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Ligands derived from the macrocycle, 1,4,7,10-tetraazacyclododecane, with differing side-chain chelating arms form complexes with the bivalent lanthanide cations that are both thermodynamically stable and kinetically inert. This short review article is intended to introduce the reader to a few potential biomedical applications of various lanthanide complexes formed with macrocyclic ligands derived from the cyclen. TmDOTP5- has proven to be quite useful as a NMR shift reagent (SR) for biological cations, capable of resolving intra- and extracellular 23Na signals in the in vivo rat kidney, liver, heart and skeletal muscle. Lanthanide complexes of the monoester derivatives of DOTP have lower net charge and differing hydrophobicities, depending upon the length of the alkyl sidechains. These complexes are handled differently in rats, with the tetra(ethyl ester) complexes clearing via renal excretion and the tetra(n-butyl ester) complexes via biliary excretion. The tetra(trifluoroethyl ester) complexes are handled more like the n-butyl ester complexes than the ethyl ester complexes. High resolution 19F NMR spectra of the latter complexes reveal that six diastereomers are present in solution at nearly equal concentrations.

To develop an inversion pulse-based, chemical exchange saturation transfer-like method for detection of (31) P magnetization exchanges among all nuclear magnetic resonance visible metabolites suitable for providing an integrated kinetic analysis of phosphorus exchange reactions in vivo.The exchange kinetics by inversion transfer (EKIT) sequence includes application of a frequency-selective inversion pulse arrayed over the range of relevant (31) P frequencies, followed by a constant delay and a hard readout pulse. A series of EKIT spectra, each given by a plot of Z-magnetization for each metabolite of interest versus frequency of the inversion pulse, can be generated from this single data set.EKIT spectra reflect chemical exchange due to known biochemical reactions, cross-relaxation effects, and relayed magnetization transfers due to both processes. The rate constants derived from EKIT data collected on resting human skeletal muscle were: ATP synthesis via ATP synthase (0.050 ± 0.016 s(-1) ), ATP synthesis via creatine kinase (0.264 ± 0.023 s(-1) ), and cross-relaxation between neighboring spin pairs within ATP (0.164 ± 0.022 s(-1) ).EKIT provides a simple, alternative method to detect chemical exchange, cross relaxation, and relayed magnetization transfer effects in human skeletal muscle at 7 T.

A Zn2+ specific GdDOTA derivative containing two bis-(3-pyrazolyl) units was prepared and characterized. Unlike a previously reported Zn2+ binding agent, the new agent binds to human albumin both in the presence and absence of Zn2+.

Tumor hypoxia is known to affect sensitivity to radiotherapy and promote development of metastases; therefore, the ability to image tumor hypoxia in vivo could provide useful prognostic information and help tailor therapy. We previously demonstrated in vitro evidence for selective accumulation of a gadolinium tetraazacyclododecanetetraacetic acid monoamide conjugate of 2-nitroimidazole (GdDO3NI), a magnetic resonance imaging T1-shortening agent, in hypoxic cells grown in tissue culture. We now report evidence for accumulation of GdDO3NI in hypoxic tumor tissue in vivo. Our data show that GdDO3NI accumulated significantly (p < 0.05) in the central, poorly perfused regions of rat prostate adenocarcinoma AT1 tumors (threefold higher concentration than for the control agent) and showed better clearance from well-perfused regions and complete clearance from the surrounding muscle tissue. Inductively coupled plasma mass spectroscopy confirmed that more GdDO3NI than control agent was retained in the central region and that more GdDO3NI was retained in the central region than at the periphery. These results show the utility of GdDO3NI to image tumor hypoxia and highlight the potential of GdDO3NI for application to image-guided interventions for radiation therapy or hypoxia-activated chemotherapy.

Amide proton transfer (APT) imaging is one of the chemical exchange saturation transfer (CEST) imaging methods which images the exchange between protons of free tissue water and the amide groups (-NH) of endogenous mobile proteins and peptides. Previous work suggested the ability of APT imaging for characterization of the tumoral grade in the brain tumor. In this study, we tested the feasibility of in-vivo APT imaging of lung tumor and investigated whether the method could differentiate the tumoral types on orthotopic tumor xenografts from two malignant lung cancer cell lines. The results revealed that APT imaging is feasible to quantify lung tumors in the moving lung. The measured APT effect was higher in the tumor which exhibited more active proliferation than the other. The present study demonstrates that APT imaging has the potential to provide a characterization test to differentiate types or grade of lung cancer noninvasively, which may eventually reduce the need invasive needle biopsy or resection for lung cancer.

Many Eu(III) complexes formed with DOTA-tetraamide ligands (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) have sufficiently slow water exchange kinetics to meet the slow-to-intermediate condition required to serve as chemical exchange saturation transfer (CEST) contrast agents for MRI. This class of MRI contrast agents offers an attractive platform for creating biological sensors because water exchange is exquisitely sensitive to subtle ligand stereochemistry and electronic effects. Introduction of carboxyl groups or carboxyl ethyl ester groups on the amide substituents has been shown to slow water exchange in these complexes, but less is known about the orientation or position of these side-chain groups relative to the inner-sphere Eu(III)-bound water molecule. In this study, a series of Eu(III) complexes having one or more carboxyl groups or carboxyl esters at the δ-position of the pendant amide side chains were prepared. Initial attempts to prepare optically pure EuDOTA-[(S)-Asp]4 resulted in a chemically pure ligand consisting of a mixture of stereochemical isomers. This was traced to racemization of (S)-aspartate diethyl ester during the synthetic procedure. Nevertheless, NMR studies of the Eu(III) complexes of this mixture revealed that each isomer had a different water exchange rate, differing by a factor of 2 or more. A second controlled synthesis and CEST study of EuDOTA-[(S)-Asp]4 and cis-EuDOTA-[(S)-Asp]2[(R)-Asp]2 confirmed that the water exchange rates in these diastereomeric complexes are controlled by the axial versus equatorial orientation of the carboxyl groups on the amide side chains. These observations provide new insights toward the development of even more slowly water exchanging systems which will be necessary for practical in vivo applications.

