In vivo NMR spectroscopy: in situ 15N pulse labelling NMR spectroscopy with photoautotrophic microorganisms

For studying the nitrogen metabolism in plants ¹⁵N NMR spectroscopy can be used. For in vivo ¹⁵N NMR (natural abundance of ¹⁵N: 0.37%) enrichment of the sample with the isotope ¹⁵N is compulsory. The detection of time courses of ¹⁵N assimilation from cells, which are enriched in culture is restricted in scope. Here, a method, the ¹⁵N pulse labelling NMR spectroscopy, is demonstrated, which permits labelling of different nitrogen compounds in photoautotrophic microorganisms during the NMR spectroscopic measurement. Using an effective illumination system it is possible to maintain photosynthesis in plant samples of high biomass densities in the magnet necessary for ammonia assimilation. The technique thus enables to directly observe ammonia assimilation pathways by application of a ¹⁵NO₃ ^? or ¹⁵NH₄ ^? pulses.

Für das Studium des Stickstoffstoffwechsels der Pflanzen kann die ¹⁵N-NMR-Spektroskopie herangezogen werden. Hierzu ist bei der in-vivo-¹⁵N-NMR (natürliche Häufigkeit von ¹⁵N: 0.37%) eine Anreicherung der Probe mit dem Isotop ¹⁵N unerläßlich. Eine Verfolgung der ¹⁵N-Assimilationskinetik mit Zellen, die in der Kultur angereichert wurden, ist jedoch nur bedingt möglich. In dieser Arbeit wird die ¹⁵N-Pulsmarkierungs-NMR-Spektroskopie als eine Methode vorgestellt, die es erlaubt, eine Markierung von Stickstoffverbindungen in photoautotrophen einzelligen Mikroorganismen während der NMR-Messung im Magneten vorzunehmen. Es wird ein spezielles Beleuchtungssystem verwendet, das eine für die Stickstoffassimilation ausreichende Photosyntheseleistung der Zellen unter NMR-Bedingungen bei hoher Biomassedichte ermöglicht. Diese Technik erlaubt durch die Applikation eines ¹⁵NO₃ ^?-oder ¹⁵NH₄ ⁺-Pulses eine direkte Verfolgung von Ammonium-Assimilationswegen.     

In vivo 1H NMR spectroscopy of individual human brain metabolites at moderate field strengths

This article reviews spectral editing techniques for in vivo ¹H NMR spectroscopy of human brain tissue at moderate field strengths of 1.5–3 Tesla. Various aspects of ¹H NMR spectroscopy are discussed with regard to in vivo applications. The parameter set [δ, J, n] (δ being the relative chemical shift, J the scalar coupling constant and n the number of coupled spins) is used to characterize the spin systems under investigation and to classify the editing techniques that are used in in vivo ¹H NMR spectroscopy.     

In vivo C magnetic resonance spectroscopy of a human brain tumor after application of C-1-enriched glucose

Objectives

As a unique tool to assess metabolic fluxes noninvasively, ¹³C magnetic resonance spectroscopy (MRS) could help to characterize and understand malignancy in human tumors. However, its low sensitivity has hampered applications in patients. The aim of this study was to demonstrate that with sensitivity-optimized localized ¹³C MRS and intravenous infusion of [1-¹³C]glucose under euglycemia, it is possible to assess the dynamic conversion of glucose into its metabolic products in vivo in human glioma tissue.

Materials and Methods

Measurements were done at 3 T with a broadband single RF channel and a quadrature ¹³C surface coil inserted in a ¹H volume coil. A ¹H/¹³C polarization transfer sequence was applied, modified for localized acquisition, alternatively in two (50 ml) voxels, one encompassing the tumor and the other normal brain tissue.

Results

After about 20 min of [1-¹³C]glucose infusion, a [3-¹³C]lactate signal appeared among several resonances of metabolic products of glucose in MR spectra of the tumor voxel. The resonance of [3-¹³C]lactate was absent in MR spectra from contralateral tissue. In addition, the intensity of [1-¹³C]glucose signals in the tumor area was about 50% higher than that in normal tissue, likely reflecting more glucose in extracellular space due to a defective blood–brain barrier. The signal intensity for metabolites produced in or via the tricarboxylic acid (TCA) cycle was lower in the tumor than in the contralateral area, albeit that the ratios of isotopomer signals were comparable.

