Probing carbohydrate metabolism using hyperpolarized 13 C-labeled molecules.

Glycolysis is a fundamental metabolic process in all organisms. Anomalies in glucose metabolism are linked to various pathological conditions. In particular, elevated aerobic glycolysis is a characteristic feature of rapidly growing cells. Glycolysis and the closely related pentose phosphate pathway can be monitored in real time by hyperpolarized 13 C-labeled metabolic substrates such as 13 C-enriched, deuterated D-glucose derivatives, [2-13 C]-D-fructose, [2-13 C] dihydroxyacetone, [1-13 C]-D-glycerate, [1-13 C]-D-glucono-δ-lactone and [1-13 C] pyruvate in healthy and diseased tissues. Elevated glycolysis in tumors (the Warburg effect) was also successfully imaged using hyperpolarized [U-13 C6 , U-2 H7 ]-D-glucose, while the size of the preexisting lactate pool can be measured by 13 C MRS and/or MRI with hyperpolarized [1-13 C]pyruvate. This review summarizes the application of various hyperpolarized 13 C-labeled metabolites to the real-time monitoring of glycolysis and related metabolic processes in normal and diseased tissues.

The influence of Ho3+ doping on 13C DNP in the presence of BDPA.

Polarization transfer from unpaired electron radicals to nuclear spins at low-temperature is achieved using microwave irradiation by a process broadly termed dynamic nuclear polarization (DNP). The resulting signal enhancement can easily exceed factors of 104 when paired with cryogenic cooling of the sample. Dissolution-DNP couples low temperature polarization methods with a rapid dissolution step, resulting in a highly polarized solution that can be used for metabolically sensitive magnetic resonance imaging (MRI). Hyperpolarized [1-13C]pyruvate is a powerful metabolic imaging agent for investigation of in vitro and in vivo cellular metabolism by means of NMR spectroscopy and MRI. Radicals (trityl OX063 and BDPA) with narrower EPR linewidths typically produce higher nuclear polarizations when carbon-13 is the target nucleus. Increased solid-state polarization is observed when narrow line radicals are doped with lanthanide ions such as Gd3+, Ho3+, Dy3+, and Tb3+. Earlier results have demonstrated an incongruence between DNP experiments with trityl and BDPA, where the optimal concentrations for polarization transfer are disparate despite similar electron spin resonance linewidths. Here, the effects of Ho-DOTA on the solid-state polarization of [1-13C]pyruvic acid were compared for 3.35 T (1.4 K) and 5 T (1.2 K) systems using BDPA as a radical. Multiple concentrations of BDPA were doped with variable concentrations of Ho-DOTA (0, 0.2, 0.5, 1, and 2 mM), and dissolved in 1 : 1 (v/v) of [1-13C] pyruvic acid/sulfolane mixture. Our results reveal that addition of small amounts of Ho-DOTA in the sample preparation increases the solid-state polarization for [1-13C] pyruvic acid, with the optimum Ho-DOTA concentration of 0.2 mM. Without Ho-DOTA doping, the optimum BDPA concentration found for 3.35 T (1.4 K) is 40 mM, and for 5 T (1.2 K) system it is about 60 mM. In both systems, inclusion of Ho-DOTA in the 13C DNP sample leads to a change in the breadth (ΔDNP) of the extrema between the P(+) and P(-) frequencies in microwave spectra. At no combination of BDPA and Ho3+ did polarizations reach those achievable with trityl. Simplified analysis of increased polarization as a function of decreased electron T1e used to explain results in trityl are insufficient to describe DNP with BDPA.

Improved Efficacy of Synthesizing *MIII-Labeled DOTA Complexes in Binary Mixtures of Water and Organic Solvents. A Combined Radio- and Physicochemical Study.

