437 - Leveraging Data from Bioenergetic Experiments to Determine the Absolute Flux of ATP in Cells from Respiration and Glycolysis

Abstract

Bioenergetic data, such as the instrumentation from Seahorse/Agilent, that examine cellular energy-production, in real-time, in live cells, has resulted in a wealth of new information on the balancing roles of mitochondrial respiration and glycolysis in the basic biology of cells. Knowing the rates of oxygen consumption and extracellular acidification by cells allows the assessment of metabolic flux, i.e. the flux of ATP, providing insight into the biology of health and disease. We have applied the concept of moles per cell to the rate of consumption of dioxygen (OCR) by cells in culture as well as the actual rate of cellular proton production (PPR) to determine the absolute flux of ATP in cells. Here we show how to gather information on buffer capacity, which can be used to make reliable estimates of the PPR. Then use of OCR and PPR in the same units, i.e. amol cell -1 s -1 , along with the known stoichiometries of biochemical processes, allows absolute determination of requirements for precursors and the quantitative determination of the flux of products, e.g. ATP. With this approach the flux of metabolic substrates and products in cells can be compared on an absolute basis, providing an entirely new window into information on cellular bioenergetic processes. Supported by NIH grants R01 CA169046, R01 GM073929, P30 CA086862, and P42 ES013661.