Dual-phase imaging of cardiac metabolism using hyperpolarized pyruvate.

PURPOSE: Previous cardiac imaging studies using hyperpolarized (HP) [1-13 C]pyruvate were acquired at end-diastole (ED). Little is known about the interaction between cardiac cycle and metabolite content in the myocardium. In this study, we compared images of HP pyruvate and products at end-systole (ES) and ED. METHODS: A dual-phase 13 C MRI sequence was implemented to acquire two sequential HP images within a single cardiac cycle at ES and ED during successive R-R intervals in an interleaved manner. Each healthy volunteer (N = 3) received two injections of HP [1-13 C]pyruvate for the dual-phase imaging on the short-axis and the vertical long-axis planes. Spatial distribution of HP 13 C metabolites at each cardiac phase was correlated to multiphase 1 H MRI to confirm the mechanical changes. Ratios of myocardial HP metabolites were compared between ES and ED. Segmental analysis was performed on the midcavity short-axis plane. RESULTS: In addition to mechanical changes, metabolic profiles of the heart detected by HP [1-13 C]pyruvate differed between ES and ED. The myocardial signal of [13 C]bicarbonate relative to [1-13 C]lactate was significantly smaller at ED than the ratio at ES (p < .05), particularly in mid-anterior and mid-inferoseptal segments. The distinct metabolic profiles in the myocardium likely reflect the technical aspects of the imaging approach such as the coronary flow in addition to the cyclical changes in metabolism. CONCLUSION: The study demonstrates that metabolic profiles of the heart, measured by HP [1-13 C]pyruvate, are affected by the cardiac cycle in which that the data are acquired.

Dynamic 13 C MR spectroscopy as an alternative to imaging for assessing cerebral metabolism using hyperpolarized pyruvate in humans.

PURPOSE: This study is to investigate time-resolved 13 C MR spectroscopy (MRS) as an alternative to imaging for assessing pyruvate metabolism using hyperpolarized (HP) [1-13 C]pyruvate in the human brain. METHODS: Time-resolved 13 C spectra were acquired from four axial brain slices of healthy human participants (n = 4) after a bolus injection of HP [1-13 C]pyruvate. 13 C MRS with low flip-angle excitations and a multichannel 13 C/1 H dual-frequency radiofrequency (RF) coil were exploited for reliable and unperturbed assessment of HP pyruvate metabolism. Slice-wise areas under the curve (AUCs) of 13 C-metabolites were measured and kinetic analysis was performed to estimate the production rates of lactate and HCO3- . Linear regression analysis between brain volumes and HP signals was performed. Region-focused pyruvate metabolism was estimated using coil-wise 13 C reconstruction. Reproducibility of HP pyruvate exams was presented by performing two consecutive injections with a 45-minutes interval. RESULTS: [1-13 C]Lactate relative to the total 13 C signal (tC) was 0.21-0.24 in all slices. [13 C] HCO3- /tC was 0.065-0.091. Apparent conversion rate constants from pyruvate to lactate and HCO3- were calculated as 0.014-0.018 s-1 and 0.0043-0.0056 s-1 , respectively. Pyruvate/tC and lactate/tC were in moderate linear relationships with fractional gray matter volume within each slice. White matter presented poor linear regression fit with HP signals, and moderate correlations of the fractional cerebrospinal fluid volume with pyruvate/tC and lactate/tC were measured. Measured HP signals were comparable between two consecutive exams with HP [1-13 C]pyruvate. CONCLUSIONS: Dynamic MRS in combination with multichannel RF coils is an affordable and reliable alternative to imaging methods in investigating cerebral metabolism using HP [1-13 C]pyruvate.

Hyperpolarized 13C MR Spectroscopy Depicts in Vivo Effect of Exercise on Pyruvate Metabolism in Human Skeletal Muscle.

