Quantification of metabolites from single-voxelin vivo 1H NMR data of normal human brain by means of time-domain data analysis

We present here a combination of time-domain signal analysis procedures for quantification of human brainin vivo ¹H NMR spectroscopy (MRS) data. The method is based on a separate removal of a residual water resonance followed by a frequency-selective time-domain line-shape fitting analysis of metabolite signals. Calculation of absolute metabolite concentrations was based on the internal water concentration as a reference. The estimated average metabolite concentrations acquired from six regions of normal human brain with a single-voxel spin-echo technique for theN-acetylaspartate, creatine, and choline-containing compounds were 11.4±1.0,6.5±0.5, and 1.7±0.2 mmol kg^–1 wet weight, respectively. The time-domain analyses ofin vivo ¹H MRS data from different brain regions with their specific characteristics demonstrate a case in which the use of frequency-domain methods pose serious difficulties.     

Choline metabolism in breast cancer;2H-,13C- and31P-NMR studies of cells and tumors

Choline metabolism in breast cancer cells and tumors has been investigated by multinuclear NMR in order to provide the biochemical basis for the presence of high phosphocholine in breast carcinoma relative to benign breast tumors and normal breast tissue. Choline was found to be transported into MCF7 human breast cancer cells and rapidly phosphorylated to phosphocholine which was then accumulated in the cells to high concentrations. The increased level of phosphocholine did not affect the rate of synthesis of phosphatidylcholine, indicating tight regulation of this pathway. The incorporation of [l,2-¹³C]choline (100 μM) into phosphocholine and phosphatidylcholine after 24 h was 69.5 and 36% of the total respective pools. Incorporation of²H₉-choline to tumors implanted in nude mice was achieved by infusing the deuterated choline to the blood circulation. The metabolism of deuterated choline was then monitored by²H localized MRS. The blood level of choline before the infusion was 58.6 ± 10.3 μM (measured by¹H-NMR of plasma samples) and increased ～ 5-fold during the infusion (measured by²H-NMR). This increase in the blood level resulted in a gradual increase of a signal at 3.2 ppm due to deuterated choline metabolites. It appears that the increased availability of choline in the blood circulation leads to accumulation of phosphocholine in the tumors by the same mechanism as in the cells. © 1998 Elsevier Science B.V. All rights reserved.     

Severity of delayed (“secondary”) cerebral energy failure after acute hypoxia-ischemia is related to the time integral of acute ATP depletion

The aim of this study was to reproduce the delayed (secondary) cerebral energy failure previously described in birth-asphyxiated newborn infants and to investigate relationships between primary insult severity and the extent of the delayed energy failure. Phosphorus (³¹P) magnetic resonance spectroscopy (MRS) at 7 T was used to study the brains of 12 newborn piglets during an acute, reversible, cerebral hypoxic-ischemic episode which continued until nucleotide triphosphates (NTP) were depleted. After reperfusion and reoxygenation, spectroscopy was continued for 48 h. High-energy metabolite concentrations returned to near normal levels after the insult, but later they fell as delayed energy failure developed. The time integral of NTP depletion in the primary insult correlated strongly with the minimum [phosphocreatine (PCr)]/[inorganic orthophosphate (P_i)] observed 24–48 h after the insult. (Linear regression analysis gave slope –8.04 h^–1; ordinate intercept=1.23;r=0.92;P<0.0001.) This model is currently being used to investigate the therapeutic potential of various cerebroprotective strategies including hypothermia.     

31P MR spectroscopy and in vitro markers of oxidative capacity in type 2 diabetes patients

