Apparent rate constant mapping using hyperpolarized [1-(13)C]pyruvate.

Hyperpolarization of [1-13C]pyruvate in solution allows real-time measurement of uptake and metabolism using MR spectroscopic methods. After injection and perfusion, pyruvate is taken up by the cells and enzymatically metabolized into downstream metabolites such as lactate, alanine, and bicarbonate. In this work, we present comprehensive methods for the quantification and interpretation of hyperpolarized 13C metabolite signals. First, a time-domain spectral fitting method is described for the decomposition of FID signals into their metabolic constituents. For this purpose, the required chemical shift frequencies are automatically estimated using a matching pursuit algorithm. Second, a time-discretized formulation of the two-site exchange kinetic model is used to quantify metabolite signal dynamics by two characteristic rate constants in the form of (i) an apparent build-up rate (quantifying the build-up of downstream metabolites from the pyruvate substrate) and (ii) an effective decay rate (summarizing signal depletion due to repetitive excitation, T1-relaxation and backward conversion). The presented spectral and kinetic quantification were experimentally verified in vitro and in vivo using hyperpolarized [1-13C]pyruvate. Using temporally resolved IDEAL spiral CSI, spatially resolved apparent rate constant maps are also extracted. In comparison to single metabolite images, apparent build-up rate constant maps provide improved contrast by emphasizing metabolically active tissues (e.g. tumors) and suppression of high perfusion regions with low conversion (e.g. blood vessels). Apparent build-up rate constant mapping provides a novel quantitative image contrast for the characterization of metabolic activity. Its possible implementation as a quantitative standard will be subject to further studies.

Metabolism of hyperpolarized [1-¹³C]pyruvate in the isolated perfused rat lung - an ischemia study.

We report studies of the effect of ischemia on the metabolic activity of the intact perfused lung and its restoration after a period of reperfusion. Two groups of rat lungs were studied using hyperpolarized 1-(13) C pyruvate to compare the rate of lactate labeling differing only in the temporal ordering of ischemic and normoxic acquisitions. In both cases, a several-fold increase in lactate labeling was observed immediately after a 25-min ischemia event as was its reversal back to the baseline after 30-40 min of resumed perfusion (n = 5, p < 0.025 for both comparisons). These results were corroborated by (31) P spectroscopy and correspond well to measured changes in lactate pool size determined by (1) H spectroscopy of freeze-clamped specimens.

Effects of pyruvate dose on in vivo metabolism and quantification of hyperpolarized ¹³C spectra.

Real-time in vivo measurements of metabolites are performed by signal enhancement of [1-(13)C]pyruvate using dynamic nuclear polarization, rapid dissolution and intravenous injection, acquisition of free induction decay signals and subsequent quantification of spectra. The commonly injected dose of hyperpolarized pyruvate is larger than typical tracer doses, with measurement before complete dilution of the injected bolus. Pyruvate is in exchange with its downstream metabolites lactate, alanine and bicarbonate. A transient exposure to high pyruvate blood concentrations may cause the saturation of cellular uptake and metabolic conversion. The goal of this study was to examine the effects of a [1-(13)C]pyruvate bolus on metabolic conversion in vivo. Spectra were quantified by three different methods: frequency-domain fitting with LCModel, time-domain fitting with AMARES and simple linear least-squares fitting in the time domain. Since the simple linear least-squares approach showed bleeding artifacts and LCModel produced noisier time signals. AMARES performed best in the quantification of in vivo hyperpolarized pyruvate spectra. We examined pyruvate doses of 0.1-0.4 mmol/kg (body mass) in male Wistar rats by acquiring slice-selective free induction decay signals in slices dominated by heart, liver and kidney tissue. Dose effects were noted in all cases, except for alanine in the cardiac slice below the dose of 0.2 mmol/kg. Our results indicate unlimited cellular uptake of pyruvate up to this dose and limited enzymatic activity of lactate dehydrogenase. In the cardiac slice above 0.2 mmol/kg and in liver and kidney slices, reflect limited cellular uptake or enzymatic activity, or a combination of both effects. The results indicate that the dose of pyruvate must be recognized as an important determinant for metabolic tissue kinetics, and saturation effects must be taken into account for the quantitative interpretation of the observed results.

Cluster formation restricts dynamic nuclear polarization of xenon in solid mixtures.

