Metabolic stability of cells for extended metabolomical measurements using NMR. A comparison between lysed and additionally heat inactivated cells.

NMR measurements for metabolic characterization of biological samples like cells, biopsies or plasma, may take several hours for advanced methods. Preanalytical issues, such as sample preparation and stability over the measurement time, may have a high impact on metabolite content, and potentially lead to misinterpretation. The aim of this study was therefore to investigate by 1H HR-MAS NMR the impact of different cell handling preparation protocols on the stability of the cell metabolite content over the measurement time. For this purpose, the metabolite content of fibroblasts and adrenal cells were measured at different time points after lysis and after additional heating. Interestingly the results showed similar metabolite concentrations between lysed and lysed-heated cells at the beginning of the measurement, but increasing differences after some hours of measurement. In lysed cells, metabolism was ongoing, producing metabolite changes over time, contrary to a stable metabolite content of the lysed-heated cells. These results were confirmed in both fibroblasts and adrenal cells. Therefore, in order to minimize metabolite content modifications over the measurement time, it is suggested to use cell lysis in combination with heat inactivation for extended HR-MAS NMR measurements.

Visibility of lipid resonances in HR-MAS spectra of brain biopsies subject to spinning rate variation.

Lipid resonances from mobile lipids can be observed by ¹H NMR spectroscopy in multiple tissues and have also been associated with malignancy. In order to use lipid resonances as a marker for disease, a reference standard from a healthy tissue has to be established taking the influence of variable factors like the spinning rate into account. The purpose of our study was to investigate the effect of spinning rate variation on the HR-MAS pattern of lipid resonances in non-neoplastic brain biopsies from different regions and visualize polar and non-polar lipids by fluorescence microscopy using Nile Red staining. ¹H HR-MAS NMR spectroscopy demonstrated higher lipid peak intensities in normal sheep brain pure white matter biopsies compared to mixed white and gray matter biopsies and pure gray matter biopsies. High spinning rates increased the visibility particularly of the methyl resonances at 1.3 and the methylene resonance at 0.89 ppm in white matter biopsies stronger compared to thalamus and brainstem biopsies, and gray matter biopsies. The absence of lipid droplets and presence of a large number of myelin sheaths observed in white matter by Nile Red fluorescence microscopy suggest that the observed lipid resonances originate from the macromolecular pool of lipid protons of the myelin sheath's plasma membranes. When using lipid contents as a marker for disease, the variable behavior of lipid resonances in different neuroanatomical regions of the brain and at variable spinning rates should be considered. The findings may open up interesting possibilities for investigating lipids in myelin sheaths.

Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study.

High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

Metabolic characteristics of cortical malformations causing epilepsy.

PURPOSE: Cortical malformations (CMs) are increasingly recognized as the epileptogenic substrate in patients with medically refractory neocortical epilepsy (NE). The aim of this study was to test the hypotheses that: 1. CMs are metabolically heterogeneous. 2. The structurally normal appearing perilesional zone is characterized by similar metabolic abnormalities as the CM. METHODS: Magnetic resonance spectroscopic imaging (MRSI) in combination with tissue segmentation was performed on eight patients with NE and CMs and 19 age matched controls. In controls, NAA, Cr, Cho,NAA/Cr and NAA/Cho of all voxels of a given lobe were expressed as a function of white matter content and thresholds for pathological values determined by calculating the 95% prediction intervals. These thresholds were used to identify metabolically abnormal voxels within the CM and in the perilesional zone. RESULTS: 30% of all voxels in the CMs were abnormal, most frequently because of decreases of NAA or increases of Cho. Abnormal voxels tended to form metabolically heterogeneous clusters interspersed in metabolically normal regions. Furthermore, 15% of all voxels in the perilesional zone were abnormal, the most frequent being decreases of NAA and Cr. CONCLUSION: In CMs metabolically normal regions are interspersed with metabolically heterogeneous abnormal regions. Metabolic abnormalities in the perilesional zone share several characteristics of CMs and might therefore represent areas with microscopic malformations and/or intrinsic epileptogenicity.

Temporal lobectomy for epilepsy: recovery of the contralateral hippocampus measured by (1)H MRS.

