivations of the false victims. This case report is a reminder that an allegation of rape can be considered only on the basis of proof and not on speculation.

The perfect crime: myth or reality?

The primum movens of a forensic autopsy is to track down the crime. The perfect crime can be defined as one which will never be suspected and/or one for which the criminal will never be arrested. We have reported several cases that have been adjudicated or are being adjudicated, and we show how actual homicides could have been taken for accidental deaths, suicides, or even natural deaths.