"Omics"-based analyses are widely used in numerous areas of research, advances in instrumentation (both hardware and software) allow investigators to collect a wealth of data and therein characterize metabolic systems. Although analyses generally examine differences in absolute or relative (fold-) changes in concentrations, the ability to extract mechanistic insight would benefit from the use of isotopic tracers. Herein, we discuss important concepts that should be considered when stable isotope tracers are used to capture biochemical flux. Special attention is placed on in vivo systems, however, many of the general ideas have immediate impact on studies in cellular models or isolated-perfused tissues. While it is somewhat trivial to administer labeled precursor molecules and measure the enrichment of downstream products, the ability to make correct interpretations can be challenging. We will outline several critical factors that may influence choices when developing and/or applying a stable isotope tracer method. For example, is there a "best" tracer for a given study? How do I administer a tracer? When do I collect my sample(s)? While these questions may seem straightforward, we will present scenarios that can have dramatic effects on conclusions surrounding apparent rates of metabolic activity.
Keywords: Flux; Isotope; Metabolic activity; Metabolomics; Pathway.