(1)H MRS imaging was obtained from 10 patients with mesial temporal lobe epilepsy before and after surgery. After surgery, metabolic recovery in the contralateral hippocampus was detected. Preoperatively, reduced N-acetylaspartate (p < 0.04) increased after surgery nonsignificantly to equal control values. Cholines increased after surgery (p < 0.02) and creatine-phosphocreatine showed a trend to higher values. The results suggest that the contralateral hippocampus is affected by repeated seizure activity in the ipsilateral hippocampus, rather than presence of bilateral mesial temporal sclerosis.

1H MRSI predicts surgical outcome in MRI-negative temporal lobe epilepsy.

1H MRS imaging (MRSI) was performed on 15 patients with MRI-negative temporal lobe epilepsy (TLE) who underwent seizure surgery. The non-seizure-free patients (NSF) ipsilateral hippocampal N-acetylaspartate (NAA)/(Cr+Cho) z scores were lower than the contralateral scores (p = 0.04), and the NSF ipsilateral z scores were lower than the seizure-free patients' (SF) ipsilateral z scores (p = 0.0049). Similarly, NSF contralateral scores were lower than contralateral SF (p = 0.02). These findings suggest NAA predicts the surgical outcome in patients with TLE without evidence of mesial temporal sclerosis on MRI.

Kinetics of phenylalanine transport at the human blood-brain barrier investigated in vivo.

In vivo proton magnetic resonance spectroscopy was used to investigate intracerebral phenylalanine (Phe) concentrations in nine patients with classical phenylketonuria (PKU). The study included serial examinations (n = 31; plasma Phe levels: 0.47-2.24 mmol/l) of patients either receiving a Phe-restricted diet (200 mg Phe per day; four patients) or a diet rich in Phe (1000 mg Phe per day; three patients). No spectrum showed metabolic abnormalities besides elevated Phe. Difference spectroscopy yielded intracerebral Phe concentrations between 0.20 and 0.76 mmol/l. Regional variations between parieto-occipital periventricular brain, frontal brain, and cerebellum were not statistically significant. Data could be fitted assuming saturable Phe transport into the brain, based on a symmetric Michaelis-Menten model (characterized by an apparent Michaelis transport constant, K(t,app), and a maximum transport velocity, Tmax) and constant Phe consumption in the brain cells (described by a velocity Vmax). Non-linear least-squares fitting of the combined data from all patients yielded K(t,app) = 0.16 +/- 0.11 mmol/l and (Tmax / Vmax) = 9.0 +/- 4.1. Carrier saturation and competitive inhibition of the influx of other large neutral amino acids can be expected at plasma Phe levels usually found in PKU patients.

Short TE in vivo (1)H MR spectroscopic imaging at 1.5 T: acquisition and automated spectral analysis.

Spectral analysis of short TE in vivo proton magnetic resonance spectroscopic imaging (MRSI) data are complicated by the presence of spectral overlap, low signal to noise and uncharacterized signal contributions. In this study, it is shown that an automated data analysis method can be used to generate metabolite images from MRSI data obtained from human brain at TE = 25 ms and 1.5 T when optimized pulse sequences and a priori metabolite knowledge are used. The analysis approach made use of computer simulation methods to obtain a priori spectral information of the metabolites of interest and utilized a combination of parametric spectral modeling and non-parametric signal characterization for baseline fitting. This approach was applied to data from optimized PRESS-SI and multi-slice spin-echo SI acquisitions, for which sample spectra and metabolite images are shown.

Kinetics of metabolism in human kidney transplants measured by dynamic 31P NMR spectroscopy.

The technique of ex vivo 31P NMR spectroscopy has been used for the investigation of metabolic turnover in 15 cadaveric human kidneys obtained from brain-dead donors for transplantation. Measurements were carried out at 1.5 T time-dependently during regular hypothermic storage in histidine-tryptophan-alpha-ketoglutarate solution. The minimum delay between explanation and spectroscopy was 2 h 41 min. In one case of a discarded organ the measurements could be extended over a period of 161 h 35 min. A detailed kinetic model describing monoexponential ischemic phosphate turnover, which accounts for various interrelations of the NMR visible metabolites, has been derived. Averaged velocity constants of the decays of nucleotides and phosphomono- and -diesters ranged from 0.0047 to 0.294h-1 at approximately 4 degrees C. Intracellular pH decreased monoexponentially with an averaged velocity constant of 0.31 h-1.

