Differentiation and characterization of healthy versus pathological tau using chemical exchange saturation transfer.

Neurofibrillary tangles of tau constitute one of the key biological hallmarks of Alzheimer's disease. Currently, the assessment of regional tau accumulation requires intravenous administration of radioactive tracers for PET imaging. A noninvasive MRI-based solution would have significant clinical implications. Herein, we utilized an MRI technique known as chemical exchange saturation transfer (CEST) to determine the imaging signature of tau in both its monomeric and pathologic fibrillated conformations. Three sets of purified recombinant full-length (4R) tau protein were prepared for collection of CEST spectra using a 9.4 T NMR spectrometer at varying temperatures (25, 37, and 42 °C) and RF intensities (0.7, 1.0, 1.5, and 2.2 µT). Monomeric and fibrillated tau were readily distinguished based on their Z-spectrum profiles. Fibrillated tau demonstrated a less prominent peak at 3.5 ppm with additional peaks near 0.5 and 1.5 ppm. No significant differences were identified between fibrillated tau prepared using heparin versus seed-competent tau. In conclusion, monomeric and fibrillated tau can be readily detected and distinguished based on their CEST-derived Z-spectra, pointing to the potential utility of CEST-MRI as a noninvasive biomarker of regional pathologic tau accumulation in the brain. Further testing and validation in vitro and in vivo will be necessary before this can be applied clinically.

Enhancing r1 Relaxivity in GdDOTA-Monoamide Complexes through Polar Group-Mediated Ordering of Second-Sphere Water Molecules.

This study was designed to test whether the single appended phosphonate group in GdDOTA-1AmP is sufficient for catalyzing the exchange of proton from the single inner-sphere water-exchanging molecule. Unlike the other phosphonate derivatives in this series, GdDOTA-1AmP showed a surprisingly smooth increase in r1 relaxivity from 3.0 to 6.3 mM-1 s-1 at 20 MHz as the pH was lowered from 9 to 2.5. In comparison to the bis-, tris-, and tetrakis-phosphonate analogues, which all show a biphasic dependence of r1 with changes in pH, the unique r1 versus pH characteristics of GdDOTA-1AmP are shown to closely parallel deprotonation of the single appended phosphonate group. Although the tissue biodistribution and clearance rates of GdDOTA-1AmP are more favorable than the other more highly charged phosphonate derivatives, the pH dependency of r1 is substantially reduced at magnetic fields typically used for small animal imaging (7 and 9.4T), so the attractiveness of this new molecule for quantitative imaging of tissue pH is diminished. However, this study provides some new insights into the feasibility of designing pH-responsive MRI contrast agents based upon fundamental acid-base prototropic mechanisms.

Prospects and limitations of paramagnetic chemical exchange saturation transfer agents serving as biological reporters in vivo.

The concept of using paramagnetic metal ion complexes as chemical exchange saturation transfer agents (paraCEST) for molecular imaging of various biological processes first appeared in the literature about 20 years ago. The first paraCEST agent was based on a highly shifted, inner-sphere, slowly exchanging water molecule that could be activated at a frequency far away from bulk water, a substantial advantage for selective activation of the agent alone. Many other paraCEST agent designs followed that were based on activation of exchanging -NH or -OH proton on the chelate itself. Both types of paraCEST designs are attractive for molecular imaging because the rates of water molecule or ligand proton exchange can be designed to be sensitive to a biological or physiological property such as pH, enzyme activity, or redox. Hence, the intensity or frequency of the resulting CEST signal provides a direct readout of that property. Many molecular designs have appeared in the literature over the past 20 years, mostly reported as proof-of-concept designs but, unfortunately, only a few reports have explored the limitations of paraCEST agents for imaging a biological process in vivo. As a community, we now know that the sensitivity of paraCEST agents is lower than one might anticipate based upon simple chemical exchange principles and, in general, it appears the sensitivity of paraCEST agents is even lower in vivo than in vitro. In this short review, we address some of the factors that contribute to the limited sensitivity of paraCEST agents in vivo, offer some thoughts on approaches that could lead to dramatically improved paraCEST sensitivity, and challenge the scientific community to perform more in vivo experiments designed to test these ideas.

Recent progress in analysis of intermediary metabolism by ex vivo 13 C NMR.