We have performed temperature-dependent electron spin resonance (ESR) measurements of the stable free radical trityl OX063, an efficient polarizing agent for dissolution dynamic nuclear polarization (DNP), at the optimum DNP concentration (15 mM). We have found that (i) when compared to the W-band electron spin-lattice relaxation rate T1e(-1) of other free radicals used in DNP at the same concentration, trityl OX063 has slower T1e(-1) than BDPA and 4-oxo-TEMPO. At T > 20 K, the T1e(-1)vs. T data of trityl OX063 appears to follow a power law dependence close to the Raman process prediction whereas at T < 10 K, electronic relaxation slows and approaches the direct process behaviour. (ii) Gd(3+) doping, a factor known to enhance DNP, of trityl OX063 samples measured at W-band resulted in monotonic increases of T1e(-1) especially at temperatures below 20-40 K while the ESR lineshapes remained essentially unchanged. (iii) The high frequency ESR spectrum can be fitted with an axial g-tensor with a slight g-anisotropy: g(x) = g(y) = 2.00319(3) and g(z) = 2.00258(3). Although the ESR linewidth D monotonically increases with field, the temperature-dependent T1e(-1) is almost unchanged as the ESR frequency is increased from 9.5 GHz to 95 GHz, but becomes faster at 240 GHz and 336 GHz. The ESR properties of trityl OX063 reported here may provide insights into the efficiency of DNP of low-γ nuclei performed at various magnetic fields, from 0.35 T to 12 T.

The chemical shifts of 31 P and 1 H in thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) (TmDOTP 5- ) are approximately two orders of magnitude more sensitive to temperature than are water proton and 19 F shifts. In the physiologically relevant pH range, the 31 P and 1 H chemical shifts of TmDOTP 5- are linear functions of temperature between 25 and 47°C. The results indicate that using Tm-DOTP 5- can provide measurements of temperature in vivo that are significantly more accurate than methods based on water and fluorocarbon chemical shifts.

These examples serve to illustrate that it is now possible to investigate metabolism in intact tissues using a variety of biophysical methods. While we have concentrated on NMR methods, reflecting our own interests in using 13C as a metabolic tracer, GC-mass spectroscopy can often provide similar metabolic information and has the advantage of increased sensitivity over NMR. Combining either or both of these technologies with cleaver 'chemical biopsy' methods offers new opportunities to examine what may seem to be old metabolic questions in a much more relevant environment, the native state.

Interactions between Li + , Na + , Cs + , Ca 2+ and Mg 2+ and the shift reagent (SR), TmDOTP 5 were studied by 7 Li, 6 Li, 23 Na and 133 Cs multinulear NMR spectroscopy. The unusually large paramagnetic shifts (⪢100 ppm) observed in the cation NMR resonances at low alkali metal ion to TmDOTP 5− ratios indicated that the cations bind near the 4-fold symmetry axis of the complex. The geometric parameters for the adducts formed between 7 Li, 6 Li, 23 Na and 133 Cs cations and TmDOTP 5− were obtained from shift and relaxation rate data and compared with those calculated using MMX energy minimization techniques. It is found that the larger Cs + ion lies closest to the 4-fold axis of symmetry and displays the largest binding constant. The smaller Li + ion deviates most from the 4-fold symmetry axis and has a less favorable binding interaction than Cs + . Na + lies somewhere between these two extremes but has the largest limiting shifts due to its favorable distance from the paramagnetic metal center. A significant reversal of the 23 Na shift upon addition of Ca 2+ and Mg 2+ indicate that these two divalent cations form very stable 1:1 adducts with TmDOTP 5 (log K (Ca 2+ ) = 5.69 and log Kt (Mg 2+ ) = 3.85). An analysis of the Mg 2+ titration data shows that the 2Mg 2+ :TmDOTP 5− adduct becomes dominant at high Mg 2+ /TmDOTP 5− ratio (log K 2 (Mg 2+ ) = 2.17). The significant influence of NH 4 + on the TmDOTP 5− -induced 6 Li, shifts suggests that the NH 4 + ion may form multiple H-bonds with the SR along the 4-fold axis of symmetry.

A simple relationship between parameters derived from a 13C NMR isotopomer analysis and O2 consumption is presented that allows measurement of the absolute rate of acetyl-CoA oxidation and anaplerotic flux in tissues oxidizing a mixture of four substrates. The method was first applied in a study of the effects of work state and beta-adrenergic stimulation on net acetate oxidation and anaplerosis in the isolated working rat heart. The results demonstrate that the anticipated ratio of 2 between O2 consumption and TCA cycle flux for hearts oxidizing only acetate holds at low workload when anaplerosis is low, but deviates toward a factor of 3 under high workload conditions when anaplerosis is increased. This analysis was also extended to hearts that oxidize a more physiological mixture of substrates including long-chain fatty acids, acetoacetate, lactate, pyruvate, and glucose. We show that the contribution each substrate makes to total TCA cycle flux can be determined by combined 13C NMR and O2 consumption measurements. The present study also demonstrates that stimulation of anaplerosis (by addition of propionate) can significantly alter the relative contribution each substrate makes to total TCA cycle flux. We conclude if 13C labeling patterns are selected appropriately, a comprehensive picture of flux through all major metabolic pathways feeding the cycle can be determined in a single experiment even when complex physiological mixtures of substrates are provided.

A series of 1,4,7-triazacyclononane-based ligands containing one, two, or three methylphosphinate (MP) side chains has been prepared. The macrocycle with three methylphosphinates, NOTMP, appeared to be a very promising ligand for monitoring MgII in biological samples by 31P NMR spectroscopy. The 31P resonances of the free ligand and the MgII complex were well resolved, in slow exchange, and well downfield of typical tissue phosphate and phosphate ester metabolite resonances. The Kd value of the Mg(NOTMP) complex was 0.35 mM at physiological pH and temperature, a value which is optimal for accurate assessment of free MgII in cells and blood plasma. The latter was confirmed experimentally. Furthermore, the selectivity of NOTMP for MgII over CaII exceeded 2 orders of magnitude. The Kd values of the MgII complexes were sensitive to pH and temperature. The pH effects could be easily predicted from potentiometric pH titration data. Examination of the MgII ligand equilibria by 31P NMR over a wide temperature ran...