Administration and (1)H MRS detection of histidine in human brain: application to in vivo pH measurement.

Measurement of histidine in vivo offers the potential for tissue pH measurement using routinely performed (1)H MR spectroscopy. In the brain, however, histidine concentrations are generally too low for reliable measurement. By using oral loading of histidine, this study demonstrates that brain concentrations can be significantly increased, enabling detection of histidine by localized (1)H MR measurements and making in vivo pH measurement possible. In studies carried out on healthy human subjects at 1.5 T, a consistent spectral quality downfield from water was achieved using a PRESS sequence at short echo times. Measurements at different TE values helped to characterize the downfield spectral region. Histidine loading of 400 mg/kg of body weight increased brain histidine levels by approximately 0.8 mM, with maximum histidine concentration reached 4 to 7 hr after consumption. The pH calculated from histidine resonances was 6.96, and a hyperventilation study demonstrated the potential for measuring altered pH.

A practical double-tuned 1H/31P quadrature birdcage headcoil optimized for 31P operation.

A double-tuned 1H/31P birdcage head coil for use with humans at 1.5 T is described. The coil was designed for proton-decoupled 31P excitation and reception and incorporated a number of practical features including optimized sensitivity for 31P, quadrature operation at 1H and 31P frequencies, and a radiofrequency (RF) mirror for improved B1 homogeneity. The design achieved similar B1 homogeneity at both 31P and 1H frequencies. Inductive matching was used to accommodate samples with large loading differences. A facile method for tuning and matching over a variety of sample loadings is presented, along with capacitively shortened bazookas for suppression of cable braid currents. The proton sensitivity, although down by approximately a factor of two compared with an optimized 1H birdcage head coil, was still ample for shimming and generation of scout images. Advantages of the design are discussed and proton-decoupled 31P spectra of human brain are presented.

Methyl tunnelling, reorientation and NMR relaxation in solid acetates.

Rotational excitations of methyl groups in six solid acetates have been investigated by 1H nuclear magnetic resonance (NMR) relaxation time measurements at 15 MHz and 30 MHz and at temperatures between 10 K and the melting point. Hindering barriers between 1.6 kJ/mol and 3.7 kJ/mol have been found that could be correlated to the tunnelling frequencies observed by inelastic neutron scattering. A consistent description of the relaxation rate dependences from the classical regime at high temperatures to the quantum-mechanical regime at low temperatures is possible by Haupt's equation. The rotational potentials are mainly determined by inter-molecular interaction with an important influence of water of crystallization, if present.

Hippocampal structures: anteroposterior N-acetylaspartate differences in patients with epilepsy and control subjects as shown with proton MR spectroscopic imaging.

PURPOSE: To determine the distribution of proton metabolites along the long axis of the hippocampus. MATERIALS AND METHODS: Proton magnetic resonance (MR) spectroscopic imaging measurements were performed in the hippocampi of 14 control subjects and nine patients with unilateral mesial temporal lobe epilepsy. RESULTS: Control subjects showed significantly lower ratios of N-acetylaspartate (NAA) to choline-containing compounds (Ch) and creatine plus phosphocreatine (CR) (NAA/[Cr + Ch]) in the anterior as compared with the posterior part of the hippocampus. Furthermore, a similar anteroposterior (AP) difference in NAA/(Cr + Ch) values was found in both ipsilateral and contralateral hippocampi of patients. In the patients compared with the control subjects, ipsilateral NAA/(Cr + Ch) levels were reduced in every part of hippocampal tissue with an average reduction of 17%, and contralateral NAA/(Cr + Ch) was reduced by about 10%. In the patients compared with the control subjects, the proportional reduction in ipsilateral NAA/(Cr + Ch) was greatest in voxels from anterior hippocampal regions. CONCLUSION: AP differences could be a result of fewer neurons in the anterior compared with the posterior hippocampus or of the increasing thickness of the hippocampus from posterior to anterior, which leads to different contributions from adjacent tissue. Measurements of T2 showed that T2 differences are probably not responsible for these changes.

