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Post-mortem changes in the metabolomic compositions of rabbit blood, aqueous and vitreous humors

ABSTRACT: Introduction: 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). It may also help in the analysis of disease-induced metabolomic changes in human tissues when the postoperational samples are compared to the post-mortem samples from healthy donors. Objectives: The goals of this study are to observe and classify the post-mortem changes occurring in the rabbit blood, aqueous and vitreous humors (AH and VH), to identify the potential PMI markers among a wide range of metabolites, and also to determine which biological fluid – blood, AH or VH – is more suitable for the PMI estimation. Methods: The quantitative metabolomic profiling of samples of the rabbit serum, AH and VH taken at different PMIs has been performed with the combined use of high-frequency NMR and high-resolution LC-MS methods. Results: The quantitative levels of 63 metabolites in the rabbit 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. Conclusion: The obtained results indicate the advantage of ocular fluids, AH and VH, 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.

INSTRUMENT(S): Bruker

SUBMITTER: Vadim Yanshole

PROVIDER: MTBLS349 | MetaboLights | 2017-08-23

REPOSITORIES: MetaboLights

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			Action	DRS
	MTBLS349	Other
	FILES	Other
	a_MTBLS349_rabpmi_metabolite_profiling_NMR_spectroscopy.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS349_rabpmi_metabolite_profiling_NMR_spectroscopy_v2_maf.tsv	Tabular

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Project description:Introduction: The optical elements of the eye – cornea, lens, and vitreous humor – are avascular tissues, and their nutrition and waste removal are provided by aqueous humor (AH). The AH production occurs through the active secretion and the passive diffusion/ultrafiltration of blood plasma. The comparison of the metabolomic profiles of aqueous humor (AH) and plasma is important for understanding of the mechanisms of biochemical processes and metabolite transport taking place in vivo in ocular tissues. Objectives: The work is aimed at the determination of concentrations of a wide range of most abundant metabolites in the human AH, the comparison of the metabolomic profiles of AH and serum, and the analysis of the post-mortem metabolomic changes in these two biological fluids. Methods: The quantitative metabolomic profiling was carried out with the use of two independent methods – high-frequency 1H NMR spectroscopy and HPLC with high-resolution ESI-MS detection (LC-MS). Results: The concentrations of 71 most abundant metabolites in blood serum and AH from living patients and human cadavers have been measured. It has been found that the level of ascorbate in AH is by two orders of magnitude higher than that in serum; the levels of other metabolites are either similar to that in serum, or differ from that by a factor of 2-5. The post-mortem metabolomic composition of both serum and AH undergoes rapid and strong changes. Conclusion: The differences between the metabolomic profiles of AH and serum for majority of metabolites can be attributed to the metabolic activity of the ocular tissues leading to the lack or excess of some metabolites, while the high concentration of ascorbate in AH demonstrates the activity of ascorbate-specific pumps at the blood-aqueous border. The post-mortem metabolomic changes are caused by the disruption of the major biochemical cycles and cell lysis. These changes should be taken into account in the analysis of disease-induced changes in post-mortem samples of the ocular tissues.

Project description:The bloodstream forms of Trypanosoma brucei (BSF), responsible for the sleeping sickness, primarily evaluate in the blood of its mammalian hosts. The skin and the adipose tissues were recently identified as additional major sites for the parasite proliferation and transmission through its insect vector blood ingestion. Glucose is the only carbon source used by the blood parasites to feed its central metabolism, however, the metabolic behavior of the tissue-adapted parasites has not yet been addressed. Since production of glycerol is an important primary function of adipocytes, we have adapted the BSF trypanosomes to glucose-depleted and glycerol-rich growth medium (CMM_Gly/GlcNAc) and compared its metabolism and proteome with standard glucose-rich growth conditions (CMM_Glu). BSF consume 2-times more oxygen per carbon consumed in CMM_Gly/GlcNAc and is 11.5-times more sensitive to SHAM, a specific inhibitor of the alternative oxidase, which is consistent with the expected absolute requirement of the mitochondrial respiratory chain activity to convert glycerol into dihydroxyacetone phosphate. Metabolomic analyses by mass spectrometry showed that 13C-labeled glycerol is incorporated into hexose phosphates through gluconeogenesis. After several weeks of growth in CMM_Gly/GlcNAc, BSF adapt their metabolism by increasing and decreasing the rate of glycerol and glucose consumption, respectively. As expected RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol degradation and is lethal for BSF growth in CMM_Gly/GlcNAc. However, the glycerol kinase activity is 7.8-fold decreased in CMM_Gly/GlcNAc, as confirms by western blotting and proteomic analyses, which suggests that the huge excess of glycerol kinase, which is not necessary for glycerol metabolism, is required for another yet undetermined non-essential function in glucose rich-conditions. Altogether these data shows that BSF trypanosomes are well adapted to glycerol-rich conditions, which could be encountered by the parasite in extravascular niches, such as skin and adipose tissues.

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Post-mortem changes in the metabolomic compositions of rabbit blood, aqueous and vitreous humors

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