Experimental strategies for in vivo 13C NMR spectroscopy
Introduction
In vivo carbon-13 (13C) magnetic resonance spectroscopy has emerged about thirty years ago as a unique technique to study metabolism in intact organisms, and since then has generated important knowledge about cellular metabolism in various organs such as skeletal muscles, the heart, the liver and, maybe most importantly, the brain, with major advancements in the understanding of cerebral energy metabolism, neuron-astrocyte compartmentation and cooperation, and glutamatergic neurotransmission (see Refs. [1], [2] for recent review about 13C MRS in the brain). The basic principles underlying the approach are quite simple. 13C, the only stable isotope of carbon having a nuclear magnetic moment, has a natural abundance of only ∼1.1%, so that administration of a highly 13C-enriched substrate will yield an unambiguous 13C MRS signal increase. Unlike techniques based on radioactive isotopes, such as positron emission tomography or autoradiography, MRS offers the unparalleled ability to chemically resolve the origin of the signal. It allows precisely tracking the metabolic fate of administered 13C-labeled substrates, i.e. specifically identifying which metabolites are being labeled, and at what atomic positions. This unique chemical specificity unfortunately comes at the expense of detection sensitivity when compared to radioisotope techniques, with detection thresholds being of the order of the millimolar (mM). This has been driving many methodological efforts to improve acquisition strategies to enhance measurement's temporal or spatial resolution. Once methodological challenges have been adequately addressed, one of the great strengths of MRS is the possibility to get a quantitative measurement of 13C labeling, either in terms of fractional enrichment (the ratio between labeled and total pools) or of absolute 13C concentration, making possible the estimation of metabolic fluxes via metabolic modeling.
In this paper we review experimental strategies for 13C MRS in vivo. We first introduce some of the enrichment strategies which have been proposed so far. Then, the various MRS acquisition paradigms to measure 13C labeling are presented. Finally, we discuss some practical aspects of 13C quantification on spectra.
Section snippets
Strategies for 13C infusion and labeling of endogenous metabolites
Before going through the variety of 13C-labeled substrates and infusion strategies which can be used to enrich metabolic byproducts, we shall briefly mention that natural abundance 13C MRS can be of interest in a limited number of cases, essentially when the increased spectral resolution compensates for the low detection sensitivity, as compared to 1H MRS. Some notable cases are fatty acids in adipose tissue [3], [4] and, due to the high glycogen concentration in these organs, glycogen
13C detection
Among the many technical considerations associated with in vivo 13C detection, one must address the actual sensitivity of the experiment, its anatomic specificity and the complexity of its set-up. Basically, two classes of MRS experiments can be considered to detect 13C: direct detection, where one acquires spectra at the 13C frequency (determined by 13C gyromagnetic ratio γC = 6.728 × 107 rad/s/T); or indirect detection, where one acquires spectra at the 1H frequency (determined by 1H
Quantification of 13C spectra
The quantification of 13C spectra, i.e. the determination of the fractional enrichment (in percent) or absolute concentration (in mM or μmol/g) of 13C-labeled metabolites, can be considered as the outcome of 13C experiments, or the last step before performing metabolic modeling.
Quantification can be achieved at the positional level with direct or indirect detection, i.e. the amount of 13C at a given atomic position is quantified irrespective of the labeling at other atomic positions. In direct
Perspectives
In this review we have provided some keys to understand and design 13C MRS experiments, from the infusion protocol to the spectral quantification. We hope that we have convinced the readers that great care should be taken at all stages, and that they will find some help in these pages to design their experiments and derive meaningful 13C enrichments with respect to the metabolic question asked. We have not addressed metabolic modeling, which goes beyond the “experimental” aspects described
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