Background Pyruvate dehydrogenase (PDH) and lactate dehydrogenase are essential for adenosine triphosphate production in skeletal muscle. At the onset of exercise, oxidation of glucose and glycogen is quickly enabled by dephosphorylation of PDH. However, direct measurement of PDH flux in exercising human muscle is daunting, and the net effect of covalent modification and other control mechanisms on PDH flux has not been assessed. Purpose To demonstrate the feasibility of assessing PDH activation and changes in pyruvate metabolism in human skeletal muscle after the onset of exercise using carbon 13 (13C) MRI with hyperpolarized (HP) [1-13C]-pyruvate. Materials and Methods For this prospective study, sedentary adults in good general health (mean age, 42 years ± 18 [standard deviation]; six men) were recruited from August 2019 to September 2020. Subgroups of the participants were injected with HP [1-13C]-pyruvate at resting, during plantar flexion exercise, or 5 minutes after exercise during recovery. In parallel, hydrogen 1 arterial spin labeling MRI was performed to estimate muscle tissue perfusion. An unpaired t test was used for comparing 13C data among the states. Results At rest, HP [1-13C]-lactate and [1-13C]-alanine were detected in calf muscle, but [13C]-bicarbonate was negligible. During moderate flexion-extension exercise, total HP 13C signals (tC) increased 2.8-fold because of increased muscle perfusion (P = .005), and HP [1-13C]-lactate-to-tC ratio increased 1.7-fold (P = .04). HP [13C]-bicarbonate-to-tC ratio increased 8.4-fold (P = .002) and returned to the resting level 5 minutes after exercise, whereas the lactate-to-tC ratio continued to increase to 2.3-fold as compared with resting (P = .008). Conclusion Lactate and bicarbonate production from hyperpolarized (HP) [1-carbon 13 {13C}]-pyruvate in skeletal muscle rapidly reflected the onset and the termination of exercise. These results demonstrate the feasibility of imaging skeletal muscle metabolism using HP [1-13C]-pyruvate MRI and the sensitivity of in vivo pyruvate metabolism to exercise states. © RSNA, 2021 Online supplemental material is available for this article.

Cardiac T2∗ measurement of hyperpolarized 13 C metabolites using metabolite-selective multi-echo spiral imaging.

PURPOSE: Noninvasive imaging with hyperpolarized (HP) pyruvate can capture in vivo cardiac metabolism. For proper quantification of the metabolites and optimization of imaging parameters, understanding MR characteristics such as T2∗ s of the HP signals is critical. This study is to measure in vivo cardiac T2∗ s of HP [1-13 C]pyruvate and the products in rodents and humans. METHODS: A dynamic 13 C multi-echo spiral imaging sequence that acquires [13 C]bicarbonate, [1-13 C]lactate, and [1-13 C]pyruvate images in an interleaved manner was implemented for a clinical 3 Tesla system. T2∗ of each metabolite was calculated from the multi-echo images by fitting the signal decay of each region of interest mono-exponentially. The performance of measuring T2∗ using the sequence was first validated using a 13 C phantom and then with rodents following a bolus injection of HP [1-13 C]pyruvate. In humans, T2∗ of each metabolite was calculated for left ventricle, right ventricle, and myocardium. RESULTS: Cardiac T2∗ s of HP [1-13 C]pyruvate, [1-13 C]lactate, and [13 C]bicarbonate in rodents were measured as 24.9 ± 5.0, 16.4 ± 4.7, and 16.9 ± 3.4 ms, respectively. In humans, T2∗ of [1-13 C]pyruvate was 108.7 ± 22.6 ms in left ventricle and 129.4 ± 8.9 ms in right ventricle. T2∗ of [1-13 C]lactate was 40.9 ± 8.3, 44.2 ± 5.5, and 43.7 ± 9.0 ms in left ventricle, right ventricle, and myocardium, respectively. T2∗ of [13 C]bicarbonate in myocardium was 64.4 ± 2.5 ms. The measurements were reproducible and consistent over time after the pyruvate injection. CONCLUSION: The proposed metabolite-selective multi-echo spiral imaging sequence reliably measures in vivo cardiac T2∗ s of HP [1-13 C]pyruvate and products.

