Abstract
Determining the postmortem interval (PMI) or time of death is often the most sought after piece of information associated with a death investigation. PMI determination not only establishes how long a person has been deceased but also assists in distinguishing antemortem pathology from post-mortem artefact, in helping to include and exclude suspects based on their whereabouts and to provide a timeframe in which remains decomposed beyond recognition can be connected with missing persons. Therefore an accurate estimation of PMI is extremely important to the death investigation and is deduced from the evidence collected. However the various methods currently used in estimating PMI yield large postmortem windows and sometimes contradict one another. Biochemical methods are currently being explored as an alternative approach for PMI estimation as new discoveries and developments in the biochemical field have the potential to provide new approaches for determining PMI.
This project sought to understand biochemical changes post-mortem by examining pH, proteins, metabolites and blood cell morphology in post-mortem blood from euthanised rats and piglets and antemortem human blood stored in a tube, using the analytical methods of mass spectroscopy, enzymatic assays, protein gels and light microscopy to identify biochemical candidates for the estimation of PMI.
Light microscopy was used to examine blood cell morphology and showed that blood cells alter in a time-dependant manner postmortem. However, the rate of change in individual cells was variable and did not occur linearly with time indicating that blood cell morphology would not be a suitable marker to estimate PMI. A pH meter was used to examine the pH of post-mortem blood and showed that post-mortem blood decreased rapidly from pH 7.3-6.0 in the first 24 hours before decreasing slowly over the next three days to pH 5.0 by 96 hours.
Protein gels were used to visualise whether the total amount of protein in plasma had altered post-mortem. The protein gels showed that numerous proteins were altered, with 12 proteins being identified by mass spectroscopy as having significantly increased or decreased compared to antemortem. Protein assays were also used to examine if the total amount of protein in plasma had altered. The total amount of protein in the bloodstream was shown to have decreased with the exception of haemoglobin which was shown to have increased due to lysed red blood cells.
Enzymatic assays were used to determine if six metabolite markers that are thought to be involved in anaerobic reactions or pathways were altered post-mortem. These six metabolite markers were lactic acid, hypoxanthine, uric acid, ammonia, NADH and formic acid and were shown to have altered concentrations compared to normal antemortem concentrations. Hypoxanthine, ammonia, NADH and formic acid all had concentrations that increased over time indicating that these metabolites could be PMI markers. The concentrations of uric acid and lactate increased and then decreased again over time with uric acid showing a different concentration profile in the rat compared to the human and piglet concentrations, indicating that these metabolites are unlikely to be useful for PMI estimations.
Gas chromatography was used to conduct a metabolic survey of post-mortem rat blood. 85 metabolites were shown to be altered post-mortem. 26 metabolites none of which were in the six assayed enzymatically, had concentrations which increased post-mortem indicating that these metabolites may have potential as PMI markers, while the remaining 59 metabolites had fluctuating concentrations post-mortem so would not be able to be useful in PMI estimations.
Therefore, this study was able to identify 30 metabolites and 12 proteins in blood during the early post-mortem interval that may be potential markers for PMI estimations.