Dynamic nuclear polarization (DNP) is a technique that uses a microwave-driven transfer of high spin alignment from electrons to nuclear spins. This is most effective at low temperature and high magnetic field, and with the invention of the dissolution method, the amplified nuclear magnetic resonance (NMR) signals in the frozen state in DNP can be harnessed in the liquid-state at physiologically acceptable temperature for in vitro and in vivo metabolic studies. A current optimization practice in dissolution DNP is to dope the sample with trace amounts of lanthanides such as Gd3+ or Ho3+, which further improves the polarization. While Gd3+ and Ho3+ have been optimized for use in dissolution DNP, other lanthanides have not been exhaustively studied for use in C13 DNP applications. In this work, two additional lanthanides with relatively high magnetic moments, Dy3+ and Tb3+, were extensively optimized and tested as doping additives for C13 DNP at 3.35 T and 1.2 K. We have found that both of these lanthanides are also beneficial additives, to a varying degree, for C13 DNP. The optimal concentrations of Dy3+ (1.5 mM) and Tb3+ (0.25 mM) for C13 DNP were found to be less than that of Gd3+ (2 mM). W-band electron paramagnetic resonance shows that these enhancements due to Dy3+ and Tb3+ doping are accompanied by shortening of electron T1 of trityl OX063 free radical. Furthermore, when dissolution was employed, Tb3+-doped samples were found to have similar liquid-state C13 NMR signal enhancements compared to samples doped with Gd3+, and both Tb3+ and Dy3+ had a negligible liquid-state nuclear T1 shortening effect which contrasts with the significant reduction in T1 when using Gd3+. Our results show that Dy3+ doping and Tb3+ doping have a beneficial impact on C13 DNP both in the solid and liquid states, and that Tb3+ in particular could be used as a potential alternative to Gd3+ in C13 dissolution DNP experiments.

The synthesis and characterization of four new DOTA-tetraamide ligands having variable alkyl chain lengths (C(1), C(12), C(14), and C(16)) and their respective europium (III) complexes are reported. The three EuL complexes having long alkyl chains spontaneously form micelles of variable size. The critical micelle concentration differed for each complex (lower for the C(16) complex than the C(12) complex) while micelle size increased with increasing alkyl chain length. Chemical exchange saturation transfer (CEST) experiments showed that all four Eu(III) complexes display slow-to-intermediate water exchange kinetics. As expected, the CEST signals in these complexes decreased with increasing temperatures due to faster water exchange but, interestingly, the CEST signals for the C(14) and C(16) complexes approached a maximum near 25°C consistent with exchange limited CEST at or near room temperature. The water residence lifetimes obtained by fitting the CEST spectra to the Bloch equations increased in parallel with an increase in alkyl carbon chain-length. By comparisons with the monomethylamide complex, which served as control, the data illustrate that micelle formation serves to slow the rate of water exchange in these systems. The complex having the largest CEST effect per unit Eu(III) concentration (the C(16) analog) had a detection limit of 5.3 μM. This represents an approximate 250-fold increase in sensitivity relative to the monomethylamide control (detection limit ~1.3 mM). These features highlight the potential of using micelle-based systems such as these as paramagnetic chemical exchange saturation transfer (PARACEST) agents for molecular imaging by MRI.

1,4,7-Triazacyclononane reacts selectively with 2-(t-butoxycarbonyloxyimino)- and 2-(benzyloxycarbonyloxyimino)-2-phenylacetonitrile to give 1,4-diprotected derivatives which could be converted to mono and disubstituted triazacyclononanes.

Tetraazamacrocycles containing the pyridine moiety as part of the cyclic backbone and acetate pendant arms were prepared. The resulting products were characterized by high resolution NMR (‘H and I3C) spectroscopy and single-crystal X-ray diffraction. Single-crystal X-ray diffraction data were obtained for the following compounds: py[l2]aneN4*HC1, C I I H I ~ N ~ C ~ , orthorhombic Pnma (No. 62), a = 10.304(6) A, b = 9.160(2) A, c = 13.552(2) A, Z = 4, Mo Ka, ;1 = 0.710 69, R = 0.037, R, = 0.036; and BP2A.2HC1-3H20, C18H22N404c ~ ~ . ~ H ~ o , triclinic pi (NO. 21, u = 10.201(0) A, b = i3.267(3) A, c = 8.612(6) A, a = 97.676(2)”, p = 105.821(0)”, I. = 86.363(7)”, Z = 2, Mo Ka, = 0.710 69, R = 0.039, R , = 0.038. In addition, protonation constants (log KH~L) and metal ion stability constants (log KML) for BP2A (log KH,-L = 9.57, 5.99, 2.59, 2.22; log KMgL = 8.9; log K c ~ L = 10.0; log KGdL = 14.5) and PC2A (log KH,-~L = 12.5, 5.75, 3,28, 2.38, -1; log KMgL = 8.4, log Kc& = 10.0, log KGdL = 16.6) were determined using potentiometric and NMR pH titrations. These values agree well with previously reported literature values for analogous ligand systems.

Four Gd{sup 3+} complexes [Gd(BP2A){sup +}, Gd(PC2A){sup +}, Gd(PCTA){sup 0}, and Gd(BPO4A){sup {minus}}] with polyazamacrocyclic ligands that contain a pyridine moiety were prepared and examined for possible use as MRI contrast enhancement agents. The authors estimated the number of inner sphere water molecules (q{sub Gd}) for the Gd{sup 3+} complexes from the values of q found for the Tb{sup 3+} and/or Eu{sup 3+} complexes by luminescence lifetime measurements. It was estimated that q{sub Gd} = 3.5, 3.3, 2.4, and 0.2 for Gd(BP2A){sup +}, Gd(PC2A){sup +}, Gd(PCTA){sup 0}, and Gd(BPO4A){sup {minus}}, respectively. The inner sphere relaxivities (r{sub 1,inner}) of these tetraaza macrocyclic complexes were higher than that of Gd(DOTA){sup {minus}} [i.e. 6.79 for Gd(BP2A){sup +}, 6.21 for Gd(PC2A){sup +}, and 4.60 for Gd(PCTA){sup 0} mM{sup {minus}1}s{sup {minus}1} at 40 MHz and 25{degrees}C], but the normalized relaxivities per q{sub Gd} (1.94, 1.88, and 1.92 mM{sup {minus}1}s{sup {minus}1}, respectively) were comparable to that of Gd(DOTA){sup {minus}}. A quantitative treatment of the NMRD profiles based on Solomon-Bloembergen-Morgan theory, using the NMRD profile of Gd(BPO4A){sup {minus}} to correct for an outer sphere contribution, showed that the complexes exhibit characteristics similar to that of Gd(DOTA){sup {minus}} but with shorter electronic relaxation times. Tissue biodistribution results usingmore » radioactive {sup 153}Sm and {sup 159}Gd complexes in rats indicate that the cationic [{sup 153}Sm-(BP2A){sup +} and {sup 153}Sm(PC2A){sup +}] complexes accumulate preferably in the bone tissue while the neutral [{sup 153}Sm-(PCTA){sup 0}] and anionic [{sup 153}Sm(BPO4A){sup {minus}}] complexes appear to have renal clearances similar to those of other low molecular weight contrast agents [i.e. Gd(DTPA){sup 2{minus}} and Gd(DOTA){sup {minus}}].« less

