Fresh vs. frozen human decomposition – A preliminary investigation of lipid degradation products as biomarkers of post-mortem interval

Highlights

• Tissue samples from two human donors were collected and biomarkers investigated.

• Significant differences were observed between the fresh and frozen lipid profiles.

• Fatty acids were changing, whereas sterols remained stable.

• Several biomarkers were identified as good indicators of time since death.

Abstract

Traditionally, the post-mortem interval (PMI) is determined using methods such as forensic entomology or forensic pathology, however, these techniques are often limited to a particular post-mortem window of up to 72 – 120 h after death. In this study, lipids extracted from decomposing human tissue were investigated as potential soft-tissue biomarkers of PMI. Tissue samples were collected from two whole human donors (n = 2), one frozen and one fresh, placed at the Australian Facility for Taphonomic Experimental Research (AFTER) over the course of 69 days post-placement. These samples were analysed using gas chromatography coupled with tandem mass spectrometry (GC–MS/MS), demonstrating statistically significant differences for most fatty acid analytes and further highlighting the well-known stability of sterol compounds over time. Differences were observed between the fresh and frozen donor, with the fresh donor displaying more distinct stages of decomposition. Chemical differences between the donors were more distinct in the fatty acids than the sterols. The fatty acid profiles over time were further investigated and target analytes comprising saturated fatty acids (stearic acid and palmitic acid) and unsaturated fatty acids (oleic acid and linoleic acid) were found to be of particular importance due to their potential as indicators of PMI.

Introduction

Despite its critical importance in forensic death investigations, a precise estimation of the post-mortem interval (PMI) remains a difficult task for investigators [1]. This is due to the complexity of decomposition chemistry and the multitude of intrinsic and extrinsic variables that impact the dynamicity of the process [2], [3], [4]. Current research in forensic taphonomy has shown great potential in the development of novel and alternative approaches for the estimation of PMI and/or time since deposition. These include (but are not limited to) the analysis of: volatile organic compounds (VOC’s) [3], [5], [6], [7], [8], the interactive effect of decomposition by-products retained in textiles [9], [10], the assessment of post-mortem proteomics [11] and lipidomics [12], and the investigation of soil chemistry related to cadaver decomposition islands (CDI’s) [13]. The human body is comprised of approximately 64% water, 20% protein, 10% lipids, 5% minerals and 1% carbohydrates [14], [15], [16], thus targeting these soft tissue biomarkers and studying their degradation patterns is a prospective method to estimate PMI. As a majority of proteins and carbohydrates are hydrophilic molecules [17], they are a poor target for the aims of this study, mainly due to naturally occuring ucontrollable extrinsic factors such as rainfall. Therefore, this research aims to investigate mostly hydrophobic lipids as a soft tissue biomarker in human tissue to assess their viability as an aid for PMI estimations.

Traditionally gas chromatography – mass spectrometry (GC–MS) has been used for the analysis of lipid products in forensic contexts [10], [18], [19]. However, a more sensitive method is gas chromatography coupled with tandem mass spectrometry (GC–MS/MS). GC–MS/MS allows for the selection of precursor ions based on a known mass-to-charge ratio in the first detection mode which can then be further fragmented into its product ions in the collision cell of the instrument [20]. This enables a selective and sensitive targeted approach to lipid analysis. Hence, GC–MS/MS is a suitable tool for the analysis of lipids and their degradation patterns of human remains as trace quantities might be present.

In spite of recent resistance in the use of whole human cadavers for taphonomic research [24], [26], the authors believe the opportunity to study decomposition chemistry on humans, for the purpose of human-related forensic casework, is invaluable. This study involves the preliminary investigation of lipids extracted from decomposing human tissue collected from two whole human donors (n = 2) over the course of 69 days post-placement. The effects of freezing a body prior to natural decomposition was observed as Donor 1 was kept frozen as a replicate for Donor 2 (fresh) to ensure both donors were studied under identical environmental conditions. This also provided a unique opportunity to investigate the effect of freezing on human decomposition and subsequent lipid products.