Background: Skeletal muscle mitochondrial function in type 2 diabetes (T2D) is currently being studied intensively. In vivo ³¹P magnetic resonance spectroscopy (³¹P MRS) is a noninvasive tool used to measure mitochondrial respiratory function (MIFU) in skeletal muscle tissue. However, microvascular co-morbidity in long-standing T2D can interfere with the ³¹P MRS methodology. Aim: To compare ³¹P MRS-derived parameters describing in vivo MIFU with an in vitro assessment of muscle respiratory capacity and muscle fiber-type composition in T2D patients. Methods: ³¹P MRS was applied in long-standing, insulin-treated T2D patients. ³¹P MRS markers of MIFU were measured in the M. vastus lateralis. Muscle biopsy samples were collected from the same muscle and analyzed for succinate dehydrogenase activity (SDH) and fiber-type distribution. Results: Several ³¹P MRS parameters of MIFU showed moderate to good correlations with the percentage of type I fibers and type I fiber-specific SDH activity (Pearson’s R between 0.70 and 0.75). In vivo and in vitro parameters of local mitochondrial respiration also correlated well with whole-body fitness levels (VO _2peak) in these patients (Pearson’s R between 0.62 and 0.90). Conclusion: Good correlations exist between in vivo and in vitro measurements of MIFU in long-standing insulin-treated T2D subjects, which are qualitatively and quantitatively consistent with previous results measured in healthy subjects. This justifies the use of ³¹P MRS to measure MIFU in relation to T2D.     

Magnetic resonance spectroscopy data analysis: time or frequency domain?

Comparisons of time and frequency domain methods are presented forin vitro ¹H andin vivo ³¹P magnetic resonance spectroscopy (MRS) data. Many distortions in the MR spectrum, introduced by applying the Fourier transform to a nonideal free-induction decay (FID), can be handled more elegantly in the time domain, where operations are carried out directly on the measured signal. It was found that if the measured signal is well conditioned—high signal-to-noise ratio (S/N), no truncation, no baseline problems—then both time and frequency domain methods give the same results within the error limits. However, distortions in the measured signal make analysis by time domain methods preferable over frequency domain methods. In all applications, the use of prior knowledge appeared to play an important role.     

PASADENA hyperpolarization of 13C biomolecules: equipment design and installation

Object  The PASADENA method has achieved hyperpolarization of 16–20% (exceeding 40,000-fold signal enhancement at 4.7 T), in liquid samples of biological molecules relevant to in vivo MRI and MRS. However, there exists no commercial apparatus to perform this experiment conveniently and reproducibly on the routine basis necessary for translation of PASADENA to questions of biomedical importance. The present paper describes equipment designed for rapid production of six to eight liquid samples per hour with high reproducibility of hyperpolarization. Materials and methods  Drawing on an earlier, but unpublished, prototype, we provide diagrams of a delivery circuit, a laminar-flow reaction chamber within a low field NMR contained in a compact, movable housing. Assembly instructions are provided from which a computer driven, semi-automated PASADENA polarizer can be constructed. Results  Together with an available parahydrogen generator, the polarizer, which can be operated by a single investigator, completes one cycle of hyperpolarization each 52 s. Evidence of efficacy is presented. In contrast to competing, commercially available devices for dynamic nuclear polarization which characteristically require 90 min per cycle, PASADENA provides a low-cost alternative for high throughput. Conclusions  This equipment is suited to investigators who have an established small animal NMR and wish to explore the potential of heteronuclear (¹³ C and ¹⁵ N) MRI, MRS, which harnesses the enormous sensitivity gain offered by hyperpolarization.     

Large increases in1H metaboliteT 2's after cerebral hypoxia-ischemia correlate with ATP depletion

In vivo proton (¹H) magnetic resonance spectroscopy (MRS) can measure cerebral metabolite concentrations and nuclear relaxation times. Function of the sodium (Na⁺)/potassium (K⁺) pump in cell membranes depends on adequate adenosine triphosphate (ATP) levels: intracellular Na⁺ is normally extruded in exchange for extracellular K⁺. Low ATP will cause pump dysfunction and loss of K⁺ accompanied by influx of Na⁺and water. Raised intracellular water may increase molecular mobility and this might be detectable as increased apparent transverse relaxation times (T ₂'s).¹H-MRS of the brains of newborn piglets during acute hypoxia-ischemia revealed enigmatic increases in the peak area of creatine + phosphocreatine (Cr) relative to those of choline-containing compounds (Cho) andN-acetylaspartate (NAA). Interleaved¹H and phosphorus (³¹P) MRS showed that theT ₂'s of both Cr and lactate (Lac) increased during acute hypoxia-ischemia and these changes correlated with reductions in nucleotide triphosphate (NTP; largely ATP). Within 50 h of metabolic recovery from the primary insult, as delayed energy failure developed, theT ₂'s of Cho, Cr, NAA, and Lac increased greatly. TheseT ₂ changes also correlated with NTP depletion. These observations demonstrate important relationships betweenT ₂'s and function of the ATP-dependent Na⁺/K⁺ pump.     