Studies of glycerol metabolism in the heart have largely emphasized its role in triglyceride synthesis. However, glycerol may also be oxidized in the citric acid cycle, and glycogen synthesis from glycerol has been reported in the nonmammalian myocardium. The intent of this study was to test the hypothesis that glycerol may be metabolized to glycogen in mammalian heart. Isolated rat hearts were supplied with a mixture of substrates including glucose, lactate, pyruvate, octanoate, [U-13C3]glycerol, and 2H2O to probe various metabolic pathways including glycerol oxidation, glycolysis, the pentose phosphate pathway, and carbon sources of stored glycogen. NMR analysis confirmed that glycogen production from the level of the citric acid cycle did not occur and that the glycerol contribution to oxidation in the citric acid cycle was negligible in the presence of alternative substrates. Quite unexpectedly, 13C from [U-13C3]glycerol appeared in glycogen in carbon positions 4–6 of glucosyl units but none in positions 1–3. The extent of [4,5,6-13C3]glucosyl unit enrichment in glycogen was enhanced by insulin but decreased by H2O2. Given that triose phosphate isomerase is generally assumed to fully equilibrate carbon tracers in the triose pool, the marked 13C asymmetry in glycogen can only be attributed to conversion of [U-13C3]glycerol to [U-13C3]dihydroxyacetone phosphate and [U-13C3]glyceraldehyde 3-phosphate followed by rearrangements in the nonoxidative branch of the pentose phosphate pathway involving transaldolase that places this 13C-enriched 3-carbon unit only in the bottom half of hexose phosphate molecules contributing to glycogen. Background: The metabolic fate of glycerol in the heart is controversial. Results: [U-13C3]Glycerol is metabolized to glycogen, but the label is only found in carbon positions 4–6 of each glucose moiety. Conclusion: This isotopomer pattern can only be ascribed to transaldolase activity. Significance: This is the first detection of transaldolase activity of an isolated organ using a stable isotope.

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.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.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.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.

The goal of this work was to test feasibility of using galvinoxyl (2,6-di-tert-butyl-α-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy) as a polarizing agent for dissolution dynamic nuclear polarization (DNP) NMR spectroscopy. We have found that galvinoxyl is reasonably soluble in ethyl acetate, chloroform, or acetone and the solutions formed good glasses when mixed together or with other solvents such as dimethyl sulfoxide. W-band electron spin resonance (ESR) measurements revealed that galvinoxyl has an ESR linewidth D intermediate between that of carbon-centered free radical trityl OX063 and the nitroxide-based 4-oxo-TEMPO, thus the DNP with galvinoxyl for nuclei with low gyromagnetic ratio γ such as 13 C and 15 N is expected to proceed predominantly via the thermal mixing process. The optimum radical concentration that would afford the highest 13 C nuclear polarization (approximately 6% for [1- 13 C]ethyl acetate) at 3.35 T and 1.4 K was found to be around 40 mM. After dissolution, large liquid-state NMR enhancements were achieved for a number of 13 C and 15 N compounds with long spin–lattice relaxation time T 1 . In addition, the hydrophobic galvinoxyl free radical can be easily filtered out from the dissolution liquid when water is used as the solvent. These results indicate that galvinoxyl can be considered as an easily available free radical polarizing agent for routine dissolution DNP-NMR spectroscopy.

Despite its importance in energy metabolism, lactate in human skeletal muscle has been difficult to detect by noninvasive (1)H-magnetic resonance spectroscopy mainly due to interference from large water and lipid signals. Long echo-time acquisitions at 7 T effectively attenuates the water and lipid signals in forearm muscle allowing direct observation of both lactate resonances, the methine at 4.09 ppm and the methyl at 1.31 ppm. Using this approach, we were able to monitor lactate dynamics at a temporal resolution of 32 s. While lactate was not detectable at rest, immediately after an acute period of exercise to fatigue the forearm muscle, lactate rose to a level comparable to that of creatine (∼30 mmol/kg wet weight). In a typical (1)H-magnetic resonance spectrum collected using a echo-time of 140 ms, the lactate methine and methyl resonances both appear as doublets with an unusually large splitting of ∼20 Hz due to residual dipolar coupling. During muscle recovery following exercise, the lactate signals decay rapidly with a time constant of t½ = 2.0 ± 0.6 min (n = 12 subjects). This fast and simple lactate detection method may prove valuable for monitoring lactate metabolism in cancer and in sports medicine applications.

Our ever-expanding knowledge about the role of zinc in biology includes its role in redox modulation, immune response, neurotransmission, reproduction, diabetes, cancer, and Alzheimers disease is galvanizing interest in detecting and monitoring the various forms of Zn(II) in biological systems. This paper reviews reported strategies for detecting and tracking of labile or "free" unchelated Zn(II) in tissues. While different bound structural forms of Zn(II) have been identified and studied in vitro by multiple techniques, very few molecular imaging methods have successfully tracked the ion in vivo. A number of MRI and optical strategies have now been reported for detection of free Zn(II) in cells and tissues but only a few have been applied successfully in vivo. A recent report of a MRI sensor for in vivo tracking of Zn(II) released from pancreatic β-cells during insulin secretion exemplifies the promise of rational design of new Zn(II) sensors for tracking this biologically important ion in vivo. Such studies promise to provide new insights into zinc trafficking in vivo and the critical role of this ion in many human diseases.

Silica nanoparticles of average diameter 53 ± 3 nm were prepared using standard water-in-oil microemulsion methods. After conversion of the surface Si-OH groups to amino groups for further conjugation, the PARACEST agent, EuDOTA-(gly)₄ (-) was coupled to the amines via one or more side-chain carboxyl groups in an attempt to trap water molecules in the inner-sphere of the complex. Fluorescence and ICP analyses showed that approximately 1200 Eu(3+) complexes were attached to each silica nanoparticle, leaving behind excess protonated amino groups. CEST spectra of the modified silica nanoparticles showed that attachment of the EuDOTA-(gly)₄ (-) to the surface of the nanoparticles did not result in a decrease in water exchange kinetics as anticipated, but rather resulted in a complete elimination of the normal Eu(3+) -bound water exchange peak and broadening of the bulk water signal. This observation was traced to catalysis of proton exchange from the Eu(3+) -bound water molecule by excess positively charged amino groups on the surface of the nanoparticles.