Typically, the synthesis of radiometal-based radiopharmaceuticals is performed in buffered aqueous solutions. We found that the presence of organic solvents like ethanol increased the radiolabeling yields of [68Ga]Ga-DOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacatic acid). In the present study, the effect of organic cosolvents [ethanol (EtOH), isopropyl alcohol, and acetonitrile] on the radiolabeling yields of the macrocyclic chelator DOTA with several trivalent radiometals (gallium-68, scandium-44, and lutetium-177) was systematically investigated. Various binary water (H2O)/organic solvent mixtures allowed the radiolabeling of DOTA at a significantly lower temperature than 95 °C, which is relevant for the labeling of sensitive biological molecules. Simultaneously, much lower amounts of the chelators were required. This strategy may have a fundamental impact on the formulation of trivalent radiometal-based radiopharmaceuticals. The equilibrium properties and formation kinetics of [M(DOTA)]- (MIII= GaIII, CeIII, EuIII, YIII, and LuIII) complexes were investigated in H2O/EtOH mixtures (up to 70 vol % EtOH). The protonation constants of DOTA were determined by pH potentiometry in H2O/EtOH mixtures (0-70 vol % EtOH, 0.15 M NaCl, 25 °C). The log K1H and log K2H values associated with protonation of the ring N atoms decreased with an increase of the EtOH content. The formation rates of [M(DOTA)]- complexes increase with an increase of the pH and [EtOH]. Complexation occurs through rapid formation of the diprotonated [M(H2DOTA)]+ intermediates, which are in equilibrium with the kinetically active monoprotonated [M(HDOTA)] intermediates. The rate-controlling step is deprotonation (and rearrangement) of the monoprotonated intermediate, which occurs through H2O (*M(HL) kH2O) and OH- (*M(HL) kOH) assisted reaction pathways. The rate constants are essentially independent of the EtOH concentration, but the M(HL) kH2O values increase from CeIII to LuIII. However, the log KM(HL)H protonation constants, analogous to the log KH2 value, decrease with increasing [EtOH], which increases the concentration of the monoprotonated M(HDOTA) intermediate and accelerates formation of the final complexes. The overall rates of complex formation calculated by the obtained rate constants at different EtOH concentrations show a trend similar to that of the complexation rates determined with the use of radioactive isotopes.

The effect of Ho3+ doping on 13C dynamic nuclear polarization at 5 T.

Dissolution dynamic nuclear polarization was introduced in 2003 as a method for producing hyperpolarized 13C solutions suitable for metabolic imaging. The signal to noise ratio for the imaging experiment depends on the maximum polarization achieved in the solid state. Hence, optimization of the DNP conditions is essential. To acquire maximum polarization many parameters related to sample preparation can be modulated. Recently, it was demonstrated that Ho3+, Dy3+, Tb3+, and Gd3+ complexes enhance the polarization at 1.2 K and 3.35 T when using the trityl radical as the primary paramagnetic center. Here, we have investigated the influence of Ho-DOTA on 13C solid state DNP at 1.2 K and 5 T. We have performed 13C DNP on [1-13C] sodium acetate in 1 : 1 (v/v) water/glycerol with 15 mM trityl OX063 radicals in the presence of a series of Ho-DOTA concentrations (0, 0.5, 1, 2, 3, 5 mM). We have found that adding a small amount of Ho-DOTA in the sample preparation not only enhances the 13C polarization but also decreases the buildup time. The optimum Ho-DOTA concentration was 2 mM. In addition, the microwave sweep spectrum changes character in a manner that suggests both the cross effect and thermal mixing are active mechanisms for trityl radical at 5 T and 1.2 K.

Stilbene vinyl sulfonamides as fluorogenic sensors of and traceless covalent kinetic stabilizers of transthyretin that prevent amyloidogenesis.