Experimental31p nmr study of the influence of ionic strength on the apparent dissociation constant of mgatp

The classical method for³¹P NMR determination of intracellular free magnesium concentration ([Mg _free ²⁺ ]) requires an accurate knowledge of the apparent dissociation constant (K _D ) of MgATP. There is a large difference between the previously determined values ofK _D . Although the value of 50 µM, determined by a³¹P NMR method, is now largely accepted, a value of 86 µM has more recently been measured with a fitting method derived from the original one, and with a different ionic strength. The purpose of our study was to assess if the cause of the difference between these two previously reportedK _D values was due to the measuring method or to the ionic strength value used.Working at pH=7.2,T=37°C, and [KCl]=0.25 M, we performedK _D measurements with the original³¹P NMR method and with the fitting method. The results (67±13 µM and 61±20 µM, respectively) were not significantly different. Then, with the first method, we measured K_D at [KCl]=0.12 M and found a value of 19±5 µM. We conclude that the main cause of difference between theK _D values measured by³¹P NMR reside in the disparity of ionic strength values used for their measurement. OurK _D measurements at [KCl]=0.25 and 0.12 M demonstrate the importance of the ionic strength value used for imitating the intracellular medium on the absolute value of ([Mg _free ²⁺ ]) measured by³¹P NMR spectroscopy.Address for correspondence: Université Catholique de Louvain, Unité CPMC, Bâtiment Lavoisier, Place Louis Pasteur n°1, B-1348 Louvain-la-Neuve, Belgium. Additional reprints of this chapter may be obtained from the Reprints Department, Chapman & Hall, One Penn Plaza, New York, NY 10119.     

Glycolytic atp production estimated from31p magnetic resonance spectroscopy measurements during ischemic exercisein vivo

In an oxygen-depleted muscle, glycolytically produced ATP is inversely related to the ([ATP] + creatine phosphate [PCr]) decrease because ATP, PCr, and glycolysis are virtually the only energy sources under these conditions. In particular, the onset of glycolysis or any appreciable increase in the rate of glycolytic ATP production will lead to a slower rate of ([ATP] + [PCr]) breakdown at a given energy consumption. To quantify this relationship, endurance athletes performed isometric foot plantar flexion (20% of a test force [TF],n=10; 50% TF,n=5) during local arterial occlusion. Parameters of energy metabolism were measured with³¹P magnetic resonance spectroscopy (³¹P-MRS). During exercise, [PCr] decreased to 80±10 (20% TF) and 11±4% (50% TF) of its resting concentration, and pH dropped from 7.04 0.01 to 6.98±0.10 (20% TF) and from 7.03±0.02 to 6.70 0.10 (50% TF). In both experiments, two phases of ([ATP] + [PCr]) decrease were observed: an initial faster decrease was followed by a slower decline. The latter phase started at about the time when the pH began to drop. The difference between a line extrapolated from the slope of the initial phase and the measured ([ATP] + [PCr]) decrease was used as an estimate for glycolytically produced ATP. This estimate and pH were significantly correlated withr=–0.97 (20% TF) andr=–0.99 (50% TF). These results indicate that glycolytically produced ATP can be estimated from the ([ATP] + [PCr]) decrease during exercise.     