The role of diet and fat consumption in the pathogenesis of breast cancer is an important subject. We report a method for noninvasive determination of lipid composition in human breast by proton magnetic resonance spectroscopy (MRS) at 7 T. Two respiratory-triggered TE-averaged stimulated echo acquisition mode (STEAM) acquisitions were performed on the adipose tissue of 10 healthy volunteers where the second acquisition had all gradients inverted. This acquisition protocol allows the suppression of modulation sidebands that complicate spectral analysis at the short TEavg = 24.5 ms. The entire acquisition takes ∼10 min. Ten lipid peaks were typically resolved. T1 and T2 were also measured and used to correct the peak intensities. The calculated average lipid composition for saturated was 28.7 ± 8.4%, monounsaturated, 48.5 ± 7.9%, and polyunsaturated, 22.7 ± 3.1%, in close agreement with reported values from subcutaneous adipose measurements. Intrasubject variability was 2.0, 1.6, and 3.6% for the saturated, monounsaturated, and polyunsaturated fractions, respectively. In conclusion, we have shown that a chemical analysis of lipids in breast tissue can be determined quite simply, quickly, and noninvasively by proton MRS at 7 T. Magn Reson Med, 2011. © 2011 Wiley-Liss, Inc.

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 B1 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 (kex >104 s−1) without interference from conventional broad-band MT. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.

Tissue hypoxia occurs in pathologic conditions, such as cancer, ischemic heart disease and stroke when oxygen demand is greater than oxygen supply. An imaging method that can differentiate hypoxic versus normoxic tissue could have an immediate impact on therapy choices. In this work, the gadolinium(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with a 2-nitroimidazole attached to one carboxyl group via an amide linkage was prepared, characterized and tested as a hypoxia-sensitive MRI agent. A control complex, Gd(DO3A-monobutylamide), was also prepared in order to test whether the nitroimidazole side-chain alters either the water proton T(1) relaxivity or the thermodynamic stability of the complex. The stabilities of these complexes were lower than that of Gd(DOTA)(-) as expected for mono-amide derivatives. The water proton T(1) relaxivity (r(1)), bound water residence lifetime (τ(M)) and rotational correlation time (τ(R)) of both complexes was determined by relaxivity measurements, variable temperature (17) O NMR spectroscopy and proton nuclear magnetic relaxation dispersion (NMRD) studies. The resulting parameters (r(1) =6.38 mM(-1) s(-1) at 20 MHz, τ(M) =0.71 μs, τ(R) =141 ps) determined for the nitroimidazole derivative closely parallel to those of other Gd(DO3A-monoamide) complexes of similar molecular size. In vitro MR imaging experiments with 9L rat glioma cells maintained under nitrogen (hypoxic) versus oxygen (normoxic) gas showed that both agents enter cells but only the nitroimidazole derivative was trapped in cells maintained under N(2) as evidenced by an approximately twofold decrease in T(1) measured for hypoxic cells versus normoxic cells exposed to this agent. These results suggest that the nitroimidazole derivative might serve as a molecular reporter for discriminating hypoxic versus normoxic tissues by MRI.

The host–guest complex formed in aqueous solution between γ-cyclodextrin (γ-CD) and the macrocyclic paramagnetic shift reagent TmDOTP5− was examined by NMR spectroscopy. Paramagnetic lanthanide-induced shifts (LIS) and spin-lattice relaxation rate enhancements in the proton resonances of γ-CD on binding of TmDOTP5− were used to define the position of the guest anion within the host cavity. A proportionality constant which relates pseudo-contact shifts induced by TmDOTP5− to the geometrical position of each ligand nucleus (previously determined from a LIS study of the TmDOTP5− complex) was used to determine a fully bound LIS value for each γ-CD proton. These values, in combination with experimental LIS data from TmDOTP5− titrations at constant γ-CD concentration, were then used to obtain the association constant for the host-guest complex (3.9–4.5 mol l−1). These constants were indicative of a weak host-guest interaction and consistent with the location of the TmDOTP5− binding site reported by the LIS values well above the center of the γ-CD cavity.

Recently Befroy and colleagues measured TCA cycle flux and anaplerosis in human liver by analyzing 13C enrichment in carbon 1 and carbon 5 of glutamate during [1-13C]acetate infusion1. The entry of [1-13C]acetyl-CoA into the cycle yields [5-13C]glutamate on each turn of the cycle but [1-13C]glutamate only on subsequent turns. Anaplerosis, for instance via pyruvate carboxylation, results in the influx of unlabeled oxaloacetate. The subsequent loss of 13C-oxaloacetate from the cycle to make phosphoenolpyruvate (PEP) decreases the enrichment in glutamate-C1 but not glutamate-C5. Hence, the steady-state ratio of [1-13C] to [5-13C]glutamate should reflect anaplerosis relative to TCA cycle activity (i.e. citrate synthase flux = 1). PEP formed from anaplerosis has at least two fates, either in gluconeogenesis (GNG) or to regenerate pyruvate via pyruvate kinase (PK). PK, and other pathways (e.g. malic enzyme), contribute to a futile pyruvate cycle whereby oxaloacetate → pyruvate → oxaloacetate. The relationship among these variables is simple: GNG = anaplerosis – PK flux. This pathway has been included in virtually all models of hepatic GNG2-9. In humans PK flux, relative to citrate synthase (CS), was 1.5 to 5.8 when determined using a [3-14C]lactate tracer with radioisotope detection,8 [3-13C]lactate tracer with mass isotope detection3 or [U-13C]propionate tracer with NMR detection6,9. In fact, pyruvate cycling accounted for the majority of anaplerosis in these human studies3,6,8,9, yet was excluded from anaplerosis by Befroy et al.1 This exclusion complicates the analysis considerably because pyruvate cycling in the presence of H13CO3-, as observed in the in vivo 13C NMR spectra1, regenerates [1-13C]glutamate. Hence, the steady-state ratio of [1-13C] to [5-13C]glutamate no longer reflects total anaplerosis. Given the implications, it is worth examining the effect of pyruvate cycling on 13C in glutamate C1 and C5 using the conditions reported by Befroy et al. Carbon-13 labeling in glutamate is a complex function of pyruvate cycling, anaplerosis and enrichment in acetyl-CoA and bicarbonate 4,10. Consequently, simulations must be used to explore these interactions10. Using published relationships10 and the conditions reported by Befroy et al., glutamate 13C1/13C5 is sensitive to total anaplerosis if 13CO2 = 0 and pyruvate cycling = 0 but much less sensitive when 13CO2 = 6% 1 and pyruvate cycling is active2-9 (Figure 1). The glutamate 13C1/13C5 ratio reported by Befroy et al. is indeed consistent with substantial pyruvate cycling (Figure 1), as previously described in human liver3,6,8,9. However, the presence of pyruvate cycling also means that a 5-fold range in anaplerosis is essentially undetectable by the glutamate 13C1/13C5 ratio. Thus, this method cannot be used to support the conclusion by Befroy et al., that other methods have overestimated anaplerosis1, since the glutamate 13C1/13C5 ratio cannot detect total anaplerosis under these conditions (Figure 1). Figure 1 The effect of pyruvate cycling on the steady-state 13C1/13C5 of glutamate. Data was simulated using program tcaSIM (see source data). All simulations were conducted using an [1-13C]acetyl-CoA fractional enrichment of 0.22 and 13CO2 fractional enrichment ... Futile cycles serve a role in the regulation of metabolism. Earlier studies, including those in humans, found that the futile cycle interconverting pyruvate and oxaloacetate is unquestionably active in liver regardless of whether lactate or propionate tracers are used2-9. Thus, it is best to assume that pyruvate cycling is always a feature of liver metabolism and to incorporate this possibility in data analysis.

