Ferrous ascorbate as a potential biomarker for diabetic retinopathy: a vitreous humour metabolomics study.

BACKGROUND: This study aimed to explore differences in vitreous humour metabolites and metabolic pathways between patients with and without diabetic retinopathy (DR) and identify potential metabolite biomarkers. METHODS: Clinical data and vitreous fluid samples were collected from 125 patients (40 without diabetes, 85 with DR). The metabolite profiles of the vitreous fluid samples were analysed using ultra-high performance liquid chromatography, Q-Exactive mass spectrometry, and multivariate statistical analysis. A machine learning model based on Least Absolute Shrinkage and Selection Operator Regularized logistic regression was used to build a risk scoring model based on selected metabolite levels. Candidate metabolites were regressed to glycated haemoglobin levels by a logistic regression model. RESULTS: Twenty differential metabolites were identified between the DR and control groups and were significantly enriched in five Kyoto Encyclopedia of Genes and Genomes pathways (arginine biosynthesis; tricarboxylic acid cycle; alanine, aspartate, and glutamate metabolism; tyrosine metabolism; and D-glutamate metabolism). Ferrous ascorbate significantly contributes to poorer glycaemic control outcomes, offering insights into potential new pathogenic pathways in DR. CONCLUSIONS: Disorders in the metabolic pathways of arginine biosynthesis, tricarboxylic acid cycle, alanine, aspartate, glutamate metabolism, tyrosine metabolism, and D-glutamate metabolism were associated with DR. Risk scores based on vitreous fluid metabolites can be used for the diagnosis and management of DR. Ferrous ascorbate can provide insights into potential new pathogenic pathways for DR.

The use of vitreous humour as a potential source of DNA for postmortem identification in forensic science.

Vitreous humour (VH) is routinely collected for toxicological analyses. However, only a few studies have examined its possible use in genotyping. Because of its isolation from the rest of the body, later onset of postmortem changes, and availability, VH could be a potential source of DNA for postmortem identification and other molecular analyses in forensic genetics. During forensic autopsies, samples of different VH volumes (5 ml, 4 ml, 3 ml, 2 ml, 1 ml, and 0.5 ml) were taken from 66 cadavers 24-48 h after death. DNA was also isolated from the samples immediately after collection and after a specific time of storage (1, 2, and 6 months). DNA was isolated using phenol‒chloroform‒isoamyl alcohol (PCI), and the yield and purity of the obtained DNA were determined spectrophotometrically using a FastGene NanoView Photometer. The integrity of the isolated DNA molecule was determined by PCR amplification of the hTERT (113 bp) gene. The results showed that VH could be a reliable source of genetic material for forensic analysis, and the method used for DNA extraction was effective. The yield of the isolated DNA ranged from 6.20 to 609.5 ng/µl, and the purity of the samples was 1.24-2.34. The isolated DNA concentration and integrity depend on the sample volume, but the DNA purity does not. It is also shown that DNA can be extracted from VH samples that have been stored for up to 6 months at - 20 °C. Therefore, using VH can be a valuable material for DNA identification.

Methanol intoxication in the central region of Saudi Arabia: Five case studies.

Outbreaks of methanol poisoning have been described in the medical literature worldwide. However, the few outbreaks that have occurred in Saudi Arabia remain undocumented. This is especially noteworthy in light of the fact that Saudi Arabia is among the countries that explicitly prohibit the usage of alcoholic beverages and recreational drugs. Herein, we describe five cases of methanol poisoning in Saudi Arabia. The first three comprise patients admitted to the emergency room (ER) with signs of methanol toxicity, such as heart palpitations, vomiting, and blurred vision; otherwise, those patients were considered medically free. The remaining two cases were examined postmortem. A headspace gas chromatography-flame ionization detector was used to test blood, vitreous humor, and urine samples for methanol. Specific lethal concentrations of methanol were defined based on published case studies as 23-740 mg/dL in blood and 12-396 mg/dL in vitreous humor. In postmortem cases of our present study, samples exhibited lethal concentrations: 118 and 257 mg/dL in blood, 116.3 and 283 mg/dL in vitreous humor. In ER cases, methanol concentrations in urine samples were lower, at 7.5, 9.1, and 20.9 mg/dL; however, toxic symptoms were still observed. These case studies indicate that it is necessary to raise community awareness about the risk of methanol poisoning in order to minimize future poisoning epidemics.

PMI estimation through metabolomics and potassium analysis on animal vitreous humour.

