Elsevier

Analytical Biochemistry

Volume 529, 15 July 2017, Pages 216-228
Analytical Biochemistry

Experimental strategies for in vivo 13C NMR spectroscopy

https://doi.org/10.1016/j.ab.2016.08.003Get rights and content

Abstract

In vivo carbon-13 (13C) MRS opens unique insights into the metabolism of intact organisms, and has led to major advancements in the understanding of cellular metabolism under normal and pathological conditions in various organs such as skeletal muscles, the heart, the liver and the brain. However, the technique comes at the expense of significant experimental difficulties. In this review we focus on the experimental aspects of non-hyperpolarized 13C MRS in vivo. Some of the enrichment strategies which have been proposed so far are described; the various MRS acquisition paradigms to measure 13C labeling are then presented. Finally, practical aspects of 13C spectral quantification are discussed.

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

References (127)

  • D.E. Befroy et al.

    Assessment of in vivo mitochondrial metabolism by magnetic resonance spectroscopy

    Methods Enzym.

    (2009)
  • I.Y. Choi et al.

    In vivo 13C NMR assessment of brain glycogen concentration and turnover in the awake rat

    Neurochem. Int.

    (2003)
  • G. Oz et al.

    Direct, noninvasive measurement of brain glycogen metabolism in humans

    Neurochem. Int.

    (2003)
  • A.J. Shaka et al.

    Evaluation of a new broad-band decoupling sequence – Waltz-16

    J. Magn. Reson.

    (1983)
  • A.J. Shaka et al.

    Broadband spin decoupling in isotropic liquids

    Prog. Nucl. Mag. Res. Sp.

    (1987)
  • M.H. Levitt et al.

    Composite pulse decoupling

    J. Magn. Reson.

    (1981)
  • M.H. Levitt

    Symmetrical composite pulse sequences for Nmr population-inversion .1. Compensation of radiofrequency field inhomogeneity

    J. Magn. Reson.

    (1982)
  • M.H. Levitt

    Symmetrical composite pulse sequences for Nmr population-inversion .2. Compensation of resonance offset

    J. Magn. Reson.

    (1982)
  • W.P. Aue et al.

    Localized C-13 Nmr-spectra with enhanced sensitivity obtained by volume-selective excitation

    J. Magn. Reson.

    (1985)
  • D.P. Burum et al.

    Net polarization transfer via a J-ordered state for signal enhancement of low-sensitivity nuclei

    J. Magn. Reson.

    (1980)
  • D.M. Doddrell et al.

    Distortionless enhancement of Nmr signals by polarization transfer

    J. Magn. Reson.

    (1982)
  • G. Adriany et al.

    A half-volume coil for efficient proton decoupling in humans at 4 Tesla

    J. Magn. Reson.

    (1997)
  • D.L. Rothman et al.

    13C MRS studies of neuroenergetics and neurotransmitter cycling in humans

    NMR Biomed.

    (2011)
  • T.B. Rodrigues et al.

    (13)C NMR spectroscopy applications to brain energy metabolism

    Front. Neuroenergetics

    (2013)
  • E.L. Thomas et al.

    Noninvasive characterization of neonatal adipose tissue by 13C magnetic resonance spectroscopy

    Lipids

    (1997)
  • J.H. Hwang et al.

    In vivo characterization of fatty acids in human adipose tissue using natural abundance 1H decoupled 13C MRS at 1.5 T: clinical applications to dietary therapy

    NMR Biomed.

    (2003)
  • R. Gruetter et al.

    13C NMR visibility of rabbit muscle glycogen in vivo

    Magn. Reson. Med.

    (1991)
  • R. Taylor et al.

    Validation of 13C NMR measurement of human skeletal muscle glycogen by direct biochemical assay of needle biopsy samples

    Magn. Reson. Med.

    (1992)
  • R. Gruetter et al.

    Validation of 13C NMR measurements of liver glycogen in vivo

    Magn. Reson. Med.

