Noninvasive Fluxomics in Mammals by Nuclear Magnetic Resonance Spectroscopy

Abstract

Metabolism is an interconnecting network of metabolite consumption and creation. Metabolomics has focused on metabolite concentrations in metabolic networks. Fluxomics is also required in the study of metabolism and quantifies the flux of substrate through each reaction step or a series of reaction steps (i.e., metabolic pathway or cycle), and ultimately is required for energy balance equations of the system. The primary noninvasive method of quantifying fluxes in living systems is by in vivo 13C nuclear magnetic resonance (NMR) spectroscopy. The present state of noninvasive in vivo NMR technology allows for just four simultaneous flux measurements of metabolic pathways: gluconeogenesis, glycogen synthesis, glycolysis, and citric acid cycle. Since the liver is the gatekeeper and metabolic center for the animal, in vivo fluxomics of liver is extensively reviewed. Additionally, other organ systems studies are discussed demonstrating interorgan cycles, such as the Cori and Randall cycles. This review discusses the basics of in vivo fluxomics focusing on the general details of the NMR experimental protocol and required hardware/software needed to analyze the data.Currently, there are two general methods for determining multiple flux rates. The dynamic method entails acquiring serial time points, whereas the static method is a single measurement in which flux through metabolic pathways is quantified by isotopomer (i.e., isotope isomers) analysis. The flux data are analyzed by mathematical models to calculate the global flux measurement (in silico fluxomics), and create a mass balance of the biosystem. Models are especially useful for inferring various metabolic states of the system, which are affected by drugs, toxicants, or pathology. As with all in silico models, increasing the number of empirically derived concentrations and fluxes into the model greatly increases the accuracy and utility of the model. NMR spectroscopy (NMRS) is inherently insensitive compared to other analytical modalities, limiting the temporal resolution of the dynamic in vivo measurements. To address the NMR sensitivity, several technological advances have been made. First, magnets are now at higher magnetic field strengths. Second, the technique of dynamic nuclear polarization (DNP) of substrates increases the signal for 13C up to five orders of magnitude.In vivo fluxomics requires a broad knowledge of biochemistry, in vivo NMRS, and metabolic modeling. Therefore, this chapter is intended as a handbook for upper division undergraduate students and graduate students in biochemistry or engineering and relates the basics of electrical and biochemical engineering and animal handling. The chapter is intended for use in an introductory graduate course on NMR-based fluxomics for physical scientist.