Conclusion

With an improved ¹³C MRS approach, the uptake of glucose and its conversion into metabolites such as lactate can be monitored noninvasively in vivo in human brain tumors. This opens the way to assessing metabolic activity in human tumor tissue.     

13C NMR spectroscopy of polycyclic aromatics. VI. Coumarin and the methylcoumarins

The ¹³C NMR spectra of coumarin and its six methyl derivatives and of 6-ethyl-8-methylcoumarin have been assigned by selective ¹³C{¹H} double resonance and by inspection of one-bond and longer-range ¹³C−¹H coupling constants. The effects, Δδ, of the methyl substituents on the chemical shifts are compared with corresponding effects in the methylnaphthalenes. Correlations between Δδ′s and INDO MO charge densities are poor. A relation between ortho- (or β−) effects of sterically unperturbed methyl groups and π bond orders (P_π) is demonstrated.     

Natural abundance (17)O NMR spectroscopy of rat brain in vivo

Oxygen is an abundant element that is present in almost all biologically relevant molecules. NMR observation of oxygen has been relatively limited since the NMR-active isotope, oxygen-17, is only present at a 0.037% natural abundance. Furthermore, as a spin 5/2 nucleus oxygen-17 has a moderately strong quadrupole moment which leads to fairly broad resonances (T(2)=1-4 ms). However, the similarly short T(1) relaxation constants allow substantial signal averaging, whereas the large chemical shift range (>300 ppm) improves the spectral resolution of (17)O NMR. Here it is shown that high-quality, natural abundance (17)O NMR spectra can be obtained from rat brain in vivo at 11.74 T. The chemical shifts and line widths of more than 20 oxygen-containing metabolites are established and the sensitivity and potential for (17)O-enriched NMR studies are estimated.     

Anoxia followed by hyperoxia: In vivo 31-P NMR of cat brain

In vivo ³¹P NMR spectroscopy was performed on a cat brain subjected to an extended period of anoxia followed by restoration of oxygen. High energy phosphate spectra were continuously obtained and pH measured. Following the onset of anoxia, phosphocreatine and ATP peaks decreased with a concomitant increase in inorganic phosphate. Following 34 min ventilation on 100% N₂, the animal was ventilated on 100% O₂. The spectral content progressively changed, inorganic phosphate decreased and ATP increased with the spectrum closely resembling that of control. Our results suggest that the absence of NMR detectable ATP signal cannot be interpreted as an irreversable change in cellular metabolic function.     

High resolution NMR spectroscopy of rat brain in vivo through indirect zero-quantum-coherence detection

The time evolution of zero-quantum-coherences (ZQCs) is insensitive to magnetic field inhomogeneity. Using a 2D indirect ZQC detection method it is shown that high-resolution (1)H NMR spectra can be obtained from rat brain in vivo at 11.74T that are immune to magnetic field inhomogeneity. Simulations based on the density matrix formalism, as well as in vitro measurements are used to demonstrate the features of 2D ZQC NMR spectra. Unique spectral information which is normally not directly available from regular (1)H NMR spectra can be extracted and used for compound identification or improved prior knowledge during spectral fitting.     

In vivo proton magnetic resonance spectroscopy studies of human brain

In vivo localized proton magnetic resonance spectroscopy (MRS) studies of brain were performed on eighteen normal subjects using the stimulated echo (STE) sequence. The absolute concentrations and proton relaxation times of N-acetyl aspartate (NAA), total creatine (Cr) and choline (Cho) were estimated. The MRS data was quantitatively analyzed for repeatability and intersubject variability. Quantitative analysis indicates excellent spectral repeatability. Significant intersubject variations in [NAA] and [Cr] have been observed while the intersubject variability in [Cho] has been found to be fairly small. Significant intensity distortions have been observed for mixing times longer than 50 msec.     