In vivo study of brain metabolism in galactosemia by 1H and 31P magnetic resonance spectroscopy.

In order to further evaluate different hypotheses concerning brain metabolism in galactosemia, six adult patients aged 18-29 years with classical galactosemia under dietary treatment underwent localized 1H and 31P magnetic resonance spectroscopy (MRS) in addition to conventional T1- and T2-weighted MRI. Galactose-1-phosphate levels in erythrocytes were 1.1-3.7 mg/dl, plasma concentrations of galactitol ranged from 8.4-14.2 mumol/l. Imaging revealed abnormal peripheral myelination in five and enlargement of lateral ventricles in two patients. Brain galactitol in parieto-occipital white matter was below detectability of 1H MRS leading to the assumption that brain concentrations of the free metabolite was well below 1 mmol/kg. Signal intensities of free myo-inositol, which was postulated to be indicative of changes in the second messenger pathway, were within the normal range. MRS data revealed a normal energy status of the brain. In summary 1H and 31P MRS indicated normal metabolite concentrations as compared to healthy controls.

Hippocampal N-acetylaspartate in neocortical epilepsy and mesial temporal lobe epilepsy.

Previous magnetic resonance spectroscopy (MRS) studies have shown that N-acetylaspartate (NAA) is reduced not only in the ipsilateral but also in the contralateral hippocampus of many patients with mesial temporal lobe epilepsy (mTLE). The reason for the contralateral damage is not clear. To test whether the hippocampus is also damaged if the focus is outside the hippocampus, we have measured patients with neocortical epilepsy (NE). Therefore, the goals of this study were to determine if hippocampal NAA is reduced in NE and if hippocampal NAA discriminates NE from mTLE. MRS imaging (MRSI) studies were performed on 10 NE patients and compared with MRSI results in 23 unilateral mTLE patients and 16 controls. The results show that, in contrast to mTLE, NAA was not reduced in the hippocampus of NE patients, neither ipsilateral nor contralateral to the seizure focus. These results suggest that repeated seizures do not cause secondary damage to the hippocampus. The absence of spectroscopic differences in NE may help to distinguish NE from mTLE.

In-vivo NMR spectroscopy in patients with phenylketonuria: changes of cerebral phenylalanine levels under dietary treatment.

Localized proton magnetic resonance spectroscopy at short echo times was used to measure phenylalanine (Phe) in parieto-occipital periventricular brain. Six treated adult patients with phenylketonuria were investigated repeatedly following reinstitution of a Phe-restricted diet. Difference spectroscopy clearly enabled the identification of elevated cerebral Phe levels by subtracting spectra obtained from healthy volunteers. Estimates of absolute brain concentrations always yielded values well below the serum levels with ratios [Phe]brain/[Phe]serum ranging from 0.27 to 0.63. A plot of [Phe]brain versus [Phe]serum could be fitted to a straight line (R = 0.90) if [Phe]serum was below 1.3 mM. Measurements at higher serum levels could only be performed in one patient and yielded brain Phe concentrations of 0.63 +/- 0.10 mM suggesting a saturation of the carrier systems. The feasibility to quantify Phe transport across the blood-brain barrier in humans non-invasively employing in-vivo proton spectroscopy can effectively improve the prognostic significance of serum data.

In vivo 31P NMR spectroscopy of human musculoskeletal tumors as a measure of response to chemotherapy.

The value of in vivo 31P NMR spectroscopy to provide indicators of response to cytostatic chemotherapy was studied in patients with malignant musculoskeletal tumors. Characteristics of untreated cancers were strong signals of PME and PDE, moderately increased Pi and low PCr. The intracellular pH was slightly alkaline. The intracellular concentration of free magnesium was 70% of that in muscle. Spectroscopic findings at different times of therapy were compared with the percentage of tumor necrosis after surgical resection in 28 patients. In follow-up studies, energy-rich phosphates declined in nonresponders, while PME, Pi and frequently PDE increased. Treatment response appeared to involve the reversal of these trends. In five responders, a biphasic pattern was observed, i.e. initially the spectrum changed into that of severely ischemic cell injury followed by a successive phase of apparent 'tumor activation'. Pretreatment levels of (PCr+Pi)/total phosphate > or = 0.35 and PCr/ alpha-NTP > or = 1.5, an accelerated increase in total low-energy phosphates/total high-energy phosphates (> or = 3.0%/day) after the initial drug application, and a long-term decrease (< or = -0.4%/day) during later therapy were highly indicative of tumor response to chemotherapy. Such spectroscopic predictors for treatment response proved to be superior to currently used indices such as tumor size.