Small molecules that react selectively with a specific non-enzyme drug-target protein in a complex biological environment without displacement of a leaving group (tracelessly) are rare and highly desirable. Herein we describe the development of a family of fluorogenic stilbene-based vinyl amides and vinyl sulfonamides that covalently modify transthyretin (TTR) tracelessly. These small molecules bind selectively to TTR in complex biological environments and then undergo a rapid and chemoselective 1,4-Michael addition with the pKa-perturbed Lys-15 ε-amino group of TTR. Replacing the vinyl amide in 2 with the more reactive vinyl sulfonamide in 4 hastens the conjugation kinetics. X-ray cocrystallography verified the formation of the secondary amine bond mediating the conjugation in the case of 2 and 4 and confirmed the expected orientation of the stilbene within the TTR binding sites. Vinyl amide 2 and vinyl sulfonamide 4 potently inhibit TTR dissociation and amyloid fibril formation in vitro. The TTR binding selectivity, modification yield, and reaction chemoselectivity of vinyl sulfonamide 4 are good enough in human plasma to serve as a starting point for medicinal chemistry efforts. Moreover, vinyl sulfonamide 4 is fluorogenic: it exhibits minimal background fluorescence in complex biological environments, remains dark upon binding to TTR, and becomes fluorescent only upon reaction with TTR. The fluorogenicity of 4 was utilized to accurately quantify the native TTR concentration in Escherichia coli lysate using a fluorescence plate reader.

MRI Methods for Imaging Beta-Cell Function in the Rodent Pancreas.

The role of Zn2+ ions in proper storage of insulin in ß-cell granules is well-established so when insulin is secreted from ß-cells stimulated by an increase in plasma glucose, free Zn2+ ions are also released. This local increase in Zn2+ can be detected in the pancreas of rodents in real time by the use of a zinc-responsive MR contrast agent. This method offers the opportunity to monitor ß-cell function longitudinally in live rodents. The methods used in our lab are fully described in this short report and some MR images of a rat pancreas showing clearly enhanced hot spots in the tail are presented.

Investigations into the Signaling Pathways Involving Glucose-Stimulated Zinc Secretion (GSZS) from Prostate Epithelial Cells In Vitro and In Vivo.

PURPOSE: Recently, we reported that exposure of prostate cells in vitro or the in vivo prostate to high glucose results in release of Zn2+ ions, a process now referred to as glucose-stimulated zinc secretion (GSZS). To our knowledge, the metabolic event(s) that trigger GSZS remain largely unknown. Here, we explore several signaling pathways both in vitro using a prostate epithelial cell line and in vivo from the rat prostate. METHODS: PNT1A cells grown to confluence were washed and tagged with ZIMIR to monitor zinc secretion by optical methods. The expression levels of GLUT1, GLUT4, and Akt in cells cultured in either zinc-rich or zinc-poor media and after exposure to high versus low glucose were determined. Zinc secretion from the rat prostate in vivo as detected by MRI was compared in control animals after injection of glucose, deoxyglucose, or pyruvate to initiate zinc secretion and in animals pre-treated with WZB-117 (a GLUT1 inhibitor) or S961 (a peripheral insulin receptor inhibitor). RESULTS: PNT1A cells exposed to high levels of glucose secrete zinc whereas cells exposed to an equivalent amount of deoxyglucose or pyruvate do not. Expression of Akt was dramatically altered by zinc supplementation of the culture media but not after exposure to glucose while GLUT1 and GLUT4 levels were less affected. Rats pre-treated with WZB-117 prior to imaging showed a reduction in GSZS from the prostate compared to controls whereas rats pre-treated with S961 showed no difference. Interestingly, in comparison to PNT1A cells, pyruvate and deoxyglucose also stimulate zinc secretion in vivo likely through indirect mechanisms. CONCLUSIONS: GSZS requires metabolism of glucose both in vitro (PNT1A cells) and in vivo (rat prostate). Pyruvate also stimulates zinc secretion in vivo but likely via an indirect pathway involving rapid production of glucose via gluconeogenesis. These combined results support the conclusion that glycolytic flux is required to trigger GSZS in vivo.

Imaging ß-Cell Function Using a Zinc-Responsive MRI Contrast Agent May Identify First Responder Islets.