The magnitude of signal errors introduced by ISIS in quantitative31P MRS

It is well known that the quality of a quantitative³¹P MRS measurement relies largely on the performance of the volume selection method, and that image selected in vivo spectroscopy (ISIS) suffers from contaminating signal caused mostly by Tl smearing. However, these signal errors and their magnitude are seldom addressed in clinical studies. The aim of this study was therefore to investigate the magnitude of signal errors in³¹P MRS when using ISIS. The results from the measurements with a homogeneous head phantom are as follows: at low TR/T1 ratios the contamination increases rapidly, especially for small (< 27 cm³) VOI sizes; at TR/T1 = 1, the signal from a 27 cm³ VOI was 20% too high, and from an 8 cm³ VOI 150% too high. The signal obtained from different VOI positions varied between 80 and 127%. The signal varied linearly with the³¹P concentration in the object. However, a too high signal was obtained when the concentration was lower in the region of interest (inner container) than in the rest of the phantom. The agreement between the simulations and measurements shows that the results of this study are generally applicable to the measurement geometry and the ISIS experiment order rather than being specific for the MR system studied. The errors obtained both experimentally and in computer simulations are too large to be ignored in clinical studies using the ISIS pulse sequence.     

Reduced purine catabolite production in the postischemic rat heart: a31P NMR assessment of cytosolic metabolites

Ischemia can cause release of adenosine and purine catabolites from the heart, through the breakdown of ATP. If repeated periods of ischemia are induced, the efflux of purines is markedly reduced, although it is not clear if this is beneficial for the long-term survival of the heart. We have investigated changes in high-energy phosphates and purine release in the isolated perfused rat heart using³¹P NMR spectroscopy and high-performance liquid chromatography. Hearts were subjected to one of the following protocols: Group A—1 min of total global ischemia (TGI) after 40 min, 60 min, and 85 min of perfusion (a total of 3 × 1 min ischemia); Group B—1 min of TGI after 40 min of perfusion, 10 min of TGI after 50 min of perfusion, and a final 1 min of TGI after 85 min of perfusion. The profile of high-energy phosphate metabolites, P_i accumulation and purine release was similar for each 1-min period of TGI in Group A, whereas phosphocreatine content was increased and ATP content reduced by an extended period of TGI in Group B, leading to a less severe acidosis and purine efflux in the final 1 min of TGI at 85 min of perfusion. In conclusion, the reduced purine release observed in Group B may be related to the preischemic ATP pool size and accessibility and the increased myocardial energy reserve in the form of phosphocreatine.     

Comparison of in vivo1H MRS of human brain tumours with1H HR-MAS spectroscopy of intact biopsy samples in vitro

High resolution magic angle spinning (MAS)¹H nuclear magnetic resonance (NMR) spectroscopy has been employed to study intact human brain tumour tissue and comparison with the corresponding in vivo spectrum has been made. Two dimensional¹H MAS-NMR measurements, including J-resolved and homonuclear shift correlation spectra, were obtained to aid metabolite signal assignment. MAS gave greatly improved line-shape and reduced line-width in comparison to conventional high resolution in vivo¹H MRS of intact tissue, permitting the simultaneous detection of cellular lipids and metabolites. The technique provides the most direct method for comparison of in vivo spectra with high resolution spectra in vitro and hence allows more reliable peak assignment of in vivo¹H MRS spectra.     

Quality assurance of PASADENA hyperpolarization for 13C biomolecules

Object  Define MR quality assurance procedures for maximal PASADENA hyperpolarization of a biological ¹³C molecular imaging reagent. Materials and methods  An automated PASADENA polarizer and a parahydrogen generator were installed. ¹³C enriched hydroxyethyl acrylate, 1-¹³C, 2,3,3-d₃ (HEA), was converted to hyperpolarized hydroxyethyl propionate, 1-¹³C, 2,3,3-d₃ (HEP) and fumaric acid, 1-¹³C, 2,3-d₂ (FUM) to hyperpolarized succinic acid, 1-¹³C, 2,3-d₂ (SUC), by reaction with parahydrogen and norbornadiene rhodium catalyst. Incremental optimization of successive steps in PASADENA was implemented. MR spectra and in vivo images of hyperpolarized ¹³C imaging agents were acquired at 1.5 and 4.7 T. Results  Application of quality assurance (QA) criteria resulted in incremental optimization of the individual steps in PASADENA implementation. Optimal hyperpolarization of HEP of P = 20% was achieved by calibration of the NMR unit of the polarizer (B ₀ field strength ± 0.002 mT). Mean hyperpolarization of SUC, P = [15.3 ± 1.9]% (N = 16) in D ₂O, and P = [12.8 ± 3.1]% (N = 12) in H ₂O, was achieved every 5–8 min (range 13–20%). An in vivo ¹³C succinate image of a rat was produced. Conclusion  PASADENA spin hyperpolarization of SUC to 15.3% in average was demonstrated (37,400 fold signal enhancement at 4.7 T). The biological fate of ¹³C succinate, a normally occurring cellular intermediate, might be monitored with enhanced sensitivity.     