During dynamic nuclear polarization (DNP) at 1.5 K and 5 T, (129)Xe nuclear magnetic resonance (NMR) spectra of a homogeneous xenon/1-propanol/trityl-radical solid mixture exhibit a single peak, broadened by (1)H neighbors. A second peak appears upon annealing for several hours at 125 K. Its characteristic width and chemical shift indicate the presence of spontaneously formed pure Xe clusters. Microwave irradiation at the appropriate frequencies can bring both peaks to either positive or negative polarization. The peculiar time evolution of (129)Xe polarization in pure Xe clusters during DNP can be modelled as an interplay of spin diffusion and T(1) relaxation. Our simple spherical-cluster model offers a sensitive tool to evaluate major DNP parameters in situ, revealing a severe spin-diffusion bottleneck at the cluster boundaries and a significant sample overheating due to microwave irradiation. Subsequent DNP system modifications designed to reduce the overheating resulted in four-fold increase of (129)Xe polarization, from 5.3% to 21%.

Tamoxifen-independent Cre-activity in SMMHC-CreER T2 mice.

Background and aims: Recent technological advances have established vascular smooth muscle cells (SMCs) as central players in atherosclerosis. Increasingly complex genetic mouse models have unveiled that 30-70% of cells in experimentally induced atherosclerotic lesions derive from a handful of medial SMCs, and that these can adopt a broad range of plaque cell phenotypes. Most of these models are based on the SMMHC-CreER T2 mouse line as Cre-driver. Importantly, Cre-activation can be controlled in time (by administration of tamoxifen, TAM), which is critical to avoid unwanted effects of premature recombination events. The aim of this study was to scrutinize an unexpected observation of TAM-independent Cre-activity in this mouse line. Methods: Cre-activity was assessed by PCR in tissues from SMMHC-CreER T2 mice crossed with mice homozygous for loxP-flanked (floxed) exon 4 of Ccn2 (our gene-of-interest), and Ccn2 protein was measured in aortas by targeted mass spectrometry. Results: We observed spontaneous near-complete excision of floxed Ccn2 in aortas from adult mice that were not treated with TAM. As a result, Ccn2 protein was significantly reduced in aortas from these mice, but not to the same extent as TAM-treated littermates. Remarkably, most of the excision was completed in 4-week-old mice. Excision was Cre-dependent, as knockout bands were negligible in heart and liver (dominated by non-SMCs) of these mice, and undetectable in the aorta in the absence of Cre. Conclusion: Our observations warrant caution, and we advocate inclusion of appropriate controls (i.e., TAM-untreated mice) in future studies.

Measurements of the persistent singlet state of N2O in blood and other solvents--potential as a magnetic tracer.

The development of hyperpolarized tracers has been limited by short nuclear polarization lifetimes. The dominant relaxation mechanism for many hyperpolarized agents in solution arises from intramolecular nuclear dipole-dipole coupling modulated by molecular motion. It has been previously demonstrated that nuclear spin relaxation due to this mechanism can be removed by storing the nuclear polarization in long-lived, singlet-like states. In the case of N(2)O, storing the polarization of the nitrogen nuclei has been shown to substantially increase the polarization lifetime. The feasibility of utilizing N(2)O as a tracer is investigated by measuring the singlet-state lifetime of the N(2)O when dissolved in a variety of solvents including whole blood. Comparison of the singlet lifetime to longitudinal relaxation and between protonated and deuterated solvents is consistent with the dominance of spin-rotation relaxation, except in the case of blood.

Direct measurement of the triple spin flip rate in dynamic nuclear polarization.

A previous study of the effect of Gadolinium doping on the dynamic polarization (DNP) of 13C using trityls showed that the rate at which the polarization builds up is almost independent of the Gadolinium concentration, while the electron spin-lattice relaxation rate varies over an order of magnitude. In this paper we analyze the polarization build-up in detail and show that in this case DNP is due to the cross-effect (CE) and that the build-up rate can be quantitatively interpreted as the rate of the triple spin flips responsible for the CE. Thus this build-up rate presents a direct measurement of this triple spin flip rate.

Basement membrane collagen IV deficiency promotes abdominal aortic aneurysm formation.