An imaging method for detecting ß-cell function in real-time in the rodent pancreas could provide new insights into the biological mechanisms involving loss of ß-cell function during development of type 2 diabetes and for testing of new drugs designed to modulate insulin secretion. In this study, we used a zinc-responsive MRI contrast agent and an optimized 2D MRI method to show that glucose stimulated insulin and zinc secretion can be detected as functionally active "hot spots" in the tail of the rat pancreas. A comparison of functional images with histological markers show that insulin and zinc secretion does not occur uniformly among all pancreatic islets but rather that some islets respond rapidly to an increase in glucose while others remain silent. Zinc and insulin secretion was shown to be altered in streptozotocin and exenatide treated rats thereby verifying that this simple MRI technique is responsive to changes in ß-cell function.

Detection of glucose-derived D- and L-lactate in cancer cells by the use of a chiral NMR shift reagent.

BACKGROUND: Excessive lactate production, a hallmark of cancer, is largely formed by the reduction of pyruvate via lactate dehydrogenase (LDH) to L-lactate. Although D-lactate can also be produced from glucose via the methylglyoxal pathway in small amounts, less is known about the amount of D-lactate produced in cancer cells. Since the stereoisomers of lactate cannot be distinguished by conventional 1H NMR spectroscopy, a chiral NMR shift reagent was used to fully resolve the 1H NMR resonances of D- and L-lactate. METHODS: The production of L-lactate from glucose and D-lactate from methylglyoxal was first demonstrated in freshly isolated red blood cells using the chiral NMR shift reagent, YbDO3A-trisamide. Then, two different cell lines with high GLO1 expression (H1648 and H 1395) were selected from a panel of over 80 well-characterized human NSCLC cell lines, grown to confluence in standard tissue culture media, washed with phosphate-buffered saline, and exposed to glucose in a buffer for 4 h. After 4 h, a small volume of extracellular fluid was collected and mixed with YbDO3A-trisamide for analysis by 1H NMR spectroscopy. RESULTS: A suspension of freshly isolated red blood cells exposed to 5mM glucose produced L-lactate as expected but very little D-lactate. To evaluate the utility of the chiral NMR shift reagent, methylglyoxal was then added to red cells along with glucose to stimulate the production of D-lactate via the glyoxalate pathway. In this case, both D-lactate and L-lactate were produced and their NMR chemical shifts assigned. NSCLC cell lines with different expression levels of GLO1 produced both L- and D-lactate after incubation with glucose and glutamine alone. A GLO1-deleted parental cell line (3553T3) showed no production of D-lactate from glucose while re-expression of GLO1 resulted in higher production of D-lactate. CONCLUSIONS: The shift-reagent-aided NMR technique demonstrates that D-lactate is produced from glucose in NSCLC cells via the methylglyoxal pathway. The biological role of D-lactate is uncertain but a convenient method for monitoring D-lactate production could provide new insights into the biological roles of D- versus L-lactate in cancer metabolism.

In Vivo ZIMIR Imaging of Mouse Pancreatic Islet Cells Shows Oscillatory Insulin Secretion.

Appropriate insulin secretion is essential for maintaining euglycemia, and impairment or loss of insulin release represents a causal event leading to diabetes. There have been extensive efforts of studying insulin secretion and its regulation using a variety of biological preparations, yet it remains challenging to monitor the dynamics of insulin secretion at the cellular level in the intact pancreas of living animals, where islet cells are supplied with physiological blood circulation and oxygenation, nerve innervation, and tissue support of surrounding exocrine cells. Herein we presented our pilot efforts of ZIMIR imaging in pancreatic islet cells in a living mouse. The imaging tracked insulin/Zn2+ release of individual islet ß-cells in the intact pancreas with high spatiotemporal resolution, revealing a rhythmic secretion activity that appeared to be synchronized among islet ß-cells. To facilitate probe delivery to islet cells, we also developed a chemogenetic approach by expressing the HaloTag protein on the cell surface. Finally, we demonstrated the application of a fluorescent granule zinc indicator, ZIGIR, as a selective and efficient islet cell marker in living animals through systemic delivery. We expect future optimization and integration of these approaches would enable longitudinal tracking of beta cell mass and function in vivo by optical imaging.