Elemental quantitation of natural organic matter by CPMAS 13C NMR spectroscopy

Cross-polarized magic-angle-spinning NMR (CPMAS-NMR) techniques are assumed to be only semi-quantitative in the assessment of carbon distribution in humic substances or natural organic matter, due to a number of interferences such as spinning side bands (SSB) in spectra, paramagnetic species in samples, and low or remote protonation of aromatic carbons. Fast rotor spin rates or direct polarization NMR techniques are normally applied to improve quantitative signal detectability. Variable contact time pulse sequences were used here to obtain CPMAS-NMR spectra of organic compounds of known structure and different humic substances. Integration of spectral areas, previously subtracted of SSB, and relative stoichiometric factors were used for mathematical elaboration to calculate the elemental content in samples. These values did not significantly differ from those obtained by direct determination of elemental content with quantitative elemental analysis. Our results showed that the carbon observed CPMAS-NMR provides a quantitative representation of the whole carbon content in humic substances.     

In vivo NMR T2 relaxation of experimental brain tumors in the cat: a multiparameter tissue characterization.

Experimental gliomas (F98) were inoculated in cat brain for the systematic study of their in vivo T₂ relaxation time behavior. With a CPMG multi-echo imaging sequence, a train of 16 echoes was evaluated to obtain the transverse relaxation time and the magnetization M(0) at time T = 0. The magnetization decay curves were analyzed for biexponentiality. All tissues showed monoexponential T₂, only that of the ventricular fluid and part of the vital tumor tissue were biexponential. Based on these NMR relaxation parameters the tissues were characterized, their correct assignment being assured by comparison with histological slices. T₂ of normal grey and white matter was 74 ± 6 and 72 ± 6 msec, respectively. These two tissue types were distinguished through M(0) which for white matter was only 0.88 of the intensity of grey matter in full agreement with water content, determined from tissue specimens. At the time of maximal tumor growth and edema spread a tissue differentiation was possible in NMR relaxation parameter images. Separation of the three tissue groups of normal tissue, tumor and edema was based on T₂ with T_2(normal) < T_2(tumor) < T_2(edema). Using M(0) as a second parameter the differentiation was supported, in particular between white matter and tumor or edema. Animals were studied at 1–4 wk after tumor implantation to study tumor development. The magnetization M(0) of both tumor and peritumoral edema went through a maximum between the second and third week of tumor growth. T₂ of edema was maximal at the same time with 133 ± 4 msec, while the relaxation time of tumor continued to increase during the whole growth period, reaching values of 114 ± 12 msec at the fourth week. Thus, a complete characterization of pathological tissues with NMR relaxometry must include a detailed study of the developmental changes of these tissues to assure correct experimental conditions for the goal of optimal contrast between normal and pathological regions in the NMR images.     

In vivo NMR imaging and T1 measurements of water protons in the human brain

Nuclear magnetic resonance (NMR) proton density images of the human brain have been made by the FONAR method. Spin-lattice relaxation times, T₁, of water hydrogen protons have been determined at random positions within frontal and temporal regions of the human brain. The primary purpose of this ongoing research is to accumulate a large data base of normal T₁ values for water protons in normal human brain tissue. Our experience to data includes 31 measurements on 18 volunteer subjects, and the mean value ± standard deviation is 215 ± 42 msec. In addition, two metastatic lesions of the brain were studied and found to have T₁ values longer than those for normal brain tissue.     

Feasibility of using hyperpolarized [1-13C]lactate as a substrate for in vivo metabolic 13C MRSI studies

The development of dynamic nuclear polarization in solution has enabled in vivo 13C MR studies at high signal-to-noise ratio following injection of prepolarized 13C substrates. While prior studies have demonstrated the ability to observe metabolism following injection of hyperpolarized 13C pyruvate, the goal of this study was to develop and test a new hyperpolarized agent for investigating in vivo metabolism, [1-13C]lactate. A preparation for prepolarized 13C lactate and the requisite dissolution media were developed to investigate the feasibility for in vivo 13C MRS/MRSI studies following injection of this hyperpolarized agent. This study demonstrated, for the first time, not only the ability to detect hyperpolarized [1-13C]lactate in vivo but also the metabolic products 13C pyruvate, 13C alanine and 13C bicarbonate following injection in normal rats. The use of 13C lactate as a substrate provided the opportunity to study the conversion of lactate to pyruvate in vivo and to detect the secondary conversions to alanine and bicarbonate through pyruvate. This study also demonstrated the potential value of this hyperpolarized agent to investigate in vivo lactate uptake and metabolism in preclinical animal models.     