Advanced imaging technologies, large-scale metabolomics, and the measurement of gene transcripts or enzyme expression all enable investigations of intermediary metabolism in human patients. Complementary information about fluxes in individual metabolic pathways may be obtained by ex vivo 13 C NMR of blood or tissue biopsies. Simple molecules such as 13 C-labeled glucose are readily administered to patients prior to surgical biopsies, and 13 C-labeled glycerol is easily administered orally to outpatients. Here, we review recent progress in practical applications of 13 C NMR to study cancer biology, the response to oxidative stress, gluconeogenesis, triglyceride synthesis in patients, as well as new insights into compartmentation of metabolism in the cytosol. The technical aspects of obtaining the sample, preparing material for analysis, and acquiring the spectra are relatively simple. This approach enables convenient, valuable, and quantitative insights into intermediary metabolism in patients.

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.

Review and consensus recommendations on clinical APT-weighted imaging approaches at 3T: Application to brain tumors.

Amide proton transfer-weighted (APTw) MR imaging shows promise as a biomarker of brain tumor status. Currently used APTw MRI pulse sequences and protocols vary substantially among different institutes, and there are no agreed-on standards in the imaging community. Therefore, the results acquired from different research centers are difficult to compare, which hampers uniform clinical application and interpretation. This paper reviews current clinical APTw imaging approaches and provides a rationale for optimized APTw brain tumor imaging at 3 T, including specific recommendations for pulse sequences, acquisition protocols, and data processing methods. We expect that these consensus recommendations will become the first broadly accepted guidelines for APTw imaging of brain tumors on 3 T MRI systems from different vendors. This will allow more medical centers to use the same or comparable APTw MRI techniques for the detection, characterization, and monitoring of brain tumors, enabling multi-center trials in larger patient cohorts and, ultimately, routine clinical use.

31 P-MRS of healthy human brain: Measurement of guanosine diphosphate mannose at 7 T.

Guanosine diphosphate mannose (GDP-Man) is the donor substrate required for mannosylation in the synthesis of glycoproteins, glycolipids and the newly discovered glycoRNA. Normal GDP-Man biosynthesis plays a crucial role in support of a variety of cellular functions, including cell recognition, cell communication and immune responses against viruses. Here, we report the detection of GDP-Man in human brain for the first time, using 31 P MRS at 7 T. The presence of GDP-Man is evidenced by the detection of a weak 31 P doublet at -10.7 ppm that can be assigned to the phosphomannosyl group (Pß) of the GDP-Man molecule. This weak but well-resolved signal lies 0.9 ppm upfield of UDP(G) Pß-multiplet from a mixture of UDP-Glc, UDP-Gal, UDP-GlcNAc and UDP-GalNAc. In reference to ATP (2.8 mM), the concentration of GDP-Man in human brain was estimated to be 0.02 ± 0.01 mM, about 15-fold lower than the total concentration of UDP(G) (0.30 ± 0.04, N = 17) and consistent with previous reports of UDP-Man in cells and brain tissue extracts measured by high-performance liquid chromatography. The reproducibility of the measured GDP-Man between test and 2-week retest was 21% ± 15% compared with 5% ± 4% for UDP(G) (N = 7). The measured concentrations of GDP-Man and UDP(G) are linearly correlated ([UDP(G)] = 4.3 [GDP-Man] + 0.02, with R = 0.66 and p = 0.0043), likely reflecting the effect of shared sugar precursors, which may vary among individuals in response to variation in nutritional intake and consumption. Given that GDP-Man has another set of doublet (Pα) at -8.3 ppm that overlaps with NAD(H) and UDP(G)-Pα signals, the amount of GDP-Man could potentially interfere with the deconvolution of these mixed signals in composition analysis. Importantly, this new finding may be useful in advancing our understanding of glycosylation and its role in the development of cancer, as well as infectious and neurodegenerative diseases.