13C-Labeled Diethyl Ketoglutarate Derivatives as Hyperpolarized Probes of 2-Ketoglutarate Dehydrogenase Activity.

The TCA cycle is a central metabolic pathway for energy production and biosynthesis. A major control point of metabolic flux through the cycle is the decarboxylation of 2-ketoglutarate by the TCA cycle enzyme 2-ketoglutarate dehydrogenase (2-KGDH). In this project, we developed 13C labeled 2-ketoglutarate derivatives to monitor 2-KGDH activity in vivo. 13C NMR analysis of liver extracts revealed that uniformly 13C labeled 2-ketogutarate, in its cell permeable ester form, was rapidly taken up and hydrolyzed in liver and underwent extensive metabolism to produce labeled glutamate, succinate, lactate and other metabolites. Diethyl [1,2-13C2]-2-ketoglutarate was successfully polarized by dynamic nuclear polarization and within seconds after injection into rats, the probe produced hyperpolarized [13C]bicarbonate in the liver reflecting flux through the TCA cycle. These experiments demonstrate that this tracer offers the possibility of directly monitoring flux through 2-KGDH in vivo.

Hyperpolarized 89Y complexes as pH sensitive NMR probes.

Hyperpolarization can increase the sensitivity of NMR/MRI experiments, but the primary limitation is the T(1) decay of magnetization. Due to its long T(1), the hyperpolarized (89)Y nucleus makes an excellent candidate as an in vivo spectroscopy/imaging probe. Here we report the (89)Y chemical shift dependence upon pH for two hyperpolarized (89)Y(III) complexes and demonstrate how such complexes can be used as sensitive spectroscopy/imaging agents to measure pH.

Hyperpolarized 15N-labeled, deuterated tris (2-pyridylmethyl)amine as an MRI sensor of freely available Zn2.

Dynamic nuclear polarization (DNP) coupled with 15N magnetic resonance imaging (MRI) provides an opportunity to image quantitative levels of biologically important metal ions such as Zn2+, Mg2+ or Ca2+ using appropriately designed 15N enriched probes. For example, a Zn-specific probe could prove particularly valuable for imaging the tissue distribution of freely available Zn2+ ions, an important known metal ion biomarker in the pancreas, in prostate cancer, and in several neurodegenerative diseases. In the present study, we prepare the cell-permeable, 15N-enriched, d6-deuterated version of the well-known Zn2+ chelator, tris(2-pyridylmethyl)amine (TPA) and demonstrate that the polarized ligand had favorable T1 and linewidth characteristics for 15N MRI. Examples of how polarized TPA can be used to quantify freely available Zn2+ in homogenized human prostate tissue and intact cells are presented.

(S)-5-(p-nitrobenzyl)-PCTA, a promising bifunctional ligand with advantageous metal ion complexation kinetics.

A bifunctional version of PCTA (3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid) that exhibits fast complexation kinetics with the trivalent lanthanide(III) ions was synthesized in reasonable yields starting from N,N',N''-tristosyl-(S)-2-(p-nitrobenzyl)-diethylenetriamine. pH-potentiometric studies showed that the basicities of p-nitrobenzyl-PCTA and the parent ligand PCTA were similar. The stability of M(NO(2)-Bn-PCTA) (M = Mg(2+), Ca(2+), Cu(2+), Zn(2+)) complexes was similar to that of the corresponding PCTA complexes, while the stability of Ln(3+) complexes of the bifunctional ligand is somewhat lower than that of PCTA chelates. The rate of complex formation of Ln(NO(2)-Bn-PCTA) complexes was found to be quite similar to that of PCTA, a ligand known to exhibit the fastest formation rates among all lanthanide macrocyclic ligand complexes studied to date. The acid-catalyzed decomplexation kinetic studies of the selected Ln(NO(2)-Bn-PCTA) complexes showed that the kinetic inertness of the complexes was comparable to that of Ln(DOTA) chelates making the bifunctional ligand NO(2)-Bn-PCTA suitable for labeling biological vectors with radioisotopes for nuclear medicine applications.