Abdominal aortic aneurysm (AAA) is a complex disease which is incompletely accounted for. Basement membrane (BM) Collagen IV (COL4A1/A2) is abundant in the artery wall, and several lines of evidence indicate a protective role of baseline COL4A1/A2 in AAA development. Using Col4a1/a2 hemizygous knockout mice (Col4a1/a2+/-, 129Svj background) we show that partial Col4a1/a2 deficiency augmented AAA formation. Although unchallenged aortas were morphometrically and biomechanically unaffected by genotype, explorative proteomic analyses of aortas revealed a clear reduction in BM components and contractile vascular smooth muscle cell (VSMC) proteins, suggesting a central effect of the BM in maintaining VSMCs in the contractile phenotype. These findings were translated to human arteries by showing that COL4A1/A2 correlated to BM proteins and VSMC markers in non-lesioned internal mammary arteries obtained from coronary artery bypass procedures. Moreover, in human AAA tissue, MYH11 (VSMC marker) was depleted in areas of reduced COL4 as assessed by immunohistochemistry. Finally, circulating COL4A1 degradation fragments correlated with AAA progression in the largest Danish AAA cohort, suggesting COL4A1/A2 proteolysis to be an important feature of AAA formation. In sum, we identify COL4A1/A2 as a critical regulator of VSMC phenotype and a protective factor in AAA formation.

PIN diode driver for NMR and MRI.

Designing custom coils for magnetic resonance systems, such as nuclear magnetic resonance (NMR) spectrometers and magnetic resonance imaging (MRI) scanners, often entails using non-standard configurations of the transmit-receive (T/R) switch and Q-spoiling circuits. The built-in drivers of commercial NMR and MRI systems are, typically, only reconfigurable within a narrow application range (if at all). Thus, the built-in driver may not be able to properly control the custom T/R switches and Q-spoiling circuits when using custom built coils. We present a PIN diode driver which functions in both an MRI scanner and NMR spectrometer. The PIN diode driver is based on readily available discrete components and achieves switching times for the reverse and forward bias states (transmit on and off) of 2 µs and 0.4 µs respectively. Hence, this work enables a higher degree of customization of the RF switching circuits in an MR system and is potentially of interest for designers of custom coils for both NMR spectrometers and MRI scanners.

Magnetic resonance imaging with optical preamplification and detection.

Magnetic resonance (MR) imaging relies on conventional electronics that is increasingly challenged by the push for stronger magnetic fields and higher channel count. These problems can be avoided by utilizing optical technologies. As a replacement for the standard low-noise preamplifier, we have implemented a new transduction principle that upconverts an MR signal to the optical domain and imaged a phantom in a clinical 3 T scanner with signal-to-noise comparable to classical induction detection.

Hyperpolarized 133Cs is a sensitive probe for real-time monitoring of biophysical environments.

133Cs NMR is a valuable tool for non-invasive analysis of biological systems, where chemical shift and relaxation properties report on changes in the physical environment. Hyperpolarization can increase the liquid-state 133Cs NMR signal by several orders of magnitude and allow real-time monitoring of physical changes in cell based systems.

1H DNP at 1.4 T of water doped with a triarylmethyl-based radical.

Recently a triarylmethyl-based (TAM) radical has been developed for research in biological and other aqueous systems, and in low magnetic fields, 10 mT or less, large (1)H dynamic nuclear polarization (DNP) enhancements have been reported. In this paper the DNP properties of this radical have been investigated in a considerably larger field of 1.4 T, corresponding to proton and electron Larmor frequencies of 60 MHz and 40 GHz, respectively. To avoid excessive microwave heating of the sample, an existing DNP NMR probe was modified with a screening coil, wound around the sample capillary and with its axis perpendicular to the electric component of the microwave field. It was found that with this probe the temperature increase in the sample after 4 s of microwave irradiation with an incident power of 10 W was only 16 degrees C. For the investigations, 10 mM of the TAM radical was dissolved in deionized, but not degassed, water and put into a 1-mm i.d. and 6-mm long capillary tube. At 26 degrees C the following results were obtained: (I) The relaxivity of the radical is 0.07 (mMs)(-1), in accordance with the value extrapolated from low-field results; (II) The leakage factor is 0.63, the saturation factor at maximum power is 0.85, and the coupling factor is -0.0187. It is shown that these results agree very well with an analysis where the electron-dipolar interactions are the dominant DNP mechanism, and where the relaxation transitions resulting from these interactions are governed by translational diffusion of the water molecules. Finally, the possibilities of combining DNP with magnetic resonance microscopy (MRM) are discussed. It is shown that at 26 degrees C the overall DNP-enhanced proton polarization should become maximal in an external field of 0.3 T and become comparable to the thermal equilibrium polarization in a field of 30 T, considerably larger than the largest high-resolution magnet available to date. It is concluded that DNP MRM in this field, which corresponds to a standard microwave frequency of 9 GHz, has the potential to significantly increase the sensitivity in NMR and MRI experiments of small aqueous samples doped with the TAM radical.

