Stop the rot. Enzyme inactivation at brain harvest prevents artifacts: A guide for preservation of the in vivo concentrations of brain constituents. Issue 5 (26th February 2021)

• Record Type: Journal Article
• Authors: Dienel, Gerald A.
• Is Part Of: Journal of neurochemistry. Volume 158:Issue 5(2021)
• Page Start: 1007
• Page End: 1031
• Publication Date: 2021-02-26
• Subjects: labile metabolites -- mass spectrometry -- metabolomics -- post‐mortem interval -- post‐mortem ischemia; Neurochemistry -- Periodicals; 616.8042
• DOI: 10.1111/jnc.15293
• Languages: English
• ISSNs: 0022-3042

Abstract

Abstract: 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. Abstract : The requirement for use of methods that inactivate brain enzymes by microwave fixation or freezing at harvest to preserve the concentrations of labile metabolites was established more than 50 years ago. Nevertheless, many studies using modern metabolomic analytical and imaging methods do not use these procedures. Interpretation of these data is complicated by artifacts arising from post‐mortem ischemia that begins at death. Labile metabolite concentrations change within seconds and energy failure occurs within minutes. Post‐mortem changes in levels of labile metabolites in many pathways persist for hours to days, and they can be prevented by appropriate brain harvest procedures.