In vivo proton magnetic resonance spectroscopy of alcohol in rhesus monkey brain

Brain alcohol was measured in rhesus monkeys (Macaca mulatta) by proton magnetic resonance spectroscopy (MRS) following acute nasogastric alcohol administration (0.8 g/kg). Monkeys were anesthetized with ketamine and xylazine. A 1.5 T whole body imager and a 3-inch surface coil were used to acquire TE 30 and 270 ms spectra from a 7.5 cc voxel localized with a stimulated echo (STEAM) sequence. Venous blood samples were collected during spectral acquisitions for gas chromatographic determination of temporally concordant blood alcohol levels (BALs). Acute alcohol administration did not alter the resonance areas of N-acetylaspartate/N-acetyl containing compounds (NAA), choline containing compounds, or total creatine. The NAA resonance was used as an internal standard to calculate approximate brain alcohol concentrations, which averaged 27 ± 3% and 27 ± 8% of temporally concordant BALs (T₂-corrected TE 30 and TE 270 ms spectra, respectively). In addition to reconfirming results from prior studies finding incomplete detection of brain alcohol with MRS, these results demonstrate the feasibility of measuring brain alcohol in anesthetized nonhuman primates to examine relationships between alcohol exposure history and MRS-visibility of brain alcohol.     

In vivo dynamic turnover of cerebral 13C isotopomers from [U-13C]glucose

An INEPT-based (13)C MRS method and a cost-effective and widely available 11.7 Tesla 89-mm bore vertical magnet were used to detect dynamic (13)C isotopomer turnover from intravenously infused [U-(13)C]glucose in a 211 microL voxel located in the adult rat brain. The INEPT-based (1)H-->(13)C polarization transfer method is mostly adiabatic and therefore minimizes signal loss due to B(1) inhomogeneity of the surface coils used. High quality and reproducible data were acquired as a result of combined use of outer volume suppression, ISIS, and the single-shot three-dimensional localization scheme built in the INEPT pulse sequence. Isotopomer patterns of both glutamate C4 at 34.00 ppm and glutamine C4 at 31.38 ppm are dominated first by a doublet originated from labeling at C4 and C5 but not at C3 (with (1)J(C4C5) = 51 Hz) and then by a quartet originated from labeling at C3, C4, and C5 (with (1)J(C3C4) = 35 Hz). A lag in the transition of glutamine C4 pattern from doublet-dominance to quartet dominance as compared to glutamate C4 was observed, which provides an independent verification of the precursor-product relationship between neuronal glutamate and glial glutamine and a significant intercompartmental cerebral glutamate-glutamine cycle between neurons and glial cells.     

Indirect 13C tomography and volume-selective spectroscopy via proton NMR. II. Imaging

Pulse sequences are presented permitting two- or three-dimensional Fourier transform imaging selectively of protons coupled to ¹³C nuclei. In this way, ¹³C images can indirectly be recorded with proton sensitivity. The maximum enhancement theoretically is a factor of 64 times the number of protons coupled to a ¹³C nucleus. The techniques are based on the heteronuclear editing sequences for volume-selective spectroscopy, which are reported in a previous article. The heteronuclear multiple-quantum filter imaging variant has been implemented on 4.7 and 2 T NMR tomographs and tested with phantoms as well as with biological objects with ¹³C in natural abundance.     

Reconstruction of randomly under-sampled spectra for in vivo 13C magnetic resonance spectroscopy