High-frequency dynamic nuclear polarization in the nuclear rotating frame.

A proton dynamic nuclear polarization (DNP) NMR signal enhancement (epsilon) close to thermal equilibrium, epsilon = 0.89, has been obtained at high field (B(0) = 5 T, nu(epr) = 139.5 GHz) using 15 mM trityl radical in a 40:60 water/glycerol frozen solution at 11 K. The electron-nuclear polarization transfer is performed in the nuclear rotating frame with microwave irradiation during a nuclear spin-lock pulse. The growth of the signal enhancement is governed by the rotating frame nuclear spin-lattice relaxation time (T(1rho)), which is four orders of magnitude shorter than the nuclear spin-lattice relaxation time (T(1n)). Due to the rapid polarization transfer in the nuclear rotating frame the experiment can be recycled at a rate of 1/T(1rho) and is not limited by the much slower lab frame nuclear spin-lattice relaxation rate (1/T(1n)). The increased repetition rate allowed in the nuclear rotating frame provides an effective enhancement per unit time(1/2) of epsilon(t) = 197. The nuclear rotating frame-DNP experiment does not require high microwave power; significant signal enhancements were obtained with a low-power (20 mW) Gunn diode microwave source and no microwave resonant structure. The symmetric trityl radical used as the polarization source is water-soluble and has a narrow EPR linewidth of 10 G at 139.5 GHz making it an ideal polarization source for high-field DNP/NMR studies of biological systems.

EPR and DNP properties of certain novel single electron contrast agents intended for oximetric imaging.

Parameters of relevance to oximetry with Overhauser magnetic resonance imaging (OMRI) have been measured for three single electron contrast agents of the triphenylmethyl type. The single electron contrast agents are stable and water soluble. Magnetic resonance properties of the agents have been examined with electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and dynamic nuclear polarization (DNP) at 9.5 mT in water, isotonic saline, plasma, and blood at 23 and 37 degreesC. The relaxivities of the agents are about 0.2-0.4 mM-1s-1 and the DNP enhancements extrapolate close to the dipolar limit. The agents have a single, narrow EPR line, which is analyzed as a Voigt function. The linewidth is measured as a function of the agent concentration and the oxygen concentration. The concentration broadenings are about 1-3 microT/mM and the Lorentzian linewidths at infinite dilution are less than 1 microT in water at room temperature. The longitudinal electron spin relaxation rate is calculated from the DNP enhancement curves. The oxygen broadening in water is about 50 microT/mM O2 at 37 degreesC. These agents have good properties for oximetry with OMRI.

Electron paramagnetic resonance and dynamic nuclear polarization of char suspensions: surface science and oximetry.

Carbon chars have been synthesized in our laboratory from a variety of starting materials, by means of a highly controlled pyrolysis technique. These chars exhibit electron paramagnetic resonance (EPR) line shapes which change with the local oxygen concentration in a reproducible and stable fashion; they can be calibrated and used for oximetry. Biological stability and low toxicity make chars good sensors for in vivo measurements. Scalar and dipolar interactions of water protons at the surfaces of chars may be utilized to produce dynamic nuclear polarization (DNP) of the 1H nuclear spin population in conjunction with electron Zeeman pumping. Low-frequency EPR, DNP and DNP-enhanced MRI all show promise as oximetry methods when used with carbon chars.

Postembryonic ontogeny of the spadefoot toad, Scaphiopus intermontanus (Anura: Pelobatidae): skeletal morphology.