The C-neu mammary carcinoma in Oncomice; characterization and monitoring response to treatment with herceptin by magnetic resonance methods

To characterize spontaneously occurring c-neu/HER2 overexpressing tumours in oncomice and their response to herceptin by non-invasive magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). Oncomice were monitored by localized ³¹P MRS during unperturbed growth and before and after treatment with 10 mg/kg herceptin (Hoffman La Roche) intraperitoneally for up to 21 days post-treatment. Vascular morphology and function was assessed by quantitation of tumour magnetic resonance (MR) relaxation rates R₂* and R₂ prior to and either during carbogen (95% O₂/5% CO₂) breathing or following administration of the blood-pool contrast agent NC100150 (Clariscan, Amersham Health). Immunohistochemistry showed strong membrane staining for HER2 protein overexpression. The ³¹P MRS showed only a significant (p<0.01) increase of phosphomonoester / total phosphate ratio over 21 days of growth. Herceptin increased the tumour volume doubling time compared to untreated tumours and significantly increased the phosphomonoester / -nucleoside triphosphate ratio 2 days after treatment (p=0.01). Tumours showed a highly heterogeneous yet significant (p<0.01) decrease or increase in R₂* in response to carbogen or NC100150 respectively. The absence of a decline in tumour bioenergetics with growth, commonly seen in ³¹P MRS studies of transplanted rodent tumour models, coupled with the heterogeneous blood volume revealed by ¹H MRI, suggest a metabolic and vascular phenotype similar to that found in human tumours.     

1H magnetic resonance spectroscopy of the brains of normal infants and after perinatal hypoxia-ischemia

The aims of this study were to define proton (¹H) metabolite peak-area ratios in the brains of normal infants and to investigate abnormalities after perinatal hypoxia-ischemia. Point-resolved spectroscopy (PRESS) data were collected at 2.4 T with an echo time (TE) of 270 ms from 8-ml voxels located in the thalamus or occipito-parietal region. Fourteen normal and 9 asphyxiated infants were studied. The gestational plus postnatal ages (GPA) of these two groups were 31–41 (median 36) and 27–41 (37) weeks, respectively, and the asphyxiated infants were studied aged 0–10 (2) days. Peak-area ratios were determined in the normal infants for choline-containing compounds (Cho), creatine plus phosphocreatine (Cr),N-acetylaspartate (NAA) and lactate (Lac). Lactate was detected in all the normal infants and Lac/NAA was higher in the occipito-parietal region than in the thalamus (p<0.005). Lac/NAA decreased with increasing GPA in both the thalamus (p=0.014) and the occipito-parietal region (p=0.033). In six of the nine asphyxiated infants, Lac/NAA was above 95% confidence intervals for either the thalamus and/or the occipito-parietal region. Of these six infants, two died and three were neurologically abnormal aged 2 months, indicating that elevated Lac/NAA after perinatal hypoxia-ischemia may convey a poor prognosis. Propan-l,2-diol (the phenobarbitone injection medium) was detected at 1.1 ppm in three infants.     

Characterization of Barium Titanate: BaTiO<Subscript>3</Subscript> (BT) Ceramics Prepared from Sol-Gel Derived BT Powders

Sol-gel synthesis was attempted at room temperature by adding drop wise a clear solution (made by reacting BaCO₃ with glacial acetic acid) to an equi-molar solution of titanium tetrabutoxide in isopropanol (IPA) and acetic acid. The gelation occurred within 30 minutes. The as-dried gel was calcined at 750^∘C/6 h in air to obtain carbon free BaTiO₃ powders. The resulting BT powders were further pressed into pellets and sintered at 1280^∘C/4 h in air to get dense ceramics (density ∼ 94%). XRD and SEM techniques were used for phase and microstructure analysis respectively. The room temperature relative permittivity (ε) of 1280 increased to 7200 (at Curie temperature of 127^∘C) at 1 kHz frequency. The dissipation factor (tanδ) ∼1.6% was observed in these samples. Also, the ferroelectric properties such as spontaneous polarization (Ps ∼ 13 μC/cm²), coercive field (E_c∼4.0 kV/cm) and dielectric strength (∼34 kV/cm) are estimated for these samples. These performance parameters are compared with the available standard data from the literature.     

