In Vivo ²H NMR Studies of Cellular Metabolism

Abstract

The development of in vivo spectroscopic methods for the study of cell suspensions, perfused organs, intact animals and humans over the past two decades has relied primarily on the observation of nuclei other than protons. In particular, ³¹P and ¹³C NMR studies of intact systems have provided information about the energetics and biochemistry of intact cells, and applications involving these nuclei have been reviewed extensively (Hollis, 1980; Iles et al.,1982; Baxter et al.,1983; London, 1988). A number of other nuclei have also proven useful, including sodium and fluorine; in vivo applications utilizing both are reviewed in this volume (Miller and Elgavish, this volume; Selinsky and Burt, this volume). Although deuterium tracers have proven useful for mapping out the biosynthetic pathways of secondary metabolites using in vitro NMR methods (Abell, 1986, and references therein), initial consideration of some of the limitations of this isotopequadrupolar broadening, significant kinetic isotope effects, and small chemical shift dispersion—suggests that it will have limited use as an in vivo tracer. Despite these limitations, several factors make deuterium a favorable isotope for in vivo metabolic NMR studies in some cases. The low natural abundance (1.56 × 10^−2%) minimizes interference from resonances of endogenous compounds. The relatively short spin-lattice relaxation times due to the quadrupolar relaxation mechanism, lead to important real-time sensitivity gains and minimize the intensity distortions which can arise from overpulsing. In contrast to ¹³C studies, it is generally not necessary to use decoupling methods which are difficult to implement in vivo. The relative ease of synthesis of many deuterated compounds and related low cost of commercially available deuterated materials, particularly in comparison with ¹³C-labeled compounds, is also an attractive advantage for metabolic studies. In general, in vivo studies with ²H may be useful for situations in which a relatively limited number of spectral lines are present, and in which the molecular size and/or rotational correlation time of labeled metabolites are sufficiently small so that quadrupolar broadening is not excessive. In the experience of this reviewer, the major advantage of using deuterium as an in vivo tracer is the extreme technical ease with which studies can be carried out. This factor has led to the suggested use of deuterated metabolites in pilot or feasibility studies even in situations in which it may ultimately prove necessary to use a different labeling strategy (Eng et al., 1990). Deuterium labels provide information about the chemistry of the labeled “proton” which in some cases cannot be obtained using ¹³C labels, as illustrated in the studies of formaldehyde metabolism (e.g., Mason and Sanders, 1989). Additionally, the presence of deuterium labels in metabolites of interest has been detected based on the perturbations of proton or carbon resonances of the molecule. Recent ^2H NMR studies have utilized D₂O as a freely diffusible tracer to measure blood flow and tissue perfusion (Ackerman et al., 1987a, b), and the potential of deuterium imaging has also been explored (Ewy et al., 1986, 1988; Muller and Seelig, 1987; Link and Seelig, 1990). The present review provides a general consideration of the use of ²H labels in metabolic studies (Section 2) as well as summarizing some recent metabolic (Section 3) and imaging and perfusion studies (Section 4). The general area of ²H NMR studies of lipids, which includes some in vivo NMR work, has been reviewed elsewhere (Jacobs and Oldfield, 1980; Seelig and Macdonald, 1987) and is not covered here. Finally, we note that for this review we have adopted the following nomenclature: the symbol “D” is used in chemical formulas, e.g., HDO, and “²H” is used to denote labeling when the common name is used, e.g., [methyl-²H₃]lactate.