In vivo assessment of increased oxidation of branched-chain amino acids in glioblastoma.

Altered branched-chain amino acids (BCAAs) metabolism is a distinctive feature of various cancers and plays an important role in sustaining tumor proliferation and aggressiveness. Despite the therapeutic and diagnostic potentials, the role of BCAA metabolism in cancer and the activities of associated enzymes remain unclear. Due to its pivotal role in BCAA metabolism and rapid cellular transport, hyperpolarized 13C-labeled α-ketoisocaproate (KIC), the α-keto acid corresponding to leucine, can assess both BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase complex (BCKDC) activities via production of [1-13C]leucine or 13CO2 (and thus H13CO3-), respectively. Here, we investigated BCAA metabolism of F98 rat glioma model in vivo using hyperpolarized 13C-KIC. In tumor regions, we observed a decrease in 13C-leucine production from injected hyperpolarized 13C-KIC via BCAT compared to the contralateral normal-appearing brain, and an increase in H13CO3-, a catabolic product of KIC through the mitochondrial BCKDC. A parallel ex vivo 13C NMR isotopomer analysis following steady-state infusion of [U-13C]leucine to glioma-bearing rats verified the increased oxidation of leucine in glioma tissue. Both the in vivo hyperpolarized KIC imaging and the leucine infusion study indicate that KIC catabolism is upregulated through BCAT/BCKDC and further oxidized via the citric acid cycle in F98 glioma.

Endoplasmic Reticulum Proteostasis Influences the Oligomeric State of an Amyloidogenic Protein Secreted from Mammalian Cells.

Transthyretin (TTR) is a tetrameric serum protein associated with multiple systemic amyloid diseases. In these disorders, TTR aggregates in extracellular environments through a mechanism involving rate-limiting dissociation of the tetramer to monomers, which then misfold and aggregate into soluble oligomers and amyloid fibrils that induce toxicity in distal tissues. Using an assay established herein, we show that highly destabilized, aggregation-prone TTR variants are secreted as both native tetramers and non-native conformations that accumulate as high-molecular-weight oligomers. Pharmacologic chaperones that promote endoplasmic reticulum (ER) proteostasis of destabilized TTR variants increase their fraction secreted as a tetramer and reduce extracellular aggregate populations. In contrast, disrupting ER proteostasis reduces the fraction of destabilized TTR secreted as a tetramer and increases extracellular aggregates. These results identify ER proteostasis as a factor that can affect conformational integrity and thus toxic aggregation of secreted amyloidogenic proteins associated with the pathology of protein aggregation diseases.

Albumin-binding PARACEST agents.

Lanthanide complexes (Eu(3+), Gd(3+) and Yb(3+)) of two different 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tetraamide derivatives containing two (2) and four (3) O-benzyl-L-serine amide substituents were synthesized and their chemical exchange saturation transfer (CEST) and relaxometric properties were examined in the presence and absence of human serum albumin (HSA). Both Eu2 and Eu3 display a significant CEST effect from a single slowly exchanging Eu(3+)-bound water molecule, making these PARACEST complexes potentially useful as vascular MRI agents. Yb2 also showed a detectable CEST effect from both the Yb(3+)-bound water protons and the exchangeable NH amide protons, making it potentially useful as a vascular pH sensor. Fluorescence displacement studies using reporter molecules indicate that both Gd2 and Gd3 displace dansylsarcosine from site II of HSA with inhibition constants of 32 and 96 microM, respectively, but neither complex significantly displaces warfarin from site I. Water proton relaxation enhancements of 135 and 171% were observed upon binding of Gd2 and Gd3 to HSA, respectively, at 298 K and pH 7.4.