PurposeOver the past decade, many techniques have been developed to reduce radiofrequency (RF) power deposition associated with proton decoupling in in vivo Carbon-13 (¹³C) magnetic resonance spectroscopy (MRS). In this work we propose a new strategy that uses data under-sampling to achieve reduction in RF power deposition.Materials and methodsEssentially, proton decoupling is required only during randomly selected segments of data acquisition. By taking advantage of the sparse spectral pattern of the carboxylic/amide region of in vivo ¹³C spectra of brain, we developed an iterative algorithm to reconstruct spectra from randomly under-sampled data. Fully sampled data were used as references. Reconstructed spectra were compared with the fully sampled references and evaluated using residuals and relative signal intensity errors.ResultsNumerical simulations and in vivo experiments at 7 Tesla demonstrated that this novel decoupling and data processing strategy can effectively reduce decoupling power deposition by greater than 30%.ConclusionThis study proposes and evaluates a novel approach to acquire ¹³C data with reduced proton decoupling power deposition and reconstruct in vivo ¹³C spectra of carboxylic/amide metabolite signals using randomly under-sampled data. Because proton decoupling is not needed over a significant portion of data acquisition, this novel approach can effectively reduce the required decoupling power and thus SAR. It opens the possibility of performing in vivo ¹³C experiments of human brain at very high magnetic fields.     

Improvement of the inverse-gated-decoupling sequence for a faster quantitative analysis of various samples by 13C NMR spectroscopy

The inverse-gated-decoupling sequence enables quantitative (1)H decoupled (13)C spectra to be obtained. We modified this sequence so as to obtain the same result in less time for molecules containing carbons with various relaxation properties. For that, we determined the optimal (13)C longitudinal-magnetization initial value for a faster relaxation while (1)H decoupler is stopped. This value can be calculated precisely via the nuclear Overhauser effects, the longitudinal relaxation times, together with the determination of the relaxation rate constants of carbons while (1)H are out of equilibrium. A supplementary delay of (1)H decoupling and/or a series of selective pulses applied at the beginning of the recovery delay allow an acceleration of (13)C longitudinal relaxation. We applied this method to the molecule of vanillin. The simultaneous quantification of all carbons was carried out with a recovery delay divided by two compared to the usual sequence.     

Absolute metabolite quantification by in vivo NMR spectroscopy: II. a multicentre trial of protocols for in vivo localised proton studies of human brain

We have performed a multicentre trial to assess the performance of three techniques for absolute quantification of cerebral metabolites using in vivo proton nuclear magnetic resonance (NMR). The techniques included were 1) an internal water standard method, 2) an external standard method based on phantom replacement, and 3) a more sophisticated method incorporating elements of both the internal and external standard approaches, together with compartmental analysis of brain water. Only the internal water standard technique could be readily implemented at all participating sites and gave acceptable precision and interlaboratory reproducibility. This method was insensitive to many of the experimental factors affecting the performance of the alternative techniques, including effects related to loading, standing waves and B₁ inhomogeneities; and practical issues of phantom positioning, user expertise and examination duration. However, the internal water standard method assumes a value for the concentration of NMR-visible water within the spectroscopic volume of interest. In general, it is necessary to modify this assumed concentration on the basis of the grey matter, white matter and cerebrospinal fluid (CSF) content of the volume, and the NMR-visible water content of the grey and white matter fractions. Combining data from 11 sites, the concentrations of the principal NMR-visible metabolites in the brains of healthy subjects (age range 20–35 years) determined using the internal water standard method were (mean ± SD): [NAA] = 10.0 ± 3.4 mM (n = 53), [tCho] = 1.9 ± 1.0 mM (n = 51), [Cr + PCr] = 6.5 ± 3.7 mM (n = 51). Evidence of system instability and other sources of error at some participating sites reinforces the need for rigorous quality assurance in quantitative spectroscopy.     

Indirect 13C tomography and volume-selective spectroscopy via proton NMR. I. Spectroscopic techniques

The sensitivity of ¹³C spectroscopy can be considerably enhanced by selectively detecting the hydrogen nuclei bound to the '3C nuclei of interest. The maximum signal enhancement is theoretically a factor of 64 times the number of protons coupled to a ¹³C nucleus. Three different pulse sequences which permit the image-guided volume-selective single-scan editing of protons bound to ¹³C are reported. The methods are partially designed to be insensitive to the pulse phases and amplitudes and as far as possible to coherent motions. The procedures are based on the same principles as the homonuclear versions previously reported. The lines are edited by multiple-quantum, split-pathway, or cyclic polarization transfer filtering. The sequences have been implemented on 4.7 and 2 T NMR tomographs. Tests have been carried out with phantom samples as well as with volunteers.