Case Study: Preservation of Nucleotides
Post-sampling, ATP-levels drop rapidly due to loss of oxygen and aerobic metabolism. Anabolic cellular activity on the other hand continues, and ATP as well as other tri-phosphate nucleotides will be consumed and the lower-energy di- and mono-phosphate equivalents created. It is essential to prevent changes during sample processing if one want to study tissue distribution of nucleotides and nucleotide analogs.
Elle Blatherwick, Ph.D. (University of Warwick), has studied and compared the distribution of ATP, ADP, and AMP in both snap-frozen and heat-stabilized mouse brain. In heat-stabilized samples, the levels of high-energy ATP were detected at higher levels compared to low energy AMP, whereas the reverse situation was found in snap-frozen samples. The lower ATP/AMP ratio in snap-frozen, compared to heat-stabilized samples, clearly indicates continued metabolic activity during sample preparation (Figure 2).
To confidently be able to study the levels of nucleotides in tissue sections has an immediate application in medical research where nucleotide analogs are used as antiviral and anticancer drugs. These are given as mono- or di-phosphorylated forms and are then further phosphorylated inside the cells. Heat stabilization in combination with MSI allows for the distribution of all phosphorylation stages to be assayed without the risk of interconversion between the forms during processing.