31 P-MRS of the healthy human brain at 7 T detects multiple hexose derivatives of uridine diphosphate glucose.

Nucleotide sugars are required for the synthesis of glycoproteins and glycolipids, which play crucial roles in many cellular functions such as cell communication and immune responses. Uridine diphosphate-glucose (UDP-Glc) was previously believed to be the only nucleotide sugar detectable in brain by 31 P-MRS. Using spectra of high SNR and high resolution acquired at 7 T, we showed that multiple nucleotide sugars are coexistent in brain and can be measured simultaneously. In addition to UDP-Glc, these also include UDP-galactose (UDP-Gal), -N-acetyl-glucosamine (UDP-GlcNAc) and -N-acetyl-galactosamine (UDP-GalNAc), collectively denoted as UDP(G). Coexistence of these UDP(G) species is evident from a quartet-like multiplet at -9.8 ppm (M-9.8 ), which is a common feature seen across a wide age range (24-64 years). Lineshape fitting of M-9.8 allows an evaluation of all four UDP(G) components, which further aids in analysis of a mixed signal at -8.2 ppm (M-8.2 ) for deconvolution of NAD+ and NADH. For a group of seven young healthy volunteers, the concentrations of UDP(G) species were 0.04 ± 0.01 mM for UDP-Gal, 0.07 ± 0.03 mM for UDP-Glc, 0.06 ± 0.02 mM for UDP-GalNAc and 0.08 ± 0.03 mM for UDP-GlcNA, in reference to ATP (2.8 mM). The combined concentration of all UDP(G) species (average 0.26 ± 0.06 mM) was similar to the pooled concentration of NAD+ and NADH (average 0.27 ± 0.06 mM, with a NAD+ /NADH ratio of 6.7 ± 2.1), but slightly lower than previously found in an older cohort (0.31 mM). The in vivo NMR analysis of UDP-sugar composition is consistent with those from tissue extracts by other modalities in the literature. Given that glycosylation is dependent on the availability of nucleotide sugars, assaying multiple nucleotide sugars may provide valuable insights into potential aberrant glycosylation, which has been implicated in certain diseases such as cancer and Alzheimer's disease.

Manganese(II)-Based Responsive Contrast Agent Detects Glucose-Stimulated Zinc Secretion from the Mouse Pancreas and Prostate by MRI.

A Mn(II)-based zinc-sensitive MRI contrast agent, MnPyC3A-BPEN, was prepared, characterized, and applied in imaging experiments to detect glucose-stimulated zinc secretion (GSZS) from the mouse pancreas and prostate in vivo. Thermodynamic and kinetic stability tests showed that MnPyC3A-BPEN has superior kinetic inertness compared to GdDTPA, is less susceptible to transmetalation in the presence of excess Zn2+ ions, and less susceptible to transchelation by albumin. In comparison with other gadolinium-based zinc sensors bearing a single zinc binding moiety, MnPyC3A-BPEN appears to be a reliable alternative for imaging ß-cell function in the pancreas and glucose-stimulated zinc secretion from the prostate.

Quantitative measurement of redox state in human brain by 31 P MRS at 7T with spectral simplification and inclusion of multiple nucleotide sugar components in data analysis.

PURPOSE: To develop a simplified method for quantitative measurement of NAD+ /NADH (nicotinamide adenine dinucleotides) levels in human brain by 31 P MRS without interference from the α-ATP signal and with inclusion of multiple UDP-sugar components. METHODS: Simple pulse-acquire 31 P MR spectra were collected at 7T with and without a frequency-selective inversion pulse to remove the dominant α-ATP signal from the underlying NAD(H) signal. Careful inspection of the 31 P signal at -9.8 ppm previously assigned to UDP-glucose revealed multiple UDP-sugar components that must also be considered when deconvoluting the NAD(H) signal to quantify NAD+ and NADH. Finally, the overlapping NAD(H) and UDP(G) resonances were deconvoluted into individual components using Voigt lineshape analysis and UDP(G) modeling. RESULTS: The inversion-based spectral editing method enabled clean separation of the NAD(H) signal from the otherwise dominant α-ATP signal. In addition, the upfield signal near -9.8 ppm appears more "quartet-like" than a simple doublet consistent with contributions from other nucleotide sugars such as UDP-galactose, UDP-N-acetyl-galactosamine, and UDP-N-acetyl-glucosamine in addition to UDP-glucose. Deconvolution of the combined NAD(H) and UDP(G) signals showed that the measured NAD+ /NAD ratio was heavily influenced by UDP(G) modeling (7.5 ± 1.8 when the UDP(G) signal was fitted as multiple doublets versus 5.3 ± 0.6 when a simplified pseudo doublet model was used). In a test/re-test experiments separated by 2 weeks, consistent NAD+ /NADH ratios were measured in the brain of seven human subjects. CONCLUSIONS: The NAD+ /NADH ratio in human brain can be measured using 31 P MR spectra simplified by spectral editing and with inclusion of multiple UDP-sugar components in the data analysis.

A Frequency-Selective pH-Responsive paraCEST Agent.