The amide proton exchange rates in various lanthanide(III) DOTA-tetraamide complexes were investigated by CEST as a function of variable chemical structures and charges on the amide substituents. Comparisons were made between YbDOTA-(gly)(4)(-) (Yb-1), YbDOTA-(NHCH(2)PO(3))(4 (5-) (Yb-2) and YbDOTA-(NHCH(2)PO(3)Et(2))(4)(3+) (Yb-3). The general shapes of the CEST vs pH profiles were similar for the three complexes but they showed maximum CEST intensities at different pH values, pH 8.3, 8.8 and 6.9 for Yb-1, Yb-2 and Yb-3, respectively. This indicates that a more negatively charged substituent on the amide helps stabilize the partial positive charge on the amide nitrogen and consequently more base is required to catalyze proton exchange. The chemical shifts of the -NH protons in Yb-1 and Yb-2 were similar (-17 ppm) while the -NH proton in Yb-3 was at -13 ppm. This shows that the crystal field produced by the amide oxygen donor atoms in Yb-3 is substantially weaker than that in the other two complexes. In an effort to expand the useful range of pH values that might be measured using these complexes as CEST agents, the shapes of the CEST vs pH curves were also determined for two thulium(III) complexes with much larger hyperfine shifted -NH proton resonances. The ratio of CEST from -NH exchange in Tm-1 compared with CEST from -NH exchange in Tm-3 was found to be linear over an extended pH range, from 6.3 to 7.4. This demonstrates a potential advantage of using mixtures of lanthanide(III) DOTA-tetraamides for mapping tissue pH by use of ratiometric CEST imaging.

The sensitivity of magnetic resonance imaging (MRI) contrast agents is highly dependent on the rate of water exchange between the inner sphere of a paramagnetic ion and bulk water. Normally, identifying a paramagnetic complex that has optimal water exchange kinetics is done by synthesizing and testing one compound at a time. We report here a rapid, economical on-bead combinatorial synthesis of a library of imaging agents. Eighty different 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid (DOTA)-tetraamide peptoid derivatives were prepared on beads using a variety of charged, uncharged but polar, hydrophobic, and variably sized primary amines. A single chemical exchange saturation transfer image of the on-bead library easily distinguished those compounds having the most favorable water exchange kinetics. This combinatorial approach will allow rapid screening of libraries of imaging agents to identify the chemical characteristics of a ligand that yield the most sensitive imaging agents. This technique could be automated and readily adapted to other types of MRI or magnetic resonance/positron emission tomography agents as well.

Hyperpolarized (89)Y complexes are attractive NMR spectroscopy and MR imaging probes due to the exceptionally long spin-lattice relaxation time (T(1) ≈ 10 min) of the (89)Y nucleus. However, in vivo imaging of (89)Y has not yet been realized because of the low NMR signal enhancement levels previously achieved for this ultra low-γ(n) nucleus. Here, we report liquid-state (89)Y NMR signal enhancements over 60,000 times the thermal signal at 298 K in a 9.4 T magnet, achieved after the dynamic nuclear polarization (DNP) of Y(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) samples at 3.35 T and 1.4 K. The (89)Y DNP was shown to proceed by thermal mixing and the liquid state (89)Y NMR signal enhancement was maximized by (i) establishing the optimal microwave irradiation frequency, (ii) optimizing the glassing matrix, (iii) choosing a radical with negligible inhomogeneous line broadening contribution to the ESR linewidth, and (iv) addition of an electron T(1e) relaxation agent. The highest enhancements were achieved using a trityl OX063 radical combined with a gadolinium relaxation agent in water-glycerol matrix. Co-polarization of (89)YDOTA and sodium [1-(13)C]pyruvate showed that both (89)Y and (13)C nuclear species acquired the same spin temperature, consistent with thermal mixing theory of DNP. This methodology may be applicable for the optimization of DNP of other low-γ(n) nuclei.

Chemical-exchange saturation transfer (CEST) agents are a new class of MRI contrast agents that offer a number of advantages over conventional Gd3+ agents. Over the past few years, a variety of small-molecule CEST agents responsive to physiological conditions, such as pH and temperature, have been designed and their imaging applications have been reported. One of the major drawbacks of current small-molecule CEST agents is their relatively low sensitivity. The advantages of using macromolecular and nanosized systems with large numbers of exchangeable groups to improve contrast sensitivity are highlighted in this brief review. Although this approach has been shown to amplify contrast sensitivity, other limitations, including relatively small chemical-shift differences between the exchanging species and bulk water and less than optimal proton exchange rates, still exist. By addressing these issues, it is anticipated that CEST agents will find useful applications in the detection of specific biomarkers of disease.

Nuclei with long T1s are optimal targets for dynamic nuclear polarization (DNP). Therefore, most of the agents used in metabolic imaging and spectroscopy studies are based on carboxylic acid moieties that lack protons, a strong source of dipolar relaxation. Metabolic flux information encoded into spectra of small molecule metabolites in the form of the 13C isotopomer data cannot be accessed using standard 13C hyperpolarization methods because protonated carbons relax too quickly through T1 dipolar relaxation. It is shown here that the longitudinal mixing sequence FLOPSY-8 can be used to transfer polarization from a long T1 storage nucleus to adjacent protonated carbons so that they may be detected with high sensitivity. We demonstrate that FLOPSY-8 allows a direct readout of isotopomer populations in butyrate and glutamate in vitro.