Localized proton NMR spectroscopy in the striatum of patients with idiopathic Parkinson's disease: a multicenter pilot study.

Single voxel proton MRS was used to study brain metabolism in the striatum of patients diagnosed with idiopathic Parkinson's disease (PD). Peak metabolite ratios in long echo time spectra were evaluated in 151 patient spectra and 97 age-matched control spectra collected at four participating institutions using identical hardware and clinical protocols. Combining data from all ages (27-83 years old) showed no significant difference between patient and control ratios. However, in an elderly subset of patients (51-70 years old), a significant decrease in striatal N-acetylaspartate (NAA)/choline (Cho) was observed. Also, a significant decrease in the mean NAA/Cho ratio was observed in patients versus controls for patients not being treated with Sinemet (Du Pont Pharm, Wilmington, DE) (hereafter referred to as levodopa/carbidopa). This result is consistent with the hypothesis that NAA may provide a reversible spectroscopic marker for neuronal dysfunction, although a prospective follow-up study will be needed to confirm this. Quantitation of MRS would be useful to exclude the possibility that a change in Cho levels affected the NAA/Cho ratios.

Metabolic characterization of AIDS dementia complex by spectroscopic imaging.

Prospective proton chemical shift imaging (CSI) of the brain was performed in 30 HIV- 1-seropositive patients and 11 healthy controls. Significant (P < 0.05) reductions in the N-acetyl-L-aspartate (NAA)/total creatine (Cr), and NAA/total choline (Cho) ratios and significant increases in Cho/Cr occurred in patients with 1) AIDS-defining diagnoses; 2) <200 CD4 lymphocyte counts/microl; 3) neurological evidence for an AIDS dementia complex (ADC); 4) magnetic resonance imaging (MRI) signs of cerebral atrophy. The basal ganglia and the insula were affected to approximately the same extent and without indications of spatial variations within these areas. Reduced NAA seems to indicate progressive neuronal injury or loss due to productive HIV infection in the brain and its clinical picture ADC. Spectroscopic abnormalities were, however, also observed in neurologically normal HIV patients or those with normal MRI results. Proton CSI may therefore serve as an early quantitative marker of central nervous system involvement in AIDS.

Temporal lobe epilepsy: qualitative reading of 1H MR spectroscopic images for presurgical evaluation.

PURPOSE: To study the feasibility and clinical potential of visual inspection of hydrogen 1 magnetic resonance (MR) spectroscopic metabolite images for the lateralization of unilateral nonlesional temporal lobe epilepsy (TLE). MATERIALS AND METHODS: MR imaging and 1H MR spectroscopic imaging were performed of the temporal lobes in 50 patients with TLE and 23 age-matched healthy volunteers. N-acetylaspartate (NAA) and creatine plus choline metabolite images were read by two neuroradiologists who determined lateralization according to the side of lower NAA signal intensity. Quantitative estimates of NAA were calculated by using an automated fitting program. RESULTS: Agreement in lateralization between readers was significant with a kappa score of 0.53 for all patients with TLE and 0.63 for patients displaying mild or marked NAA asymmetry. Among the 50 patients with TLE, lateralization was determined correctly by reader 1 in 38 (76%) patients and by reader 2 in 31 (62%) patients. If limited to patients with mild or marked NAA asymmetry, correct lateralization improved to 30 (77%) of 39 and 16 (80%) of 20 patients, respectively. Combined qualitative reading and quantitative spectral fitting enabled lateralization in 34 (85%) of 40 patients with TLE for reader 1 and 30 (77%) of 39 for reader 2, including nine of 14 patients with TLE with negative MR images. CONCLUSION: Reading of metabolite images is a feasible and fast means for noninvasive evaluation of patients with TLE who are candidates for surgery and enables lateralization in some patients with negative MR images.