INTRODUCTION: The estimation of post-mortem interval (PMI) remains a major challenge in forensic science. Most of the proposed approaches lack the reliability required to meet the rigorous forensic standards. OBJECTIVES: We applied 1H NMR metabolomics to estimate PMI on ovine vitreous humour comparing the results with the actual scientific gold standard, namely vitreous potassium concentrations. METHODS: Vitreous humour samples were collected in a time frame ranging from 6 to 86 h after death. Experiments were performed by using 1H NMR metabolomics and ion capillary analysis. Data were submitted to multivariate statistical data analysis. RESULTS: A multivariate calibration model was built to estimate PMI based on 47 vitreous humour samples. The model was validated with an independent test set of 24 samples, obtaining a prediction error on the entire range of 6.9 h for PMI < 24 h, 7.4 h for PMI between 24 and 48 h, and 10.3 h for PMI > 48 h. Time-related modifications of the 1H NMR vitreous metabolomic profile could predict PMI better than potassium up to 48 h after death, whilst a combination of the two is better than the single approach for higher PMI estimation. CONCLUSION: The present study, although in a proof-of-concept animal model, shows that vitreous metabolomics can be a powerful tool to predict PMI providing a more accurate estimation compared to the widely studied approach based on vitreous potassium concentrations.

Preparation of endothelial keratoplasty lamellae from donated whole eyes post vitreous humour aspiration at the central eye bank of Iran.

To compare the endothelial parameters and thickness profiles of endothelial keratoplasty (EK) lamellae from donated whole eyes post vitreous humour aspiration (VHA) with those prepared from their mate control eyes (without VHA). Between March 2019 and March 2020, a few steps were added when aspirating the vitreous humour and also before dissecting the corneal tissue with microkeratome. EK lamellae were prepared from whole eyes that underwent VHA and their corresponding endothelial and thickness profiles were compared with those prepared from their respective fellow control eyes. Post-operative data in terms of graft attachment and clarity and the rate of reoperation were also reviewed. 115 eyes that underwent VHA and 115 of their respective fellow eyes were enrolled. No significant difference was noted in the endothelial parameters between the two groups. Mean central thickness of the EK lamellae and increase of thickness towards the periphery were not significantly different between the groups. Both groups did not show a significant difference with respect to the anticipated dissection depth, post-operative graft clarity, graft attachment, and the rate of regraft. This study demonstrates that aspirating vitreous humour has no adverse effect on the endothelial and thickness profiles of the EK lamellae that are prepared from these donated whole eyes, once the specific steps outlined by the eye bank are adhered to when aspirating the vitreous humor before performing the microkeratome dissection. The success rate of the grafted lamellae was comparable between the study groups.

The biochemistry of the vitreous humour in estimating the post-mortem interval-a review of the literature, and use in forensic practice in Galicia (northwestern Spain).

The K+ and hypoxanthine (Hx) concentrations of the vitreous humour (VH) rise gradually after death, providing a means of estimating the post-mortem interval (PMI). The correlation between these analytes and the PMI is good since the vitreous chamber is partially isolated from autolytic events occurring elsewhere; the [K +] and [Hx] recorded is thus the result of changes within the eye. The present work provides a systematic review, following PRISMA recommendations, of 36 articles (3 reviews and 33 retrospective cohort studies) discussing the many procedures and regression models that have been developed for improving PMI estimates involving VH analytes. The results of a descriptive study are also provided, highlighting the causes and distribution of mortality as registered in medico-legal autopsies performed in 2019 in Galicia (northwestern Spain), and revealing the use of these PMI estimation methods in real forensic practice. Great heterogeneity was detected in the collection of VH samples, the treatments to which they were subjected before examination, and in their conservation and analysis. A lack of reproducibility in the analytical methods employed to estimate [K +] and [Hx] was noted, as well as an absence of external validation for most of the regression formulae used to determine the PMI from analyte values. The use of methods based on high-performance liquid chromatography, focal electrophoresis, or thermogravimetric/chemometric procedures might solve the problems encountered with traditional analytical techniques, offering reliable results more quickly and effectively (even when samples are contaminated). This study recommends using flexible multiple regression models that combine physical and chemical variables, and that population databases be constructed so that models can be properly validated.

Metabolic characterization of human intraocular fluid in patients with pathological myopia.