The role of diet and fat consumption in the pathogenesis of breast cancer is an important subject. We report a method for noninvasive determination of lipid composition in human breast by proton magnetic resonance spectroscopy (MRS) at 7 T. Two respiratory-triggered TE-averaged stimulated echo acquisition mode (STEAM) acquisitions were performed on the adipose tissue of 10 healthy volunteers where the second acquisition had all gradients inverted. This acquisition protocol allows the suppression of modulation sidebands that complicate spectral analysis at the short TE(avg) = 24.5 ms. The entire acquisition takes ∼10 min. Ten lipid peaks were typically resolved. T(1) and T(2) were also measured and used to correct the peak intensities. The calculated average lipid composition for saturated was 28.7 ± 8.4%, monounsaturated, 48.5 ± 7.9%, and polyunsaturated, 22.7 ± 3.1%, in close agreement with reported values from subcutaneous adipose measurements. Intrasubject variability was 2.0, 1.6, and 3.6% for the saturated, monounsaturated, and polyunsaturated fractions, respectively. In conclusion, we have shown that a chemical analysis of lipids in breast tissue can be determined quite simply, quickly, and noninvasively by proton MRS at 7 T.

Lanthanide(III) chelates of DOTA-tetraamide ligands have been an area of particular interest since the discovery that water exchange kinetics are dramatically affected by the switch from acetate to amide side-chain donors. More recently these chelates have attracted interest as potential PARACEST agents for use in MRI. In this paper we report the results of studies using chemical exchange saturation transfer (CEST) and some more recently reported chelates to re-examine the exchange processes in this class of chelate. We find that the conclusions of Parker and Aime are, for the most part, solid; water exchange is slow and a substantial amount of prototropic exchange occurs in aqueous solution. The extent of prototropic exchange increases as the pH increases above 8, leading to higher relaxivities at high pH. However, amide protons are found to contribute only a small amount to the relaxivity at high pH.

Lanthanide DOTA-tetraglycinate (LnDOTA-(gly)4 −) complexes contain four magnetically equivalent amide protons that exchange with protons of bulk water. The rate of this base catalyzed exchange process has been measured using chemical exchange saturation transfer (CEST) NMR techniques as a function of solution pH for various paramagnetic LnDOTA-(gly)4 − complexes to evaluate the effects of lanthanide ion size on this process. Complexes with Tb(III), Dy(III), Tm(III) and Yb(III) were chosen because these ions induce large hyperfine shifts in all ligand protons, including the exchanging amide protons. The magnitude of the amide proton CEST exchange signal differed for the four paramagnetic complexes in order, Yb>Tm>Tb>Dy. Although the Dy(III) complex showed the largest hyperfine shift as expected, the combination of favorable chemical shift and amide proton CEST linewidth in the Tm(III) complex was deemed most favorable for future in vivo applications where tissue magnetization effects can interfere. TmDOTA-(gly)4 − at various concentrations was encapsulated in the core interior of liposomes to yield lipoCEST particles for molecular imaging. The resulting nanoparticles showed less than 1% leakage of the agent from the interior over a range of temperatures and pH. The pH versus amide proton CEST curves differed for the free versus encapsulated agents over the acidic pH regions, consistent with a lower proton permeability across the liposomal bilayer for the encapsulated agent. Nevertheless, the resulting lipoCEST nanoparticles amplify the CEST sensitivity by a factor of ∼104 compared to the free, un-encapsulated agent. Such pH sensitive nano-probes could prove useful for pH mapping of liposomes targeted to tumors.

A new 13C NMR technique for measuring substrate utilization by the citric acid cycle based on an analysis of succinate 13C isotopomers is presented. The relative contribution of up to three different labeling patterns in acetyl-CoA entering the citric acid cycle may be determined under non-steady-state conditions. We present experimental data from perfused rat hearts subjected to a brief period of ischemia, where both succinate and glutamate resonances were observed in the 13C spectrum. The contributions of labeled exogenous acetate and lactate and unlabeled sources to the acetyl-CoA pool were compared using this succinate analysis and a previously published glutamate analysis [Malloy et al. (1990) Biochemistry 29, 6756-6761], and the two methods give identical results. This indicates that the succinate and glutamate isotopomers originated from a common alpha-ketoglutarate pool, verifying that glutamate is in isotopomeric equilibrium with alpha-ketoglutarate under these conditions.

Off-resonance saturation (ORS) is a new magnetic resonance imaging (MRI) method that has shown greatly improved contrast sensitivity for the detection of cancer-specific biomarkers by superparamagnetic nanoprobes in vivo. However, quantitative understanding of the ORS contrast mechanism and its dependence on the structural parameters of superparamagnetic nanoprobes are still lacking. Here we propose a quantitative model of ORS contrast and its experimental validation by superparamagnetic polymeric micelles (SPPM) with precisely controlled structural properties. Size selected, monodisperse Fe3O4 nanoparticles (6.1 ± 0.2 nm) were used to form a series of SPPM nanoprobes with specifically controlled corona thickness using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxypoly(ethylene glycol) (DSPE-PEG) with different PEG molecular weights. Transmission electron microscopy and dynamic light scattering showed that SPPM were uniform in size. The average hydrodynamic diameters of SPPM with PEG lengths of 0.55, 1, 2, and 5 kD were 16.6 ± 2.8, 18.4 ± 2.9, 24.1 ± 3.4, and 28.9 ± 3.4 nm, respectively. MRI experiments at 7T determined that r2 values of SPPM with 0.55, 1, 2, and 5 kD PEG as corona were 201 ± 3, 136 ± 8, 107 ± 5, and 108 ± 8 Fe mM−1 s−1, respectively. ORS intensity from Z-spectra of SPPM showed a significant correlation with the inverse of T2 relaxation rates (1/T2, s−1) of the SPPM nanoprobes regardless of the PEG corona thickness. These data provide the fundamental understanding of the structure–property relationships between the SPPM nanostructures and ORS sensitivity, which offers useful mechanistic insights for the future improvement of SPPM nanoprobes in cancer molecular imaging applications.

In this review we describe the status of development for a new class of magnetic resonance (MR) contrast agents, based on chemical exchange saturation transfer (CEST). The mathematics and physics relevant to the description of the CEST effect in MR are presented in an appendix published in the online version only. We discuss the issues arising when translating in vitro results obtained with CEST agents to using these MR agents in in vivo model studies and in humans. Examples are given on how these agents are imaged in vivo. We summarize the status of development of these CEST agents, and speculate about the next steps that may be taken towards the demonstration of CEST MR imaging in clinical applications.