Paramagnetic chemical exchange saturation transfer (paraCEST) agents are well-suited for imaging tissue pH because the basis of CEST, chemical exchange, is inherently sensitive to pH. Several previous pH-sensitive paraCEST agents were based on an exchanging Ln3+ -bound water molecule as the CEST antenna but this design often added additional line-broadening to the bulk water signal due to T2 exchange. We report herein a pH-sensitive paraCEST agent that lacks an inner-sphere water molecule but contains one Ln-bound -OH group for CEST activation. The Yb3+ complex, Yb(1), displayed a single, highly shifted CEST peak originating from the exchangeable Yb-OH proton, the frequency of which changed over the biologically relevant pH range. CEST images of phantoms ranging in pH from 6 to 8 demonstrate the potential of this agent for imaging pH. Initial rodent imaging studies showed that Gd(1) remains in the vascular system much longer than anticipated but is cleared slowly via renal filtration.

A novel inhibitor of pyruvate dehydrogenase kinase stimulates myocardial carbohydrate oxidation in diet-induced obesity.

The pyruvate dehydrogenase complex (PDC) is a key control point of energy metabolism and is subject to regulation by multiple mechanisms, including posttranslational phosphorylation by pyruvate dehydrogenase kinase (PDK). Pharmacological modulation of PDC activity could provide a new treatment for diabetic cardiomyopathy, as dysregulated substrate selection is concomitant with decreased heart function. Dichloroacetate (DCA), a classic PDK inhibitor, has been used to treat diabetic cardiomyopathy, but the lack of specificity and side effects of DCA indicate a more specific inhibitor of PDK is needed. This study was designed to determine the effects of a novel and highly selective PDK inhibitor, 2((2,4-dihydroxyphenyl)sulfonyl) isoindoline-4,6-diol (designated PS10), on pyruvate oxidation in diet-induced obese (DIO) mouse hearts compared with DCA-treated hearts. Four groups of mice were studied: lean control, DIO, DIO + DCA, and DIO + PS10. Both DCA and PS10 improved glucose tolerance in the intact animal. Pyruvate metabolism was studied in perfused hearts supplied with physiological mixtures of long chain fatty acids, lactate, and pyruvate. Analysis was performed using conventional 1H and 13C isotopomer methods in combination with hyperpolarized [1-13C]pyruvate in the same hearts. PS10 and DCA both stimulated flux through PDC as measured by the appearance of hyperpolarized [13C]bicarbonate. DCA but not PS10 increased hyperpolarized [1-13C]lactate production. Total carbohydrate oxidation was reduced in DIO mouse hearts but increased by DCA and PS10, the latter doing so without increasing lactate production. The present results suggest that PS10 is a more suitable PDK inhibitor for treatment of diabetic cardiomyopathy.

A Responsive Magnetic Resonance Imaging Contrast Agent for Detection of Excess Copper(II) in the Liver In Vivo.

The design, synthesis, and properties of a new gadolinium-based copper-responsive magnetic resonance imaging (MRI) contrast agent is presented. The sensor (GdL1) has high selectivity for copper ions and exhibits a 43% increase in r1 relaxivity (20 MHz) upon binding to 1 equiv of Cu2+ in aqueous buffer. Interestingly, in the presence of physiological levels of human serum albumin (HSA), the r1 relaxivity is amplified further up to 270%. Additional spectroscopic and X-ray absorption spectroscopy (XAS) studies show that Cu2+ is coordinated by two carboxylic acid groups and the single amine group on an appended side chain of GdL1 and forms a ternary complex with HSA (GdL1-Cu2+-HSA). T1-weighted in vivo imaging demonstrates that GdL1 can detect basal, endogenous labile copper(II) ions in living mice. This offers a unique opportunity to explore the role of copper ions in the development and progression of neurological diseases such as Wilson's disease.

Modular 31 P wideband inversion transfer for integrative analysis of adenosine triphosphate metabolism, T1 relaxation and molecular dynamics in skeletal muscle at 7T.