    (1994)
  • T.B. Price

    Regulation of glycogen metabolism in muscle during exercise

  • G.F. Mason et al.

    A comparison of C-13 NMR measurements of the rates of glutamine synthesis and the tricarboxylic acid cycle during oral and intravenous administration of [1-C-13]glucose (vol 10, pg 181, 2003)

    Brain Res. Protoc.

    (2003)
  • B. Ross et al.

    Clinical experience with 13C MRS in vivo

    NMR Biomed.

    (2003)
  • F. Boumezbeur et al.

    The contribution of blood lactate to brain energy metabolism in humans measured by dynamic C-13 nuclear magnetic resonance spectroscopy

    J. Neurosci.

    (2010)
  • S.M. Cohen et al.

    Effects of ethanol on alanine metabolism in perfused mouse liver studied by 13C NMR

    Proc. Natl. Acad. Sci. U. S. A.

    (1979)
  • C. Pahl-Wostl et al.

    Metabolic pathways for ketone body production. 13C NMR spectroscopy of rat liver in vivo using 13C-multilabeled fatty acids

    Biochemistry

    (1986)
  • A.D. Sherry et al.

    Propionate metabolism in the rat heart by 13C n.m.r. spectroscopy

    Biochem. J.

    (1988)
  • V. Lebon et al.

    Astroglial contribution to brain energy metabolism in humans revealed by 13C nuclear magnetic resonance spectroscopy: elucidation of the dominant pathway for neurotransmitter glutamate repletion and measurement of astrocytic oxidative metabolism

    J. Neurosci.

    (2002)
  • F. Boumezbeur et al.

    Altered brain mitochondrial metabolism in healthy aging as assessed by in vivo magnetic resonance spectroscopy

    J. Cereb. Blood Flow Metab.

    (2010)
  • B. Kunnecke et al.

    Cerebral metabolism of [1,2-13C2]glucose and [U-13C4]3-hydroxybutyrate in rat brain as detected by 13C NMR spectroscopy

    NMR Biomed.

    (1993)
  • S.V. Gonzalez et al.

    Brain metabolism of exogenous pyruvate

    J. Neurochem.

    (2005)
  • A.K. Bouzier et al.

    The metabolism of [3-C-13]lactate in the rat brain is specific of a pyruvate carboxylase-deprived compartment

    J. Neurochem.

    (2000)
  • J.M.N. Duarte et al.

    Brain energy metabolism measured by C-13 magnetic resonance spectroscopy in vivo upon infusion of [3-C-13]lactate

    J. Neurosci. Res.

    (2015)
  • P. Bagga et al.

    Characterization of cerebral glutamine uptake from blood in the mouse brain: implications for metabolic modeling of 13C NMR data

    J. Cereb. Blood Flow Metab.

    (2014)
  • J.R. Alger et al.

    In vivo carbon-13 nuclear magnetic resonance studies of mammals

    Science

    (1981)
  • K.F. Petersen et al.

    Mechanism by which glucose and insulin inhibit net hepatic glycogenolysis in humans

    J. Clin. InvestInvestig.

    (1998)
  • I.Y. Choi et al.

    Noninvasive measurements of [1-(13)C]glycogen concentrations and metabolism in rat brain in vivo

    J. Neurochem.

    (1999)
  • G. Oz et al.

    Human brain glycogen content and metabolism: implications on its role in brain energy metabolism

    Am. J. Physiol. Endocrinol. Metab.

    (2007)
  • F.D. Morgenthaler et al.

    Non-invasive quantification of brain glycogen absolute concentration

    J. Neurochem.

    (2008)
  • R.B. van Heeswijk et al.

    Quantification of brain glycogen concentration and turnover through localized C-13 NMR of both the C1 and C6 resonances

    NMR Biomed.

    (2010)
  • N. Tesfaye et al.

    Noninvasive measurement of brain glycogen by nuclear magnetic resonance spectroscopy and its application to the study of brain metabolism

    J. Neurosci. Res.

    (2011)
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