The square antiprism/twisted square antiprism ratio in LnDOTA-tetraamide chelates is a critical parameter in governing water-exchange kinetics and ultimately the utility of a chelate as a PARACEST MRI contrast agent. In LnDOTA-tetraamide chelates with tertiary amides, this ratio and the rate of interconversion between these two structural isomers are found to be dramatically dependent upon the solvent and possibly other local environmental factors.

Ln(S-THP)(3+) complexes are paramagnetic chemical exchange saturation transfer (PARACEST) agents for magnetic resonance imaging (MRI; S-THP = (1S,4S,7S,10S)-1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane, Ln(III) = Ce(III), Eu(III), Yb(III)). CEST spectra at 11.7 T show that the PARACEST effect of these complexes is enhanced at neutral pH in buffered solutions containing 100 mM NaCl upon the addition of 1-2 equiv of diethylphosphate (DEP). CEST images of phantoms at 4.7 T confirm that DEP enhances the properties of Yb(S-THP)(3+) as a PARACEST MRI agent in buffered solutions at neutral pH and 100 mM NaCl. Studies using (1)H NMR, direct excitation Eu(III) luminescence spectroscopy, and UV-visible spectroscopy show that DEP is an outersphere ligand. Dissociation constants for [Ln(S-THP)(OH(2))](DEP) are 1.9 mM and 2.8 mM for Ln(III) = Yb(III) at pH 7.0 and Eu(III) at pH 7.4. Related ligands including phosphorothioic acid, O,O-diethylester, ethyl methylphosphonate, O-(4-nitrophenylphosphoryl)choline, and cyclic 3,5-adenosine monophosphate do not activate PARACEST. BNPP (bis(4-nitrophenyl phosphate) activates PARACEST of Ln(S-THP)(3+) (Ln(III) = Eu(III), Yb(III)), albeit less effectively than does DEP. These data show that binding through second coordination sphere interactions is selective for phosphate diesters with two terminal oxygens and two identical ester groups. A crystal structure of [Eu(S-THP)(OH(2))]((O(2)NPhO)(2)PO(2))(2)(CF(3)SO(3)) x 2 H(2)O x iPrOH has two outersphere BNPP anions that form hydrogen bonds to the alcohol groups of the macrocycle and the bound water ligand. This structure supports (1)H NMR spectroscopy studies showing that outersphere interactions of the phosphate diester with the alcohol protons modulate the rate of alcohol proton exchange to influence the PARACEST properties of the complex. Further, DEP interacts only with the nonionized form of the complex, Ln(S-THP)(OH(2))(3+) contributing to the pH dependence of the PARACEST effect.

The popular short echo time 1H MR spectroscopy acquisition method for detection of intramyocellular lipids suffers from spectral overlap due to the large, broad, and asymmetric extramyocellular lipid signals, the time-consuming practice of selecting “lean” voxels for spectroscopy, and the overlap of the extramyocellular lipid signal with the creatine methyl 1H signal at ∼3 parts per million (ppm), commonly used as an internal standard. Using an alternative acquisition strategy, spectra with well-resolved intramyocellular lipids resonances were acquired from large volumes (10 to 15 mL) of human soleus muscle in less than 5 min by single-voxel 7-T 1H MR spectroscopy, using an echo time of 280 ms. From the high-resolution spectra, an average intramyocellular lipid concentration of 7.7 ± 3.5 mmol/kg muscle was found for 25 healthy subjects (male/female 17/8; age 29.4 ± 6.6 years). Since water suppression was not required, the 1H signals from unsaturated intracellular triglycerides at about 5.3 ppm were easily detected, which, in combination with the well-determined (CH2)n/CH3 intensity ratio at long echo time, enabled assessment of the composition of triglycerides in the intramyocellular lipids compartment. Long-echo single-voxel spectroscopy at 7 T offers rapid and convenient acquisition of high-resolution spectra from human soleus muscle. Magn Reson Med, 2010. © 2010 Wiley-Liss, Inc.

Reactive surface lysine groups on the chimeric monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4 – 10.1 chelates were added per 3G4 molecule and between 5.6 – 5.9 chelates per molecule of HSA, depending upon which conjugation chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only 17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water lifetime of Eu-1 increased only marginally, up from 53 μs for the non-conjugated chelate to 65–77 μs for conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted in little or no change in bound water lifetime (73–75 μs for both the conjugated and non-conjugated forms). These data indicate that exchange of water molecules protons between the inner-sphere site on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment of the agent to the protein and likely its chemical surroundings on the surface of the protein.

Cyclic pentamine 1 was synthesized in good yields from fragments containing two and three nitrogen atoms.

The extracellular pH (pH(e) ) of solid tumors is acidic, and there is evidence that an acidic pH(e) is related to invasiveness. Herein, we describe an MRI single-infusion method to measure pH(e) in gliomas using a cocktail of contrast agents (CAs). The cocktail contained gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaminophosphonate (GdDOTA-4AmP) and dysprosium-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetrakis(methylenephosphonic acid) (DyDOTP), whose effects on relaxation are sensitive and insensitive to pH, respectively. The Gd-CA dominated the spin-lattice relaxivity ΔR(1) , whereas the Dy-CA dominated the spin-spin relaxivity ΔR(2)*. The ΔR(2)* effects were used to determine the pixel-wise concentration of [Dy] which, in turn, was used to calculate a value for [Gd] concentration. This value was used to convert ΔR(1) values to the molar relaxivity Δr(1) and, hence, pH(e) maps. The development of the method involved in vivo calibration and measurements in a rat brain glioma model. The calibration phase consisted of determining a quantitative relationship between ΔR(1) and ΔR(2)* induced by the two pH-independent CAs, gadolinium-diethylenetriaminepentaacetic acid (GdDTPA) and DyDOTP, using echo planar spectroscopic imaging (EPSI) and T(1) -weighted images. The intensities and linewidths of the water peaks in EPSI images were affected by CA and were used to follow the pharmacokinetics. These data showed a linear relationship between inner- and outer-sphere relaxation rate constants that were used for CA concentration determination. Nonlinearity in the slope of the relationship was observed and ascribed to variations in vascular permeability. In the pH(e) measurement phase, GdDOTA-4AmP was infused instead of GdDTPA, and relaxivities were obtained through the combination of interleaved T(1) -weighted images (R(1) ) and EPSI for ΔR(2)*. The resulting r(1) values yielded pH(e) maps with high spatial resolution.

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.