PURPOSE: For efficient and integrative analysis of de novo adenosine triphosphate (ATP) synthesis, creatine-kinase-mediated ATP synthesis, T1 relaxation time, and ATP molecular motion dynamics in human skeletal muscle at rest. METHODS: Four inversion-transfer modules differing in center inversion frequency were combined to generate amplified magnetization transfer (MT) effects in targeted MT pathways, including Pi ↔ γ-ATP, PCr ↔ γ-ATP, and 31 Pγ(α)ATP ↔ 31 PßATP . MT effects from both forward and reverse exchange kinetic pathways were acquired to reduce potential bias and confounding factors in integrated data analysis. RESULTS: Kinetic data collected using 4 wideband inversion modules (8 minutes each) yielded the forward exchange rate constants, kPCr→γATP = 0.31 ± 0.05 s-1 and kPi→γATP = 0.064 ± 0.012 s-1 , and the reverse exchange rate constants, kγATP→Pi = 0.034 ± 0.006 s-1 and kγATP→PCr = 1.37 ± 0.22 s-1 , respectively. The cross-relaxation rate constant, σγ(α) ↔ ßATP was -0.20 ± 0.03 s-1 , corresponding to ATP rotational correlation time τc of 0.8 ± 0.1 × 10-7 seconds. The intrinsic T1 relaxation times were Pi (9.2 ± 1.4 seconds), PCr (6.2 ± 0.4 seconds), γ-ATP (1.8 ± 0.1 seconds), α-ATP (1.4 ± 0.1 seconds), and ß-ATP (1.1 ± 0.1 seconds). Muscle ATP T1 values were found to be significantly longer than those previously measured in the brain using a similar method. CONCLUSION: A combination of multiple inversion transfer modules provides a comprehensive and integrated analysis of ATP metabolism and molecular motion dynamics. This relatively fast technique could be potentially useful for studying metabolic disorders in skeletal muscle.

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.

Metabolism of hyperpolarized 13 C-acetoacetate to ß-hydroxybutyrate detects real-time mitochondrial redox state and dysfunction in heart tissue.

Mitochondrial dysfunction is considered to be an important component of many metabolic diseases yet there is no reliable imaging biomarker for monitoring mitochondrial damage in vivo. A large prior literature on inter-conversion of ß-hydroxybutyrate and acetoacetate indicates that the process is mitochondrial and that the ratio reflects a specifically mitochondrial redox state. Therefore, the conversion of [1,3-13 C]acetoacetate to [1,3-13 C]ß-hydroxybutyrate is expected to be sensitive to the abnormal redox state present in dysfunctional mitochondria. In this study, we examined the conversion of hyperpolarized (HP) 13 C-acetoacetate (AcAc) to 13 C-ß-hydroxybutyrate (ß-HB) as a potential imaging biomarker for mitochondrial redox and dysfunction in perfused rat hearts. Conversion of HP-AcAc to ß-HB was investigated using 13 C magnetic resonance spectroscopy in Langendorff-perfused rat hearts in four groups: control, global ischemic reperfusion, low-flow ischemic, and rotenone (mitochondrial complex-I inhibitor)-treated hearts. We observed that more ß-HB was produced from AcAc in ischemic hearts and the hearts exposed to complex I inhibitor rotenone compared with controls, consistent with the accumulation of excess mitochondrial NADH. The increase in ß-HB, as detected by 13 C MRS, was validated by a direct measure of tissue ß-HB by 1 H nuclear magnetic resonance in tissue extracts. The redox ratio, NAD+ /NADH, measured by enzyme assays of homogenized tissue, also paralleled production of ß-HB from AcAc. Transmission electron microscopy of tissues provided direct evidence for abnormal mitochondrial structure in each ischemic tissue model. The results suggest that conversion of HP-AcAc to HP-ß-HB detected by 13 C-MRS may serve as a useful diagnostic marker of mitochondrial redox and dysfunction in heart tissue in vivo.

Synchrotron Radiation X-ray Fluorescence Elemental Mapping in Healthy versus Malignant Prostate Tissues Provides New Insights into the Glucose-Stimulated Zinc Trafficking in the Prostate As Discovered by MRI.

Prostatic zinc content is a known biomarker for discriminating normal healthy tissue from benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Given that zinc content is not readily measured without a tissue biopsy, we have been exploring noninvasive imaging methods to detect these diagnostic differences using a zinc-responsive MRI contrast agent. During imaging studies in mice, we observed that a bolus of glucose stimulates secretion of zinc from the prostate of fasted mice. This discovery allowed the use of a Gd-based zinc sensor to detect differential zinc secretion in regions of healthy versus malignant prostate tissue in a transgenic adenocarcinoma mouse model of PCa. Here, we used a zinc-responsive MRI agent to detect zinc release across the prostate during development of malignancy and confirm the loss of total tissue zinc by synchrotron radiation X-ray fluorescence (µSR-XRF). Quantitative µSR-XRF results show that the lateral lobe of the mouse prostate uniquely accumulates high concentrations of zinc, 1.06 ± 0.08 mM, and that the known loss of zinc content in the prostate is only observed in the lateral lobe during development of PCa. Additionally, we confirm that lesions identified by a loss of zinc secretion indeed represent malignant neoplasia and that the relative zinc concentration in the lesion is reduced to 0.370 ± 0.001 mM. The µSR-XRF data also provided insights into the mechanism of zinc secretion by showing that glucose promotes movement of zinc pools (∼1 mM) from the glandular lumen of the lateral lobe of the mouse prostate into the stromal/smooth muscle surrounding the glands. Co-localization of zinc and gadolinium in the stromal/smooth muscle areas as detected by µSR-XRF confirm that glucose initiates secretion of zinc from intracellular compartments into the extracellular spaces of the gland where it binds to the Gd-based agent and albumin promoting MR image enhancement.