Postembryonic skeletal ontogeny of the pelobatid frog Scaphiopus intermontanus is described based on a developmental series of cleared-and-stained, whole-mount specimens. The focus is on laboratory-reared individuals fed a herbivorous diet as larvae. Although there is variation in the timing of ossification of individual skeletal elements relative to developmental stages based on external morphological criteia, the sequence of skeletal development generally is conservative. Compared with its close relative, S. bombifrons, ossifications that occur during prometamorphosis tend to be slightly delayed in S. intermontanus; however, cranial bones that ossify during late metamorphic climax in S. intermontanus are delayed until post-metamorphosis in S. bombifrons. The differences in timing between the two species are consistent, however, with differences observed between two developmental series of S. intermontanus raised at two different temperatures. Noteworthy features of skeletal development in S. intermontanus include: 1) presence of palatine ossifications that form from independent centers of ossification and soon fuse with the postnarial portion of the vomers to form the compound vomeropalatine bones; 2) compound sphenethmoid that may arise from four or more endochondral centers of ossification and one dorsal, dermal center of ossification; and 3) presence of transverse processes and vestigal prezygapophyses on the first postsacral vertebra. The morphology of the larval orbitohyoideus and interhyoideus muscles is compared. The record of skeletal ontogeny and muscle morphology presented herein for the herbivorous larval morph can serve as a baseline for comparisons with the ontogeny of the carnivorous larval morph of Scaphiopus.

[Attitude of Danish patients and physicians to information on cancer. A questionnaire study]. / Danske patienters og laegers holdning til information ved kraeftsygdom. En spørgeskemaundersøgelse.

This survey reports the attitude towards information of cancer patients as regards diagnosis and prognosis among gastroenterologists and patients (not suffering from cancer). The questions were based on a case history (a patient with colonic cancer). Most doctors informed their patients openly, and most patients expected that. Younger doctors in particular were more restrictive than patients as regards information of the spouse.

Integrated health work with 'the new family'.

In this Copenhagen practice couples receive antenatal and postnatal care in group sessions. The health care professionals believe they provide a better service. Parents learn from shared experiences and benefit from the social network that is established.

Hyperpolarized Metabolic MR in the Study of Cardiac Function and Disease.

Several diseases of the heart have been linked to an insufficient ability to generate enough energy (ATP) to sustain proper heart function. Hyperpolarized magnetic resonance (MR) is a novel technique that can visualize and quantify myocardial energy metabolism. Hyperpolarization enhances the MR signal from a biological molecule of interest by more than 10,000 times, making it possible to measure its cellular uptake and conversion in specific enzymatic pathways in real time. We review the role of hyperpolarized MR in identifying changes in cardiac metabolism in vivo, and present the extensive literature on hyperpolarized pyruvate that has been used to characterize cardiac disease in various in vivo models, such as myocardial ischemia, hypertension, diabetes, hyperthyroidism and heart failure. The technical aspects of the technique are presented as well as the challenges of translating the technique into clinical practice. Hyperpolarized MR has the prospect of transforming diagnostic cardiology by offering new insights into cardiac disease and potentially even to contribute to personalized therapy based on a thorough understanding of the individual intracellular metabolism.

Field dependence of T1 for hyperpolarized [1-13C]pyruvate.

In vivo metabolism of hyperpolarized pyruvate has been demonstrated to be an important probe of cellular glycolysis in diseases such as cancer. The usefulness of hyperpolarized (13)C imaging is dependent on the relaxation rates of the (13)C-enriched substrates, which in turn depend on chemical conformation and properties of the dissolution media such as buffer composition, solution pH, temperature and magnetic field. We have measured the magnetic field dependence of the spin-lattice relaxation time of hyperpolarized [1-(13)C]pyruvate using field-cycled relaxometry. [1-(13)C]pyruvate was hyperpolarized using dynamic nuclear polarization and then rapidly thawed and dissolved in a buffered solution to a concentration of 80 mmol l(-1) and a pH of ~7.8. The hyperpolarized liquid was transferred within 8 s to a fast field-cycling relaxometer with a probe tuned for detection of (13)C at a field strength of ~0.75 T. The magnetic field of the relaxometer was rapidly varied between relaxation and acquisition fields where the sample magnetization was periodically measured using a small flip angle. Data were recorded for relaxation fields varying between 0.237 mT and 0.705 T to map the T(1) dispersion of the C-1 of pyruvate. Using similar methods, we also determined the relaxivity of the triarylmethyl radical (OX063; used for dynamic nuclear polarization) on the C-1 of pyruvate at field strengths of 0.001, 0.01, 0.1 and 0.5 T using 0.075, 1.0 and 2.0 mmol l(-1) concentrations of OX063 in the hyperpolarized pyruvate solution.