Noninvasive Fluxomics in Mammals by Nuclear Magnetic Resonance Spectroscopy
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.
Key wordsFluxomics hyperpolarization in vivo NMR modeling
Funding for the studies was provided by NIH grants GM075941-01, CA114365-01A1, P30ES10126, and RR-000046.
Usually refers to an enzyme whose activity is modified by the presence of a ligand that binds reversibly to a site other than the active site. In the context of multisite enzyme, such allosteric effectors change the state of the enzyme to enhance or diminish substrate affinity and/or turnover number.
In cell biology comprise all closed parts within a cell whose lumen is usually surrounded by a single or double lipid layer membrane. Most organelles are compartments like mitochondria, chloroplasts (in photosynthetic organisms), peroxisomes, lysosomes, the endoplasmic reticulum, the cell nucleus, or the Golgi apparatus. Smaller elements like vesicles, and sometimes even microtubules can also be counted as compartments. In general, there are three main cellular compartments, they are: (1) The nuclear compartment comprising the nucleus; (2) The intercisternal space which comprises the space between the membranes of the endoplasmic reticulum (which is continuous with the nuclear envelope); and (3) The cytosol. Metabolically, the mitochondria comprise a very important compartment, even though they are by volume fraction small (from Wikipedia).
Finding a curve, which matches a series of data points and possibly other constraints. This section is an introduction to both interpolation (where an exact fit to constraints is expected) and regression analysis. Both are sometimes used for extrapolation. Regression analysis allows for an approximate fit by minimizing the difference between the data points and the curve (from Wikipedia).
The measurement of isotope steady state being reached by a time series measure. In the context of this chapter, it is the serial measurement of an NMR-observable tracer, such as 13C, 15N, 19F, 3H, or 2H, and its incorporation into a metabolite.
Transfer of polarization from electrons to nuclei, which occurs in a magnetic field, close to absolute zero. This is done by inducing transitions, via oscillating microwave RF pulses, between the allowed energy states of the electrons and nuclei, thus transferring polarization from the electrons to the nuclei. This results in more nuclei aligned with the magnetic field, thus resulting in enhancements of the signal-to-noise on the order of several magnitudes.
The net rate or the difference between the (unidirectional) rates of the forward and reverse reactions.
A volume coil design for an NMR radiofrequency probe consisting of two connecting loops spanning 120° and separated by 60°.
The measuring of multiple fluxes in live biosystems using noninvasive analytical instrumentation.
Use of isotopically labeled compounds administered at levels that do not affect steady state and can be used to trace flow of atoms through reaction networks.
The distribution of a single or multiple isotopes within metabolites of a biosystem.
An application of conservation of mass to the analysis of physical systems. By accounting for material entering and leaving a system, mass flows can be identified. The exact conservation law used in the analysis of the system depends on the context of the problem, but all revolve around mass conservation, i.e., that matter cannot disappear or be created spontaneously (from Wikipedia).
A method for modeling metabolism in which equations are used to describe metabolite concentration and flux, signaling pathways, and genetic pathways.
An analysis where metabolites are measured such that the amount of metabolite formation can be measured over a given period of time. This allows for the calculation of the rate of formation, or flux, of a metabolite.
The mathematical curve fitting of residuals in a time series dataset whereby the various components represent time constants for multiple rates. In the context of this chapter, this relates to metabolite turnover rate within the biosystem.
Net flux is sometimes used to define the total flux through a metabolic pathway as some of the individual fluxes can be in reverse of the net flux direction for the entire metabolic pathway.
This is usually a description of covalent modification of proteins (cf. posttranslational modification) such as phosphorylation that changes the properties of the target protein, e.g., recognition in signal transduction pathways or in chromatin remodeling.
Like a compartment, a pool is completely defined by the model describing the system. A pool can contain all the members of a particular chemical species for instance, even if they are in several different compartments. A pool can also describe those molecules in a particular physical environment, such as pyruvate in plasma, mitochondria, or cytosol. It can also be a purely theoretical physical space; when a tracer is infused into the bloodstream of an intact animal, the volume of its initial distribution (in plasma, intracellular space, etc.) is thought to describe its pool of entry (20).
A situation in which all state variables remain effectively constant, at least on the macroscopic scale (from Wikipedia).
A dimensionless parameter describing the efficiency of a transceiver, and in context to this chapter, specifically the efficiency of an NMR radiofrequency probe and a measure of the amount of power transmitted to the amount received.
Speed of a reaction under specified conditions. For simple chemical kinetics, the rate is the first derivative of the product concentration with respect to time, dp/dt, and is equal to a rate constant multiplied by reactant concentrations.
Rate is the product of a rate constant and concentration (strictly, activity). Thus, the units of a rate constant depend on the order of the reaction. For a first order reaction, the rate is v = k 1 a, and the first order rate const k 1 has dimensions in seconds. For a second order reaction, v = ka.b and k has dimensions in M/s. Enzymes saturated with substrate show zero order kinetics with respect to the substrate, v = V m.
Redox (shorthand for reduction/oxidation reaction) describes all chemical reactions in which atoms have their oxidation number (oxidation state) changed (from Wikipedia).
The ratio of signal peak to the average noise factor. Specifically, the ratio of NMR peak heights of peaks from measurable compounds to noise peaks. To improve the signal-to-noise ratio by a factor of 2, one must acquire a factor of 4 more transients.
The mathematical curve fitting of residuals in a time series dataset whereby a single component represents a time constant for a rate. In the context of this chapter, this relates to metabolite turnover rate within the biosystem.
A volume coil design for an NMR radiofrequency probe consisting a wound wire or coil, like a spring, whereby the optimum spacing f loops is equal to the wire diameter.
A method of flux analysis whereby 13C fractional enrichment or isotopomers are quantified in a single sample at steady state.
A situation in which all state variables are constant in spite of ongoing processes that strive to change them. For an entire system to be at steady state, i.e., for all state variables of a system to be constant, there must be a flow through the system (compare mass balance) (from Wikipedia).
A value used to describe the distribution of a xenobiotic between blood plasma and the rest of the body. Xenobiotics with higher volumes of distribution are generally lipid-soluble and thus have low blood plasma concentrations after dosing. Those with lower volumes of distribution are generally more polar and thus have high blood plasma concentrations after dosing.
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