Pathological myopia (PM) and its associated complications can lead to permanent vision loss. However, the cellular mechanisms underlying PM development remain unclear. To identify the metabolic alterations that may contribute to the pathophysiology of PM, we performed non-targeted metabolomics analysis using ultra-high-performance liquid chromatography with tandem mass spectrometry in age- and sex-matched patients with PM (n = 30) and individuals without myopia as controls (n = 30). Targeted metabolomics and insulin microarray were used to validate the results. We identified 508 metabolites in the aqueous humour (AH) and 601 in the vitreous humour (VH). Statistical evaluation revealed that 104 metabolites in AH and 114 metabolites in VH were significantly different between the two groups (variable important for the projection >1, fold change >1.5, or < 0.667, and P < 0.05). The four metabolic pathways enriched in both AH and VH identified to be associated with PM were: bile secretion, insulin secretion, thyroid hormone synthesis, and cGMP-PKG signaling pathway. The concentration of 10 amino acids was significantly higher in the PM than in the controls. Insulin microarray analysis showed that insulin, insulin-like growth factor 2 (IGF-2), IGF-2R, insulin-like growth factor binding protein 1 (IGFBP-1), IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-6 levels were significantly higher in PM patients compared to that in the controls. Thus, this study identified potential metabolite biomarkers for PM and provided novel insights into the mechanisms underlying this disorder.

Lactate determination in human vitreous humour by capillary electrophoresis and time of death investigation.

Forensic inquests, particularly, in assessing time since death currently recognize the importance of the analysis of vitreous humour (VH) biomarkers. Present research, studies, and validates the determination of lactate (La) in VH by CZE with indirect UV detection. The BGE (pH 8.9) consisted of Tris buffer (37 mM) containing 4-methoxybenzoic acid (4 mM) and alkyl-trimethyl-ammonium bromide (1.2 mM). Each VH specimen was diluted with a butyric acid solution (internal standard 0.057 mM) and La and butyrate were separated within 3-5 min (30 kV). The La LOQ and LOD were 4 and 2 mM, respectively. The calibration curve linearity ranged from 4 to 80 mM; intra- and interruns precisions were less than 10% for standard as well as for VH specimen, respectively. To investigate postmortem interval (PMI) and VH lactate level correlation, human VH specimens were collected during autopsy (n = 40) and stored at -20°C until assay. La levels ranged from 16 to 42 mM; PMI values ranged from 10 to 141 h. La (mM) and PMI (h) correlation was statistically significant (r2 = 0.527; p < 0.05). In conclusion, the present CZE analysis is efficacious to determine VH La as a biomarker for PMI investigation.

Relationship between rheological properties and transverse relaxation time (T2) of artificial and porcine vitreous humour.

Vitreous liquefactive processes play an integral role in ocular health. Knowledge of the degree of liquefaction would allow better monitoring of ocular disease progression and enable more informed therapeutic dosing for an individual patient. Presently this process cannot be monitored in a non-invasive manner. Here, we evaluated whether magnetic resonance imaging (MRI) could predict the viscoelasticity and in turn liquefactive state of artificial and biological vitreous humour. Gels comprising identical concentrations of hyaluronic acid and agar ranging from 0.125 to 2.25 mg/ml of each polymer were prepared and their T2 was measured using a turbo-spin echo sequence via 3T clinical MRI. The gels were subsequently subjected to rheological frequency and flow sweeps and trends between T2 and rheological parameters were assessed. The relationship between T2 and vitreous humour rheology was further assessed using ex vivo porcine eyes. An optimised imaging technique improved homogeneity of obtained artificial vitreous humour T2 maps. Strong correlations were observed between T2 and various rheological parameters of the gels. Translation to porcine vitreous humour demonstrated that the T2 of biological tissue was related to its viscoelastic properties. This study shows that T2 can be correlated with various rheological parameters within gels. Future investigations will assess the translatability of these findings to live models.

The challenge of post-mortem GHB analysis: storage conditions and specimen types are both important.

