Studying enzymes by in vivo 13C magnetic resonance spectroscopy

Abstract

Magnetic resonance spectroscopy (MRS) allows noninvasive detection of specific biologically relevant molecules in vivo. It has become a very useful and versatile tool for both clinical and basic science studies because it can measure concentrations of many important endogenous and exogenous molecules such as the putative neuronal marker N-acetylaspartate [1], the 19F-containing selective serotonin reuptake inhibitor Prozac [2], glycogen[3], and adenosine triphosphate [4]. For an endogenous molecule, its concentration measured by MRS is usually the result of a complex balance among various metabolic fluxes with each of the fluxes controlled by a host of different enzymes. By introducing exogenous 13C-labeled substrates certain metabolic pathways can be measured using 13C MRS (e.g., the glutamate-glutamine cycling flux in the brain [5, 6]). In addition to concentrations and metabolic fluxes, an exceptional feature of MRS is its ability to measure the rate of an exchange reaction catalyzed by a specific enzyme in vivo using the technique of magnetization (or saturation) transfer. When kinetically relevant reporter molecules are spin labeled and spin transferred, their exchange rate can be quantified based on the competition between chemical exchange and the longitudinal relaxation time (T1). The theory of chemical exchange magnetization transfer was developed by chemists more than fifty years ago [7–12]. The phenomenon of in vivo enzyme-specific chemical exchange magnetization transfer was discovered approximately thirty years ago for adenosine triphosphate (ATP)-related exchange reactions [13] including the exchange reactions catalyzed by creatine kinase [14] and the invertebrate-originated arginine kinase [14]. The ability of noninvasively extracting information from specific enzymes using in vivo MRS is highly significant and has generated a great deal of enthusiasm [14–21]. In particular, creatine kinase-catalyzed magnetization transfer effect has been demonstrated to be a useful magnetic resonance reporter of gene expression [22]. Obviously, it would be highly desirable if more enzymes were accessible to in vivo MRS-based magnetization transfer spectroscopy methods. However, since the early discoveries of the above-mentioned enzymes involved in catalyzing the transfer of phosphate groups no new enzymes exhibiting detectable in vivo magnetization transfer effects had been found until our recent discovery of in vivo 13C magnetization transfer effects [23, 24]. Our interests in magnetization transfer started with the long-standing controversies on the rate of exchange between brain cytosolic glutamate/aspartate and mitochondrial α-ketoglutarate/oxaloacetate pools extracted from metabolic modeling of in vivo 13C MRS data. We hypothesized that if this exchange rate is very rapid it should be directly measurable using magnetization transfer. This line of research first led to the discovery of the in vivo magnetization transfer effect catalyzed by aspartate aminotransferase (AAT), then by lactate dehydrogenase (LDH) [25], malate dehydrogenase (MDH) [26], and carbonic anhydrase (CA) [27]. We demonstrated that the chemical exchange processes of these enzymes could be measured by 13C saturation transfer with 13C detection [24–27] and/or 13C saturation transfer with 1H detection techniques [28]. We also found that the exchange between 13C-labeled mitochondrial and cytosolic pools in brain is much faster than the tricarboxylic acid (TCA) cycle flux [29]. Here we endeavor to first give a brief overview of the early work in the field of in vivo 31P magnetization transfer spectroscopy because it is beyond the scope of this article to comprehensively review all in vivo magnetization transfer studies conducted on ATP-related enzymes using 31P MRS (the interested readers are referred to several excellent reviews on this topic [19–21]), Previous in vitro studies of enzyme systems using 13C NMR spectroscopy are also discussed. Then we will present the theoretical analyses and the experimental methods associated with detecting in vivo 13C magnetization transfer effects of a rapid chemical exchange process between small and large substrate pools, and review the current applications of in vivo 13C magnetization transfer spectroscopy to the study of enzymes. The chemical shifts of chemicals involved in enzyme-specific 13C magnetization transfer effects discovered so far are given in Table 1. Table 1 13C and 1H chemical shifts of molecules involved in enzyme-specific 13C magnetization transfer effects 2. Overview Creatine kinase (CK) has proven to be particularly amenable - in conjunction with 31P magnetization transfer spectroscopy - for elucidating rapid chemical exchange processes. CK catalyzes the phosphate of phosphocreatine (PCr) exchanges with the adenosine triphosphate (ATP) reaction, and is a key enzyme for maintaining cellular energy supplies: