RESUMEN
Human blood is the most widely used biospecimen in the clinic and the metabolomics field. While both mass spectrometry and NMR spectroscopy are the two premier analytical platforms in the metabolomics field, NMR exhibits several unsurpassed characteristics for blood metabolite analysis, the most important of which are its ability to identify unknown metabolites and its quantitative nature. However, the relatively small number of metabolites accessible by NMR has restricted the scope of its applications. Enhancing the limit of identified metabolites in blood will therefore greatly impact NMR-based metabolomics. Continuing our efforts to address this major issue, our current study describes the identification of 12 metabolites, which expands the number of quantifiable blood metabolites by ~15%. These results, in combination with our earlier efforts, now provide access to nearly 90 metabolites, which is the highest to date for a simple 1D 1H NMR experiment that is widely used in the metabolomics field. Metabolites were identified based on the comprehensive investigation of human blood and plasma using 1D/2D NMR techniques. The newly identified metabolites were validated based on chemical shift databases, spectra of authentic compounds obtained under conditions identical to blood/plasma, and, finally, spiking experiments using authentic compounds. Considering the high reproducibility of NMR and the sensitivity of chemical shifts to altered sample conditions, experimental protocols and peak annotations are provided for the newly identified metabolites, which serve as a template for identification of blood metabolites for routine applications. Separately, the identified metabolites were evaluated for their sensitivity to preanalytical conditions. The results reveal that among the newly identified metabolites, inosine monophosphate (IMP) and nicotinamide are associated with labile coenzymes and their levels are sensitive to preanalytical conditions. The study demonstrates the expansion of quantifiable blood metabolites using NMR to a new height and is expected to greatly impact blood metabolomics.
RESUMEN
Post-mortem metabolism is widely recognized to cause rapid and prolonged changes in the concentrations of multiple classes of compounds in brain, that is, they are labile. Post-mortem changes from levels in living brain include components of pathways of metabolism of glucose and energy compounds, amino acids, lipids, signaling molecules, neuropeptides, phosphoproteins, and proteins. Methods that stop enzyme activity at brain harvest were developed almost 50 years ago and have been extensively used in studies of brain functions and diseases. Unfortunately, these methods are not commonly used to harvest brain tissue for mass spectrometry-based metabolomic studies or for imaging mass spectrometry studies (IMS, also called mass spectrometry imaging, MSI, or matrix-assisted laser desorption/ionization-MSI, MALDI-MSI). Instead these studies commonly kill animals, decapitate, dissect out brain and regions of interest if needed, then 'snap' freeze the tissue to stop enzymatic activity after harvest, with post-mortem intervals typically ranging from ~0.5 to 3 min. To increase awareness of the importance of stopping metabolism at harvest and preventing the unnecessary complications of not doing so, this commentary provides examples of labile metabolites and the magnitudes of their post-mortem changes in concentrations during brain harvest. Brain harvest methods that stop metabolism at harvest eliminate post-mortem enzymatic activities and can improve characterization of normal and diseased brain. In addition, metabolomic studies would be improved by reporting absolute units of concentration along with normalized peak areas or fold changes. Then reported values can be evaluated and compared with the extensive neurochemical literature to help prevent reporting of artifactual data.
Asunto(s)
Encéfalo/enzimología , Encéfalo/patología , Metabolómica/métodos , Preservación de Órganos/métodos , Cambios Post Mortem , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Metabolismo Energético/fisiología , Humanos , Metabolómica/normas , Preservación de Órganos/normas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/normas , Factores de TiempoRESUMEN
Metabolomic technologies including imaging mass spectrometry (IMS; also called mass spectrometry imaging, MSI, or matrix-assisted laser desorption/ionization-mass spectrometry imaging, MALDI MSI) are important methods to evaluate levels of many compounds in brain with high spatial resolution, characterize metabolic phenotypes of brain disorders, and identify disease biomarkers. ATP is central to brain energetics, and reports of its heterogeneous distribution in brain and regional differences in ATP/ADP ratios reported in IMS studies conflict with earlier studies. These discordant data were, therefore, analyzed and compared with biochemical literature that used rigorous methods to preserve labile metabolites. Unequal, very low regional ATP levels and low ATP/ADP ratios are explained by rapid metabolism during postmortem ischemia. A critical aspect of any analysis of brain components is their stability during and after tissue harvest so measured concentrations closely approximate their physiological levels in vivo. Unfortunately, the requirement for inactivation of brain enzymes by freezing or heating is not widely recognized outside the neurochemistry discipline, and procedures that do not prevent postmortem autolysis, including decapitation, brain removal/dissection, and 'snap freezing' are commonly used. Strong emphasis is placed on use of supplementary approaches to calibrate metabolite abundance in units of concentration in IMS studies and comparison of IMS results with biochemical data obtained by different methods to help identify potential artifacts.