BACKGROUND: For the interpretation of concentrations of gamma-hydroxybutyrate (GHB) in post-mortem specimens, a possible increase due to post-mortem generation in the body and in vitro has to be considered. The influence of different storage conditions and the specimen type was investigated. METHOD AND MATERIAL: Post-mortem GHB concentrations in femoral venous blood (VB), heart blood (HB), serum (S) from VB, urine (U), cerebrospinal fluid (CSF) and vitreous humour (VH) were determined by gas chromatography-mass spectrometry after derivatisation. Various storage conditions, that is 4 °C or room temperature (RT) and the addition of sodium fluoride (NaF), were compared during storage up to 30 days. Additionally, bacterial colonisation was determined by mass spectrometry fingerprinting. RESULTS: Twenty-six cases without involvement of exogenous GHB were examined. GHB concentrations (by specimen) at day 0 were 3.9-22.1 mg/L (VB), 6.6-33.3 mg/L (HB), < 0.5-18.1 mg/L (U), 1.1-10.4 mg/L (CSF) and 1.7-22.0 mg/L (VH). At 4 °C, concentrations increased at day 30 to 5.6-74.5 mg/L (VB), 4.6-76.5 mg/L (HB) and < 0.5-21.3 mg/L (U). At RT, concentrations rose to < 0.5-38.5 mg/L (VB), 1.2-94.6 mg/L (HB) and < 0.5-37.5 mg/L (U) at day 30. In CSF, at RT, an increase up to < 0.5-21.2 mg/L was measured, and at 4 °C, a decrease occurred (< 0.5-6.5 mg/L). GHB concentrations in VH remained stable at both temperatures (1.2-20.9 mg/L and < 0.5-26.2 mg/L). The increase of GHB in HB samples with NaF was significantly lower than that without preservation. No correlation was found between the bacterial colonisation and extent of GHB concentration changes. CONCLUSION: GHB concentrations can significantly increase in post-mortem HB, VB and U samples, depending on storage time, temperature and inter-individual differences. Results in CSF, VH, S and/or specimens with NaF are less affected.

A new method for the determination of ammonium in the vitreous humour based on capillary electrophoresis and its preliminary application in thanatochemistry.

Background Although the post-mortem increase of ammonium in biological fluids is well known, ammonium analysis in vitreous humour has never been used in recent times for the determination of the post-mortem interval. The present work represents a new application of capillary electrophoresis with indirect UV detection in the field of forensic analysis. Methods The electrophoretic separation was carried out in a running buffer made of 5 mM imidazole, 5 mM 18-crown-6 ether and 6 mM d,l-α-hydroxybutyric acid (HIBA). To overcome the lack of optical absorption of ammonium, indirect UV detection was applied. The used wavelength was 214 nm. Results The method showed good linearity in the concentration range from 0.16 to 5.0 mM. The limit of detection, 0.039 mmol/L, was established on the basis of the linearity curve. Precision and bias studies carried out on the pure ammonium solutions and in real biological samples, revealed %RSDs well below 20%. A preliminary application to real cases where the death time was precisely known (14 bodies) was carried out plotting vitreous humour ammonium vs. post-mortem interval with a resulting good linear correlation until 100 h post-mortem. Conclusions After validation in real cases, the present method can become a powerful tool to unravel one of the most challenging issues of forensic investigation: determination of the time of death.

Diet-induced obesity leads to pro-inflammatory alterations to the vitreous humour of the eye in a rat model.

OBJECTIVE AND DESIGN: The purpose of this study was to investigate if diet-induced obesity (DIO) and subsequent low-level systemic inflammation would result in local increases in pro-inflammatory mediators in the vitreous humour (VH) of the eyes of rats. METHODS: Sixteen male Sprague-Dawley rats were fed a high-fat/high-sucrose (n = 9) or chow control-diet (n = 7) for 12-weeks. RT-qPCR was conducted on RNA from VH cells and a 27-plex Luminex® Assay was conducted on VH fluid and serum. RESULTS: Increased protein levels for IL-1ß, IL-6, and IL-18 in both serum and VH fluid were observed. VH protein levels for IL-13 and IL-17 were also increased. All mediators significantly increased in VH fluid were also positively correlated with percent body fat. Increased mRNA levels in VH cells for an oxidative stress molecule were accompanied by decreased mRNA levels for an antioxidant scavenger, suggesting an antioxidant/oxidant imbalance in the VH with DIO. In addition, decreased mRNA levels for TRAIL, FAS-L and TGF-ß, molecules associated with immune privilege, were also significantly depressed. CONCLUSIONS: DIO-related metabolic disturbances disrupt VH homeostasis in a manner that reflects development of a pro-inflammatory environment. Prolonged exposure to such an environment may lead to overt pathologies with compromised eye function.

Vitreous humour as an alternative material for the determination of alcohol concentration in human corpses.