Engineering a pH-Sensitive Liposomal MRI Agent by Modification of a Bacterial Channel.

MscL is a bacterial mechanosensitive channel that serves as a cellular emergency release valve, protecting the cell from lysis upon a drop in external osmolarity. The channel has an extremely large pore (30 Å) and can be purified and reconstituted into artificial membranes. Moreover, MscL is modified to open in response to alternative external stimuli including changes in pH. These properties suggest this channel's potential as a triggered "nanopore" for localized release of vesicular contents such as magnetic resonance imaging (MRI) contrast agents and drugs. Toward this end, several variants of pH-triggered MscL nanovalves are engineered. Stealth vesicles previously been shown to evade normal in vivo clearance and passively accumulate in inflamed and malignant tissues are reconstituted. These vesicles are loaded with 1,4,7,10-tetraazacyclododecane tetraacetic acid gadolinium complex (Gd-DOTA), an MRI contrast reagent, and the resulting nanodevices tested for their ability to release Gd-DOTA as evidenced by enhancement of the longitudinal relaxation rate (R1 ) of the bulk water proton spins. Nanovalves that are responsive to physiological pH changes are identified, but differ in sensitivity and efficacy, thus giving an array of nanovalves that could potentially be useful in different settings. These triggered nanodevices may be useful in delivering both diagnostic and therapeutic agents.

Conditions for (13)C NMR detection of 2-hydroxyglutarate in tissue extracts from isocitrate dehydrogenase-mutated gliomas.

(13)C NMR (nuclear magnetic resonance) spectroscopy of extracts from patient tumor samples provides rich information about metabolism. However, in isocitrate dehydrogenase (IDH)-mutant gliomas, (13)C labeling is obscured in oncometabolite 2-hydroxyglutaric acid (2 HG) by glutamate and glutamine, prompting development of a simple method to resolve the metabolites. J-coupled multiplets in 2 HG were similar to glutamate and glutamine and could be clearly resolved at pH 6. A cryogenically cooled (13)C probe, but not J-resolved heteronuclear single quantum coherence spectroscopy, significantly improved detection of 2 HG. These methods enable the monitoring of (13)C-(13)C spin-spin couplings in 2 HG expressing IDH-mutant gliomas.

In vivo imaging of paraCEST agents using frequency labeled exchange transfer MRI.

PURPOSE: A main obstacle to in vivo applications of paramagnetic chemical exchange saturation transfer (paraCEST) is interference from endogenous tissue magnetization transfer contrast (MTC). The suitability of excitation-based frequency labeled exchange transfer (FLEX) to separate out such MTC effects in vivo was tested. METHODS: The FLEX sequence measures modulation of the water signal based on the chemical shift evolution of solute proton magnetization as a function of evolution time. Time-domain analysis of this water signal allows identification of different solute components and provides a mechanism to separate out the rapidly decaying MTC components with short effective transverse relaxation time ( T2*) values. RESULTS: FLEX imaging of paraCEST agents was possible in vitro in phantoms and in vivo in mouse kidneys and bladder. The results demonstrated that FLEX is capable of separating out the MTC signal from tissues in vivo while providing a quantitative exchange rate for the rapidly exchanging paraCEST water protons by fitting the FLEX time-domain signal to FLEX theory. CONCLUSIONS: The first in vivo FLEX images of a paraCEST agent were acquired, which allowed separation of the tissue MTC components. These results show that FLEX imaging has potential for imaging the distribution of functional paraCEST agents in biological tissues.

Using frequency-labeled exchange transfer to separate out conventional magnetization transfer effects from exchange transfer effects when detecting ParaCEST agents.

