A. Dean Sherry

Alternatives to Gadolinium-Based Metal Chelates for Magnetic Resonance Imaging†

Magnetic Resonance Imaging (MRI) has been immensely valuable in diagnostic clinical imaging over the last few decades owing to its exceptional spatial and anatomical resolution. The signal in MRI is generated by relaxation of the transverse component of the net magnetization of protons present in the body, predominantly from bulk water. Thus, any agent or process that affects the net magnetization of the water protons in body tissues will also influence image contrast. Gd3+-based contrast agents shorten both the longitudinal and transverse relaxation times (T1 and T2) of water protons to approximately the same extent, in essence by relaxing all nearby proton spins. This effect is detected as increased signal intensity in T1 weighted MRI images when the appropriate pulse sequence is applied. Over the past 25 years Gd3+-complexes have been spectacularly successful as extracellular or blood pool T1 agents but their relative insensitivity to changes in environment coupled with the fact that they are never completely silent limits their applicability in the design of responsive MRI agents. A conceptually different approach to contrast enhancement is based on chemical exchange saturation transfer (CEST). This technique relies on dynamic chemical exchange processes inherent in biological tissues to transfer saturated 1H spins into the bulk water proton pool, which leads to a decrease of net magnetization and is detected as a negative contrast (darkening of the image) in MRI. Originally exchangeable -NH and -OH protons of various biomolecules were used to generate CEST contrast (DIACEST). However, these agents suffer from a few drawbacks, particularly in association with the small, usually less than 6 ppm, chemical shift difference between the two exchanging pools. The great benefit of using paramagnetic hyperfine shifting lanthanide complexes as CEST agents (PARACEST) is that the chemical shift difference between the two exchanging pools can potentially be much larger, up to several hundred ppm, facilitating easy saturation of one of the exchangeable spin pools without partial saturation of the bulk water pool. Another advantage of PARACEST is that the exchangeable sites are not limited to -NH or -OH protons but sites with faster exchange rates such as a Ln3+-bound H2O molecule, in particular, can also be considered. Since the water exchange rate on lanthanide complexes is extremely sensitive to the chemical environment, this has created unprecedented opportunities in the design of responsive PARACEST agents. In addition, multi-frequency MRI imaging is inherent to PARACEST: multiple agents present in the body can be imaged in one experiment by selectively turning on and off each agent by applying the appropriate saturation frequency.

MRI detection of glycogen in vivo by using chemical exchange saturation transfer imaging (glycoCEST).

Detection of glycogen in vivo would have utility in the study of normal physiology and many disorders. Presently, the only magnetic resonance (MR) method available to study glycogen metabolism in vivo is 13C MR spectroscopy, but this technology is not routinely available on standard clinical scanners. Here, we show that glycogen can be detected indirectly through the water signal by using selective radio frequency (RF) saturation of the hydroxyl protons in the 0.5- to 1.5-ppm frequency range downfield from water. The resulting saturated spins are rapidly transferred to water protons via chemical exchange, leading to partial saturation of the water signal, a process now known as chemical exchange saturation transfer. This effect is demonstrated in glycogen phantoms at magnetic field strengths of 4.7 and 9.4 T, showing improved detection at higher field in adherence with MR exchange theory. Difference images obtained during RF irradiation at 1.0 ppm upfield and downfield of the water signal showed that glycogen metabolism could be followed in isolated, perfused mouse livers at 4.7 T before and after administration of glucagon. Glycogen breakdown was confirmed by measuring effluent glucose and, in separate experiments, by 13C NMR spectroscopy. This approach opens the way to image the distribution of tissue glycogen in vivo and to monitor its metabolism rapidly and noninvasively with MRI.

Numerical solution of the Bloch equations provides insights into the optimum design of PARACEST agents for MRI.