Zinc-sensitive MRI contrast agent detects differential release of Zn(II) ions from the healthy vs. malignant mouse prostate.

Many secretory tissues release Zn(II) ions along with other molecules in response to external stimuli. Here we demonstrate that secretion of Zn(II) ions from normal, healthy prostate tissue is stimulated by glucose in fasted mice and that release of Zn(II) can be monitored by MRI. An ∼50% increase in water proton signal enhancement is observed in T1-weighted images of the healthy mouse prostate after infusion of a Gd-based Zn(II) sensor and an i.p. bolus of glucose. Release of Zn(II) from intracellular stores was validated in human epithelial prostate cells in vitro and in surgically exposed prostate tissue in vivo using a Zn(II)-sensitive fluorescent probe known to bind to the extracellular surface of cells. Given the known differences in intracellular Zn(II) stores in healthy versus malignant prostate tissues, the Zn(II) sensor was then evaluated in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model in vivo. The agent proved successful in detecting small malignant lesions as early as 11 wk of age, making this noninvasive MR imaging method potentially useful for identifying prostate cancer in situations where it may be difficult to detect using current multiparametric MRI protocols.

Imaging Insulin Secretion from Mouse Pancreas by MRI Is Improved by Use of a Zinc-Responsive MRI Sensor with Lower Affinity for Zn2+ Ions.

It has been demonstrated that divalent zinc ions packaged with insulin in ß-cell granules can be detected by MRI during glucose-stimulated insulin secretion using a gadolinium-based Zn2+-sensitive agent. This study was designed to evaluate whether a simpler agent design having single Zn2+-sensing moieties but with variable Zn2+ binding affinities might also detect insulin secretion from the pancreas. Using an implanted MR-compatible window designed to hold the pancreas in a fixed position for imaging, we now demonstrate that focally intense "hot spots" can be detected in the tail of the pancreas using these agents after administration of glucose to stimulate insulin secretion. Histological staining of the same tissue verified that the hot spots identified by imaging correspond to clusters of islets, perhaps reflecting first-responder islets that are most responsive to a sudden increase in glucose. A comparison of images obtained when using a high-affinity Zn2+ sensor versus a lower-affinity sensor showed that the lower-affinity sensors produced the best image contrast. An equilibrium model that considers all possible complexes formed between Zn2+, the GdL sensor, and HSA predicts that a GdL sensor with lower affinity for Zn2+ generates a lower background signal from endogenous Zn2+ prior to glucose-stimulated insulin secretion (GSIS) and that the weaker binding affinity agent is more responsive to a further increase in Zn2+ concentration near ß-cells after GSIS. These model predictions are consistent with the in vivo imaging observations.

Unveiling a hidden 31 P signal coresonating with extracellular inorganic phosphate by outer-volume-suppression and localized 31 P MRS in the human brain at 7T.

PURPOSE: The study was undertaken to demonstrate that there is more than 1 component in the extracellular Pi31 P signal ( Piex) acquired from human head using nonlocalized 31 P MRS. METHODS: Outer-volume-suppression (OVS) saturation and 1D/2D 31 P CSI were utilized to reveal the presence of an additional component in the Piex signal. RESULTS: 67% of the head extracellular Pi signal was attenuated upon OVS saturation of the peripheral meningeal tissues, likely reflecting elimination of the Pi signal in the meningeal fluids (the blood and CSF). Localized 1D/2D CSI data provided further support for this assignment. Upon correction for the meningeal contribution, the extracellular Pi concentration was 0.51 ± 0.07 mM, whereas the intracellular Pi was 0.85 ± 0.10 mM. The extracellular pH was measured as 7.32 ± 0.04 when using OVS, as compared to 7.39 ± 0.03 when measured without OVS (N = 7 subjects). CONCLUSION: The extracellular Pi signal acquired from the human head using nonlocalized 31 P MRS contains a significant component likely contributed by peripheral blood and CSF in meninges that must be removed in order to use this signal as an endogenous probe for measuring extracellular pH and other properties in the brain.