Paramagnetic chemical exchange saturation transfer agents combine the benefits of a large chemical shift difference and a fast exchange rate for sensitive MRI detection. However, the in vivo detection of these agents is hampered by the need for high B(1) fields to allow sufficiently fast saturation before exchange occurs, thus causing interference of large magnetization transfer effects from semisolid macromolecules. A recently developed approach named frequency-labeled exchange transfer utilizes excitation pulses instead of saturation pulses for detecting the exchanging protons. Using solutions and gel phantoms containing the europium (III) complex of DOTA tetraglycinate (EuDOTA-(gly)(-) (4) ), it is shown that frequency-labeled exchange transfer allows the separation of chemical exchange effects and magnetization transfer (MT) effects in the time domain, therefore allowing the study of the individual resonance of rapidly exchanging water molecules (k(ex) >10(4) s(-1) ) without interference from conventional broad-band MT.

A concentration-independent method to measure exchange rates in PARACEST agents.

The efficiency of chemical exchange dependent saturation transfer (CEST) agents is largely determined by their water or proton exchange kinetics, yet methods to measure such exchange rates are variable and many are not applicable to in vivo measurements. In this work, the water exchange kinetics of two prototype paramagnetic agents (PARACEST) are compared by using data from classic NMR line-width measurements, by fitting CEST spectra to the Bloch equations modified for chemical exchange, and by a method where CEST intensity is measured as a function of applied amplitude of radiofrequency field. A relationship is derived that provides the water exchange rate from the X-intercept of a plot of steady-state CEST intensity divided by reduction in signal caused by CEST irradiation versus 1/omega(1)(2), referred to here as an omega plot. Furthermore, it is shown that this relationship is independent of agent concentration. Exchange rates derived from omega plots using either high-resolution CEST NMR data or CEST data obtained by imaging agree favorably with exchange rates measured by the more commonly used Bloch fitting and line-width methods. Thus, this new method potentially allows access to a direct measure of exchange rates in vivo, where the agent concentration is typically unknown.

Hyperpolarized 89Y-EDTMP complex as a chemical shift-based NMR sensor for pH at the physiological range.

Yttrium (III) complexes are interesting due to the similarity of their chemistry with gadolinium complexes that are used as contrast agents in nuclear magnetic resonance (NMR) spectroscopy or imaging (MRI). While most of the paramagnetic Gd3+-based MRI contrast agents are T1 or T2 relaxation-based sensors such as Gd3+-complexes for zinc or pH detection, a number of diamagnetic Y3+-complexes rely on changes in the chemical shift for potential quantitative MRI in biological milieu. 89Y, however, is a challenging nucleus to work with in conventional NMR or MRI due to its inherently low sensitivity and relatively long T1 relaxation time. This insensitivity problem in 89Y-based complexes can be circumvented with the use of dissolution dynamic nuclear polarization (DNP) which allows for several thousand-fold enhancement of the NMR or MRI signal relative to thermal equilibrium signal. Herein, we report on the feasibility of using hyperpolarized 89Y-complexes with phosphonated open-chain ligands, 89Y-EDTMP and 89Y-DTPMP, as potential chemical shift-based pH NMR sensors. Our DNP-NMR data show that hyperpolarized 89Y-DTPMP has an apparent pKa ~ 7.01 with a 4 ppm-wide chemical shift dispersion with the signal disappearing at pH below 6.2. On the other hand, pH titration data on hyperpolarized 89Y-EDTMP show that it has an apparent pKa of pH 6.7 and a 16-ppm wide chemical shift dispersion at pH 5-9 range. In comparison, the previously reported hyperpolarized pH NMR sensor 89Y-DOTP has a pKa of 7.64 and ~ 10-ppm wide chemical shift dispersion at pH 4-9 range. Overall, our data suggest that hyperpolarized 89Y-EDTMP is better than hyperpolarized 89Y-DOTP in terms of pH sensing capability at the physiological range.

A new gadolinium-based MRI zinc sensor.

The properties of a novel Gd(3+)-based MRI zinc sensor are reported. Unlike previously reported Gd(3+)-based MRI contrast agents, this agent (GdL) differs in that the agent alone binds only weakly with human serum albumin (HSA), while the 1:2 GdL:Zn(2+) ternary complex binds strongly to HSA resulting in a substantial, 3-fold increase in water proton relaxivity. The GdL complex is shown to have a relatively strong binding affinity for Zn(2+) (K(D) = 33.6 nM), similar to the affinity of the Zn(2+) ion with HSA alone. The agent detects as little as 30 microM Zn(2+) in the presence of HSA by MRI in vitro, a value slightly more than the total Zn(2+) concentration in blood (approximately 20 microM). This combination of binding affinity constants and the high relaxivity of the agent when bound to HSA suggests that this new agent may be useful for detection of free Zn(2+) ions in vivo without disrupting other important biological processes involving Zn(2+).

