Estimation of the Post Mortem Interval via Metabolomic Approach Lyudmila Yanshole (Presenter) International Tomography Center SB RAS Presenter Bio: Lyudmila Yanshole obtained M.Sc. at the Novosibirsk State University (Novosibirsk, Russia, 2003-2008), Faculty of natural sciences. In 2011 she received Ph.D. in physical chemistry. Since 2006 she is working at the International Tomography Center in the fields of proteomics, metabolomics and mass-spectrometry, photochemistry earlier. Now she is one of the leading specialists in the institute in these fields. Lyudmila numerously attended mass-spectrometry courses and is experienced in MALDI-TOF and high-resolution LC-MS techniques, high frequency NMR also. Current research interest is mainly devoted to the quantitative metabolomic profiling of different human tissues and liquids. One of the main interests is the search of metabolomic biomarkers of different diseases. Recent investigations include the metabolomic profiling of normal and keratoconic cornea and aqueous humor.

Authors: Lyudmila V. Yanshole (1,2), Ekaterina A. Zelentsova (1,2), Vadim V. Yanshole (1,2), Olga A. Snytnikova (1,2), Yuri P. Tsentalovich (1,2)
(1) International Tomography Center SB RAS, Novosibirsk, Russia (2) Novosibirsk State University, Novosibirsk, Russia

The analysis of post-mortem metabolomic changes in biological fluids opens the way to develop new methods for the estimation of post-mortem interval (PMI) which appears to be an actual problem in forensic medicine. The combination of modern research methods - high frequency NMR and high-performance liquid chromatography with high-resolution mass spectrometry (HPLC-MS) was used in this work. Metabolomic profiling of rabbit and human biological fluids obtained at different times after death is performed in this work: the quantitative levels of 61 metabolites have been measured. The obtained results suggest that the ocular fluids AH and VH may have some advantages over blood serum for the search of potential biochemical markers for the PMI estimation. Among the compounds studied in the present work, hypoxanthine, choline and glycerol give the biggest promise as the potential PMI biomarkers.

Introduction

There are many dynamic biological processes in the tissues of the human body. For understanding these processes the quantitative information on the maximum possible variety of organic compounds that are present in the tissue at a particular time is needed. To date, metabolomic research is one of the most promising for personalized medicine, medical biology, toxicology and forensic medicine.

It is worth mentioning that the work on the analysis of metabolomic changes in pathological tissues has been widely disseminated. For humans, autopsy materials are often used as control tissues due to the inaccessibility of the tissues of a healthy patient (brain, eye tissues and others). In tissues, where all living processes are terminated, there is a significant change in the metabolic composition, which affects the reliability of the results when comparing such tissues with the pathological tissues of living donors. For the correct use of autopsy tissues as control, a detailed description of the changes in the metabolomic content of autopsy tissues should be investigated, and the cause and post-mortem interval (PMI) of death should be taken into account.

The metabolomic profiling of rabbit and human autopsy tissues, as well as the creation of regulations for the correct use of autopsy materials as control in experiments on the study of various pathologies is carried out in this work. A quantitative analysis of the metabolomic composition of autopsy fluids – aqueous humor, vitreous humor and blood plasma - is carried out for the first time.

Methods

The quantitative metabolomic profiling of samples of the rabbit and human serum, aqueous humor (AH) and vitreous humor (VH) taken at different PMIs has been performed with the combined use of high frequency 1H nuclear magnetic resonance (NMR) and high-resolution LC–MS methods. NMR-based quantification was achieved with the use of one internal standard (DSS) for all metabolites under study, while LC-MS quantification required the construction of the calibration curves for each metabolite under study using the commercially-available chemical standards.

Special attention was paid to the collection and sample preparation for metabolomic analysis. Since the conditions of biological sample collection and storage can strongly affect the composition of low-molecular compounds in tissues, it is necessary to unify the protocols of sample preparation for analysis, as well as to identify groups of metabolites for which storage is unacceptable. This is necessary for the correct comparison of metabolomic profiles of tissues obtained in different conditions.

Results

The quantitative levels of 61 metabolites in the rabbit [1] and human serum, AH and VH at different PMIs have been measured. It has been found that the post-mortem metabolomic changes in AH and VH proceed slower than in blood, and the data scattering is lower. Among the metabolites whose concentrations increase with time, the most significant and linear growth is found for hypoxanthine, choline and glycerol. Some other compounds, including glycine, glutamate, taurine also demonstrate monotonous growth with the PMI increase, but the effect is less pronounced than for hypoxanthine, choline and glycerol. The comparison was carried out both within the blood plasma-aqueous humor-vitreous humor group from one donor and between biological fluids of the same type obtained from donors with different PMIs. The high potential of choline and glycerol as biomarkers for the PMI estimation is revealed in the present work for the first time.

Conclusions & Discussion

The obtained results indicate the advantage of ocular fluids, aqueous and vitreous humors, over blood serum for the search of potential biochemical markers for the PMI estimation: the post-mortem metabolomic changes in the ocular fluids proceed slower than in blood, and the data scattering is lower. These changes are governed by two major factors: the degradation of the ocular tissues and the diffusion of metabolites from the body vascular systems into ocular fluids. Among the compounds studied in the present work, hypoxanthine, choline and glycerol give the biggest promise as the potential PMI biomarkers: the concentrations of these metabolites almost linearly grow post-mortem, the scale of changes is large, and the reproducibility of the effect is at acceptable range.

1. Zelentsova E.A., Yanshole L.V., Snytnikova O.A., Yanshole V.V., Tsentalovich Y.P., Sagdeev R.Z., Post-mortem changes in the metabolomic compositions of rabbit blood, aqueous and vitreous humors. Metabolomics. 12 (2016): 172.

Acknowledgements: The work was supported by the Russian Scientific Foundation (Project № 18-73-10014) in LC-MS measurements, by the Russian Foundation for Basic Research (Projects № 18-34-00137, 18-415-543006) in NMR measurements.