Paramagnetic lanthanide complexes that display unusually slow water exchange between an inner sphere coordination site and bulk water may serve as a new class of MRI contrast agents with the use of chemical exchange saturation transfer (CEST) techniques. To aid in the design of paramagnetic CEST agents for reporting important biological indices in MRI measurements, we formulated a theoretical framework based on the modified Bloch equations that relates the chemical properties of a CEST agent (e.g., water exchange rates and bound water chemical shifts) and various NMR parameters (e.g., relaxation rates and applied B1 field) to the measured CEST effect. Numerical solutions of this formulation for complex exchanging systems were readily obtained without algebraic manipulation or simplification. For paramagnetic CEST agents of the type used here, the CEST effect is relatively insensitive to the bound proton relaxation times, but requires a sufficiently large applied B1 field to highly saturate the Ln3+‐bound water protons. This in turn requires paramagnetic complexes with large Ln3+‐bound water chemical shifts to avoid direct excitation of the exchanging bulk water protons. Although increasing the exchange rate of the bound protons enhances the CEST effect, this also causes exchange broadening and increases the B1 required for saturation. For a given B1, there is an optimal exchange rate that results in a maximal CEST effect. This numerical approach, which was formulated for a three‐pool case, was incorporated into a MATLAB nonlinear least‐square optimization routine, and the results were in excellent agreement with experimental Z‐spectra obtained with an aqueous solution of a paramagnetic CEST agent containing two different types of bound protons (bound water and amide protons). Magn Reson Med 53:790–799, 2005.

Chemical Exchange Saturation Transfer Contrast Agents for Magnetic Resonance Imaging

Magnetic resonance imaging (MRI) contrast agents have become an important tool in clinical medicine. The most common agents are Gd(3+)-based complexes that shorten bulk water T(1) by rapid exchange of a single inner-sphere water molecule with bulk solvent water. Current gadolinium agents lack tissue specificity and typically do not respond to their chemical environment. Recently, it has been demonstrated that MR contrast may be altered by an entirely different mechanism based on chemical exchange saturation transfer (CEST). CEST contrast can originate from exchange of endogenous amide or hydroxyl protons or from exchangeable sites on exogenous CEST agents. This has opened the door for the discovery of new classes of responsive agents ranging from MR gene reporter molecules to small molecules that sense their tissue environment and respond to biological events.

13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS).

Cellular metabolism of glucose is required for stimulation of insulin secretion from pancreatic β cells, but the precise metabolic coupling factors involved in this process are not known. In an effort to better understand mechanisms of fuel-mediated insulin secretion, we have adapted 13C NMR and isotopomer methods to measure influx of metabolic fuels into the tricarboxylic acid (TCA) cycle in insulinoma cells. Mitochondrial metabolism of [U-13C3]pyruvate, derived from [U-13C6]glucose, was compared in four clonal rat insulinoma cell 1-derived cell lines with varying degrees of glucose responsiveness. A 13C isotopomer analysis of glutamate isolated from these cells showed that the fraction of acetyl-CoA derived from [U-13C6]glucose was the same in all four cell lines (44 ± 5%, 70 ± 3%, and 84 ± 4% with 3, 6, or 12 mM glucose, respectively). The 13C NMR spectra also demonstrated the existence of two compartmental pools of pyruvate, one that exchanges with TCA cycle intermediates and a second pool derived from [U-13C6]glucose that feeds acetyl-CoA into the TCA cycle. The 13C NMR spectra were consistent with a metabolic model where the two pyruvate pools do not randomly mix. Flux between the mitochondrial intermediates and the first pool of pyruvate (pyruvate cycling) varied in proportion to glucose responsiveness in the four cell lines. Furthermore, stimulation of pyruvate cycling with dimethylmalate or its inhibition with phenylacetic acid led to proportional changes in insulin secretion. These findings indicate that exchange of pyruvate with TCA cycle intermediates, rather than oxidation of pyruvate via acetyl-CoA, correlates with glucose-stimulated insulin secretion.