Elucidating the structural organization of a novel low-density lipoprotein nanoparticle reconstituted with docosahexaenoic acid.

Low-density lipoprotein nanoparticles reconstituted with unesterified docosahexaenoic acid (LDL-DHA) is promising nanomedicine with enhanced physicochemical stability and selective anticancer cytotoxic activity. The unique functionality of LDL-DHA ultimately relates to the structure of this nanoparticle. To date, however, little is known about the structural organization of this nanoparticle. In this study chemical, spectroscopic and electron microscopy analyses were undertaken to elucidate the structural and molecular organization of LDL-DHA nanoparticles. Unesterified DHA preferentially incorporates into the outer surface layer of LDL, where in this orientation the anionic carboxyl end of DHA is exposed to the LDL surface and imparts an electronegative charge to the nanoparticles surface. This negative surface charge promotes the monodisperse and homogeneous distribution of LDL-DHA nanoparticles in solution. Further structural analyses with cryo-electron microscopy revealed that the LDL-DHA nanostructure consist of a phospholipid bilayer surrounding an aqueous core, which is distinctly different from the phospholipid monolayer/apolar core organization of plasma LDL. Lastly, apolipoprotein B-100 remains strongly associated with this complex and maintains a discrete size and shape of the LDL-DHA nanoparticles similar to plasma LDL. This preliminary structural assessment of LDL-DHA now affords the opportunity to understand the important structure-function relationships of this novel nanoparticle.

Hepatic gluconeogenesis influences (13)C enrichment in lactate in human brain tumors during metabolism of [1,2-(13)C]acetate.

(13)C-enriched compounds are readily metabolized in human malignancies. Fragments of the tumor, acquired by biopsy or surgical resection, may be acid-extracted and (13)C NMR spectroscopy of metabolites such as glutamate, glutamine, 2-hydroxyglutarate, lactate and others provide a rich source of information about tumor metabolism in situ. Recently we observed (13)C-(13)C spin-spin coupling in (13)C NMR spectra of lactate in brain tumors removed from patients who were infused with [1,2-(13)C]acetate prior to the surgery. We found, in four patients, that infusion of (13)C-enriched acetate was associated with synthesis of (13)C-enriched glucose, detectable in plasma. (13)C labeled glucose derived from [1,2-(13)C]acetate metabolism in the liver and the brain pyruvate recycling in the tumor together lead to the production of the (13)C labeled lactate pool in the brain tumor. Their combined contribution to acetate metabolism in the brain tumors was less than 4.0%, significantly lower than the direct oxidation of acetate in the citric acid cycle in tumors.

Redox- and hypoxia-responsive MRI contrast agents.

The development of responsive or "smart" magnetic resonance imaging (MRI) contrast agents that can report specific biomarker or biological events has been the focus of MRI contrast agent research over the past 20 years. Among various biological hallmarks of interest, tissue redox and hypoxia are particularly important owing to their roles in disease states and metabolic consequences. Herein we review the development of redox-/hypoxia-sensitive T1 shortening and paramagnetic chemical exchange saturation transfer (PARACEST) MRI contrast agents. Traditionally, the relaxivity of redox-sensitive Gd(3+) -based complexes is modulated through changes in the ligand structure or molecular rotation, while PARACEST sensors exploit the sensitivity of the metal-bound water exchange rate to electronic effects of the ligand-pendant arms and alterations in the coordination geometry. Newer designs involve complexes of redox-active metal ions in which the oxidation states have different magnetic properties. The challenges of translating redox- and hypoxia-sensitive agents in vivo are also addressed.