AIM OF THE STUDY: The aim of the study was to assess the suitability of vitreous humour (VH) for determining alcoholaemia in deceased bodies. The study presents a correlation between the content of ethyl alcohol in vitreous humour and the concentration of ethyl alcohol in venous blood (femoral blood - FB). MATERIAL AND METHODS: The study material consisted of blood and vitreous humour samples taken from 62 individuals who died in the period 2012-2016 and whose post-mortem examinations were carried out at the Department of Forensic Medicine, Medical University of Bialystok. The content of alcohol was determined using the method of gas chromatography (GC). The findings of the study were analyzed statistically with MF Excel and Statistica 12.5, and then presented in a descriptive form, including figures and charts. RESULTS: Statistical analysis did not reveal significant differences between the concentration of ethanol in VH and FB. Spearman's correlation between the concentration of ethanol in the materials under study was r = 0.96, p < 0.01. CONCLUSIONS: The study demonstrated no statistically significant differences between the concentration of alcohol in VH and in FB. Consequently, vitreous humour can be used as a reliable alternative material in cases where there is no possibility of collecting venous blood for toxicological tests.

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.

"Not everything that can be counted counts" in ethanol toxicological results: an antemortem and postmortem technical interpretation focusing on driving under the influence.

Ethanol blood analysis is the most common request in forensic toxicology, and some studies point to positive results in approximately one-third of all unnatural deaths. However, distinguishing sober deaths from drunk deaths is not as simple as it may seem. This technical, clinical, and forensic interpretation is proposed to interpret the ethanol toxicological results, discussing several artefacts and pitfalls that must be considered, namely focusing on driving under the influence. This work is presented with a practical and objective approach, aiming to alleviate the complexities associated with clinical, physiological, pathophysiological, and toxicological aspects to enhance comprehension, practicality, and applicability of its content, especially to courts. Particularly the physical integrity of the body, the postmortem interval, putrefactive signs, anatomic place of blood collection, alternative samples such as vitreous humour and urine, the possibility of postmortem redistribution, the inclusion of preservatives in containers, and optimal temperature conditions of shipment are among some of the aspects to pay attention. Although several biomarkers related to postmortem microbial ethanol production have been proposed, their translation into forensic routine is slow to be implemented due to the uncertainties of their application and analytical difficulties. Specifically, in the interpretation of ethanol toxicological results, "not everything that can be counted counts and not everything that counts can be counted" (attributed to Albert Einstein).

Hyaluronic acid from bluefin tuna by-product: Structural analysis and pharmacological activities.

The fishing and aquaculture industries generate a huge amount of waste during processing and preservation operations, especially those of tuna. Recovering these by-products is a major economic and environmental challenge for manufacturers seeking to produce new active biomolecules of interest. A new hyaluronic acid was extracted from bluefin tuna's vitreous humour to assess its antioxidant and pharmacological activities. The characterization by infrared spectroscopy (FT-IR), nuclear magnetic resonance ((1D1H) and 2D (1H COSY, 1H/13C HSQC)) and size exclusion chromatography (SEC/MALS/DRI/VD) revealed that the extracted polysaccharide was a hyaluronic acid with high uronic acid content (55.8 %) and a weight average molecular weight of 888 kDa. This polymer possesses significant anti-radical activity and ferrous chelating capacity. In addition, pharmacological evaluation of its anti-inflammatory and analgesic potential, using preclinical models, in comparison with reference drugs (Dexamethasone, diclofenac, and acetylsalicylate of lysine), revealed promising anti-inflammatory activity as well as interesting peripheral and central antinociceptive activity. Therefore, our new hyaluronic acid compound may therefore serve as a potential drug candidate for the treatment of pain sensation and inflammation of various pathological origins.

Estimation of Time Since Death From Potassium Levels in Vitreous Humor in Cases of Unnatural Death: A Facility-Based Cross-Sectional Study.

Background Estimation of time since death/postmortem interval (PMI) forms a crucial component for all autopsy surgeons. With the subjectivity that is prevalent with conventional morphological and physical signs of death, newer-age modalities such as chemical analysis provide better precision. The easy accessibility and the resistance to putrefaction make vitreous humor the best choice for such chemical analysis. Thus, the present study's aim is to estimate the time since death in cases of unnatural death by investigating the potassium level changes in the vitreous humor. Methodology This is a facility-based cross-sectional study conducted in the mortuary under the Department of Forensic Medicine in a public tertiary healthcare teaching hospital in South India between August and September 2022. Deceased individuals fulfilling the inclusion and exclusion criteria were recruited in the study. Vitreous samples were collected from a single eye and analyzed using an autoanalyzer for potassium values. After due derivations, postmortem intervals were calculated from potassium values, then they were compared with the PMIs estimated using physical signs and those determined using official police records. Data were entered using MS Excel 20 (Microsoft Corporation, Redmond, Washington) and analyzed using the Statistical Package for the Social Sciences (SPSS) software, version 20 (IBM Corp., Armonk, NY). Results Of the 100 deceased individuals included in the study, 68% were men, and the majority (24%) belonged to the age group of 53-62 years. A linear relationship is said to exist between vitreous potassium concentration and postmortem interval. No correlation was seen between the ambient temperature and the potassium levels of the vitreous humor. PMI confirmed by the potassium levels seconded the PMI given by the police records and physical signs (Rigor mortis) (Spearman's rho was statistically significant at the two-tailed level or at the 0.01 level with a kappa value of 0.88). Conclusion Potassium measurements in the vitreous humor to estimate the PMI are associated with improved accuracy and precision in determining the time since death. They are not affected by external factors, making them a reliable marker for the same.

Investigation on the Influence of Synovial Fluid and Vitreous Humour on Avulsion Wounds Healing in Rabbits.

In the present study, we investigated the efficacy of vitreous humour and synovial fluid on avulsion wound healing in a rabbit's model. The vitreous humour is a fluid that resembles gel consisting of approximately 98-99% water, little hyaluronic acid, glucose, collagen, anions, cations and ions. It is in the posterior eye chamber for the comparison with synovial fluid that consisted of hyaluronin, lubricin, proteinase, prostaglandins and collagenase. In this study, both synovial fluid and vitreous humour were collected from rabbits by aspiration of vitreous humour from the eye (postmortem) and arthrocentesis procedure was applied for collection of synovial fluid. Twelve adult rabbits were used in this study, they divided into three groups each group consisted of four animals wounded experimentally (an avulsion wound). Our results showed the influence of vitreous humour (group B) on healing of the wound is better than the synovial fluid (group C) in the clinical evaluation of shrinkage of the wound. The histo-pathologically changes also revealed that in the vitreous humour treated group (group B), the wound healing process proceeded better than other groups (control and synovial fluid groups). In conclusion, the histopathological and clinical observations demonstrated that application of vitreous humour on wound might be pivotal in improving the healing of avulsion wounds and establish a new tissue in rabbits.

Estimation of the post-mortem interval: Effect of storage conditions on the determination of vitreous humour [K+].

Obtaining a reliable estimate of the post-mortem interval (PMI) has been a long-running challenge in forensic medicine. Several more or less successful techniques for making such estimates have been developed, but in recent years important advances have been made thanks to the detailed study of the relationship between the PMI and the analytes - in particular K+ - of the vitreous humour (VH). The extraction and pre-treatment of VH samples has been standardized, the influence of certain environmental factors on analytical results has been quantified, and some of the circumstances under which techniques become unreliable have been identified. The present work examines how the conditions to which VH samples are subject in routine practice may alter the results of their analysis. Exposure to light and ambient temperature was found to alter the values returned in determinations of VH [K+], [Na+] and [Cl-], while exposure to several freezing/thawing cycles (even with final heating) led to no significant modifications in determinations of VH [K+] and [Na+]. It is recommended that if analysis has to be delayed, VH should be frozen for storage in a refrigerator before bringing to room temperature for processing. It is also recommended that samples not be exposed to ambient light and temperature.

Detection of intact insulin analogues in post-mortem vitreous humour-Application to forensic toxicology casework.

The application of proteomic techniques to forensic science widens the range of analytical capabilities available to forensic laboratories when answering complex toxicology problems. Currently, these techniques are underutilised in post-mortem toxicology because of the historic focus on smaller (<1,000 amu) drug molecules. Definitive confirmation of an insulin overdose by analysis of post-mortem biological matrices is rare and challenging, however can assist coronial investigations pertaining to accidental or intentional overdoses in both diabetic and nondiabetic populations. A semiautomated micro-solid phase extraction paired with mass spectrometry-based insulin methodology was developed and validated for routine use in a Forensic Coronial Toxicology Laboratory. This resulting work reports the first Australian cases where synthetic insulins were confirmed by mass spectrometry in the vitreous humour of Type 1 diabetics who intentionally or accidentally overdosed on their prescription medication glargine and aspart. The detection of glargine M1 in Case 1, aspart in Case 2 and glargine M1 was indicated in Case 3. This paper highlights advancements in forensic coronial toxicology and the promising potential of proteomic analysis in a forensic context.