Composition of adipose tissue and marrow fat in humans by 1H NMR at 7 Tesla

Proton NMR spectroscopy at 7 Tesla (7T) was evaluated as a new method to quantify human fat composition noninvasively. In validation experiments, the composition of a known mixture of triolein, tristearin, and trilinolein agreed well with measurements by 1H NMR spectroscopy. Triglycerides in calf subcutaneous tissue and tibial bone marrow were examined in 20 healthy subjects by 1H spectroscopy. Ten well-resolved proton resonances from triglycerides were detected using stimulated echo acquisition mode sequence and small voxel (∼0.1 ml), and T1 and T2 were measured. Triglyceride composition was not different between calf subcutaneous adipose tissue and tibial marrow for a given subject, and its variation among subjects, as a result of diet and genetic differences, fell in a narrow range. After correction for differential relaxation effects, the marrow fat composition was 29.1 ± 3.5% saturated, 46.4 ± 4.8% monounsaturated, and 24.5 ± 3.1% diunsaturated, compared with adipose fat composition, 27.1 ± 4.2% saturated, 49.6 ± 5.7% monounsaturated, and 23.4 ± 3.9% diunsaturated. Proton spectroscopy at 7T offers a simple, fast, noninvasive, and painless method for obtaining detailed information about lipid composition in humans, and the sensitivity and resolution of the method may facilitate longitudinal monitoring of changes in lipid composition in response to diet, exercise, and disease.

Hyperpolarized 13C allows a direct measure of flux through a single enzyme-catalyzed step by NMR

13C NMR is a powerful tool for monitoring metabolic fluxes in vivo. The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing 13C nuclei now offers the potential to measure metabolic fluxes through select enzyme-catalyzed steps with substantially improved sensitivity. Here, we investigated the metabolism of hyperpolarized [1-13C1]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved 13CO2, the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of ≈1 s along with H13CO3−, the hydrated form of 13CO2 generated catalytically by carbonic anhydrase. In hearts presented with the medium-chain fatty acid octanoate in addition to hyperpolarized [1-13C1]pyruvate, production of 13CO2 and H13CO3− was suppressed by ≈90%, whereas the signal from [1-13C1]lactate was enhanced. In separate experiments, it was shown that O2 consumption and tricarboxylic acid (TCA) cycle flux were unchanged in the presence of added octanoate. Thus, the rate of appearance of 13CO2 and H13CO3− from [1-13C1]pyruvate does not reflect production of CO2 in the TCA cycle but rather reflects flux through pyruvate dehydrogenase exclusively.

Metabolic cycling in control of glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) is central to normal control of metabolic fuel homeostasis, and its impairment is a key element of beta-cell failure in type 2 diabetes. Glucose exerts its effects on insulin secretion via its metabolism in beta-cells to generate stimulus/secretion coupling factors, including a rise in the ATP/ADP ratio, which serves to suppress ATP-sensitive K(+) (K(ATP)) channels and activate voltage-gated Ca(2+) channels, leading to stimulation of insulin granule exocytosis. Whereas this K(ATP) channel-dependent mechanism of GSIS has been broadly accepted for more than 30 years, it has become increasingly apparent that it does not fully describe the effects of glucose on insulin secretion. More recent studies have demonstrated an important role for cyclic pathways of pyruvate metabolism in control of insulin secretion. Three cycles occur in islet beta-cells: the pyruvate/malate, pyruvate/citrate, and pyruvate/isocitrate cycles. This review discusses recent work on the role of each of these pathways in control of insulin secretion and builds a case for the particular relevance of byproducts of the pyruvate/isocitrate cycle, NADPH and alpha-ketoglutarate, in control of GSIS.

Primer on gadolinium chemistry

Gadolinium is widely known by all practitioners of magnetic resonance imaging (MRI) but few appreciate the basic solution chemistry of this trivalent lanthanide ion. Given the recent linkage between gadolinium contrast agents and nephrogenic systemic fibrosis, some basic chemistry of this ion must be more widely understood. This short primer on gadolinium chemistry is intended to provide the reader the background principles necessary to understand the basics of chelation chemistry, water hydration numbers, and the differences between thermodynamic stability and kinetic stability or inertness. We illustrate the fundamental importance of kinetic dissociation rates in determining gadolinium toxicity in vivo by presenting new data for a novel europium DOTA‐tetraamide complex that is relatively unstable thermodynamically yet extraordinarily inert kinetically and also quite nontoxic. This, plus other literature evidence, forms the basis of the fundamental axiom that it is the kinetic stability of a gadolinium complex, not its thermodynamic stability, that determines its in vivo toxicity. J. Magn. Reson. Imaging 2009;30:1240–1248.

A Pyruvate Cycling Pathway Involving Cytosolic NADP-dependent Isocitrate Dehydrogenase Regulates Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet β-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-regulated K+ (KATP) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in β-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.