Citrate Content of Bone: A Potential Measure of Post Mortem Interval

Based on submission by proposer: The reliable and accurate determination of the post mortem interval (PMI) continues to be a major problem in forensic science. The PMI is a crucial piece of information for forensic scientists and medicolegal investigators, since knowledge of the PMI can lead to identification of victim(s) and/or suspects in criminal cases by establishing the time dimension in investigations. For practical purposes, remains with a PMI <50 years old are typically deemed forensically relevant, while older remains would be considered to be historic. Current chemical methods lack the accuracy and/or precision necessary to provide reliable PMI values. Schwarcz et al. (2010) have suggested that citrate in bone decreases in concentration with an increase in PMI and that the rate does not depend significantly on storage conditions. This method may provide a promising approach to PMI determination of skeletal remains; therefore it warrants further investigation. Our main objective is to further optimize and develop the method of Schwarcz et al. and apply it to the analysis of (known PMI) human bone samples. Analysis of fresh and aged pig bone samples under controlled conditions is ongoing, to obtain a composite (fresh) baseline for citrate content (i.e., mass) and test citrate decrease (aged). Schwarcz et al.'s enzymatic citrate assay method is being replicated. Results will be compared to those of previous researchers. Two improvements to the method will be attempted. First, a second analytical technique will be used to determine citrate content, high performance liquid chromatography (HPLC). This two-method approach will provide an additional assessment of method accuracy and reliability. Second, unlike other researchers who have used "mass %" to present citrate content, we propose a ratio of total citrate mass to surface area of the sample. Dicken's (1941) work and that of Hu et al. (2010) has shown that citrate is primarily bound to the surface of bone, so it is expected that surface area will provide a means to normalize between bone samples with different densities. "Mass %" will still be calculated, but will be used primarily for comparison purposes. Data collection will be performed by both project directors. Prior to analysis, sample information will be entered into a specimen log database, with the following information: accession number, origin (UTK, OCME), PMI, and any other relevant information regarding taphonomic history. Prior to analysis, surface area data for samples will be determined by high resolution digital scanning or optical microscopy and logged into a second results database. Following instrumental analysis and data treatment, citrate content and normalized results will be entered into the results database. All method files and raw data will be stored locally on PC workstations. Databases, spreadsheets, and other electronic files will be stored in the project directors' laboratory and personal PC workstations. All files will be backed up at least monthly to a second physical non-bootable storage drive and to the College at Brockport's "Filecity2" remote data server, (encrypted, behind local and campus firewalls, with antivirus protection). We expect to accomplish the following: demonstrate/not demonstrate external validity of the Schwarcz et al. study, demonstrate utility of a second method to determine bone citrate content, demonstrate utility of adding sample surface area to calculation of citrate content, and demonstrate/not demonstrate the usefulness of citrate in determination of PMI. Expected products include: at least one presentation at a national conference, at least two presentations at local conferences, at least one manuscript submission to a peer-reviewed journal, training of at least two undergraduate students, optimized methods for citrate determination in bone, and results databases made available to the scientific community. ca/ncf