Tissue oxygen tension measurements in the Shionogi model of prostate cancer using 19 F MRS and MRI

Objectives: To investigate changes in tumour tissue oxygenation throughout the tumour growth–regression–relapse cycle in an androgen-dependent animal tumour model. Materials and methods: ¹⁹F T₁ relaxometry of Perfluoro-15-Crown-5-Ether was used to measure in vivo partial oxygen pressure (pO₂) of Shionogi tumours on a 2.35-T MR scanner. Perfluoro-15-Crown-5-Ether was administered as an emulsion injected intravenously or as a neat compound injected directly into the tumour. Non-localized, tumour ¹⁹F T₁ measurements, made at multiple time points throughout the tumour cycle, were translated into pO₂ levels. Results: No correlation between tumour size and pO₂ values was found. Values of pO₂ for growing tumours (50 ± 30 torr) were significantly lower than for regressing and relapsing tumours after 9 days post-castration (70 ± 10 torr, p<0.05). Maximum pO₂ values (90 ± 30 torr) were reached between fifth and eighth day post-castration, when tumour pO₂ was significantly higher than both pre-castration (p<0.001) and after 9 days post-castration (p<0.05). Conclusion: We demonstrate that longitudinal pO₂ measurements in vivo are feasible. Values of pO₂ for growing androgen-dependent tumours were significantly lower than for regressing and relapsing androgen-independent tumours. These results have potential clinical importance in optimizing the timing of chemotherapy and/or radiotherapy of hormone dependent tumours.     

A cell perfusion system for MR spectroscopic investigations with horizontal magnets

Magnetic resonance spectroscopy (MRS) of perfused cell systems is a useful method to investigate a variety of physiological problems. With the growing importance ofin vivo MRS in biomedical studies, complementary examination of perfused cell systems can contribute to a better understanding ofin vivo data. Up to now, perfused cells were almost exclusively studied using vertical high-field magnets for two reasons. On the one hand, no tomographs suitable for spectroscopy were available when initial studies of this kind were conducted. On the other hand, vertical magnets are generally better suited for this purpose due to spectral dispersion, field homogeneity and adjustability of probe temperature. We present a cell perfusion system for MRS using MR tomographs (horizontal magnets). The compact construction of the system secures easy handling. A constant perfusion temperature (±0.1°C) was achieved by integrating all necessary elements into a temperature-adjusted unit, while a solenoid coil was used to improve the S/N ratio, compared with vertical magnet systems. Address for correspondence: Department of Radiology, Steglitz Medical Centre, FU Berlin, Hindenburgdamm 30, D-1000 Berlin 45, Germany.     

An iron-based T 1 contrast agent made of iron-phosphate complexes: In vitro and in vivo studies

A new iron-based T ₁ contrast agent consisting of a complex of iron ions coordinated to phosphate and amine ligands (Fe_(phos) in short) has been characterized by spectroscopic and magnetic measurements. NMR relaxation studies showed r ₁ values to be dependent on the phosphate salt concentration, K₂HPO₄, present in the medium. r ₁ reaches a maximum value of 2.5 mM^?1 s^?1 for measurements carried out at 7 T and 298 K. ³¹P MRS, Mössbauer spectroscopy and magnetic measurements of Fe_(phos) solutions suggest paramagnetic Fe³⁺ ions present in the studied iron–phosphate complex. In vitro and in vivo toxicity experiments with C6 cells and CD1 mice, respectively, demonstrated lack of toxicity for Fe_(phos) at the highest dose tested in the MRI experiments (12 mM iron for C6 cells and 0.32 mmol iron/kg for mice). Finally, T ₁ weighted images of brain tumours in mice have shown positive contrast enhancement of Fe_(phos) for tumour afflicted regions in the brain.