NMR functional imaging using a tailored RF gradient echo sequence: A true susceptibility measurement technique

Measurement of the resonances of glucose between 3.2 and 3.9 ppm in ¹H NMR spectra from the human brain is difficult due to spectral overlap with peaks from more concentrated metabolites. The H1 resonance of α-D-glucose at 5.23 ppm is resolved from other metabolite peaks, but potentially overlaps with the intense water signal at 4.72 ppm. This paper demonstrates that the increased resolution at 4 Tesla permits to suppress the water signal sufficiently to reliably detect glucose directly at 5.23 ppm by ¹H MRS and the estimated peak intensity is consistent with previous ¹³C NMR quantification.     

Observed BOLD effects on cerebral metabolite resonances in human visual cortex during visual stimulation: a functional (1)H MRS study at 4 T.

Using the localized spin-echo (1)H MRS technique, the water resonance and methyl resonance peaks of the cerebral metabolites N-acetylaspartate (NAA at 2.0 ppm) and phosphocreatine/creatine (Cr at 3.0 ppm) were studied in the human visual cortex to detect and quantify the blood oxygenation level dependent (BOLD) effect during visual stimulation at 4 T. Significant BOLD effects, which reflect the increases of spectral peak height (H) accompanied by the decreases of spectral linewidth (Deltaupsilon(1/2)), were observed in NAA (H: 2.5%; Deltaupsilon(1/2): -1.7%) and Cr (H: 3.1%; Deltaupsilon(1/2): -1.8%) as well as in water (H: 3.1%; Deltaupsilon(1/2): -2.3%). Because NAA and Cr mainly exist in the brain cells, the BOLD effects on these cerebral metabolite resonances only measure the susceptibility component spreading into the extravascular cellular compartment. In contrast, water is affected in the intra- and the extravascular compartments. Therefore, the water signal measures the BOLD effects in both compartments. BOLD responses in water were similar to those observed in metabolites. The similarity indicates that the susceptibility spreading into the extravascular parenchyma contributed significantly to the observed BOLD effects at 4 T. Finally, taking advantage of the higher NMR sensitivity at 4 T, the feasibility of measuring BOLD effects on cerebral metabolites by localized (1)H MRS is demonstrated.     

The carnosine C-2 proton's chemical shift reports intracellular pH in oxidative and glycolytic muscle fibers.

The appearance of new peaks in the 7.7-8.6 and 6.8-7.4 ppm regions of the postexercise (1)H spectrum of frog muscle is reported. These new peaks result from the splitting of single pre-exercise carnosine C-2 and C-4 peaks into two peaks, representing the intracellular pH (pH(I)) of oxidative and glycolytic fibers. The following data support this conclusion: 1) comparison of means and regression analysis indicates equivalence of the pH(I) measurements by (1)H and (31)P NMR; 2) the pre- and poststimulation concentrations of carnosine are equal; 3) in ischemic rat hindlimb muscles, the presence of a single, more acidic peak in the plantaris; a single, less acidic peak in the soleus; and two peaks (more and less acidic) in the gastrocnemius correspond to published values for the fiber-type composition of these muscles; and 4) in muscles treated with iodoacetate prior to and during stimulation, a second peak never appears. These data indicate that it is feasible to measure separately the pH(I) of oxidative and glycolytic fibers using (1)H NMR spectroscopy.     

Metabolite signature of developmental foregut cyst on in vivo and in vitro (1)H MR spectroscopy

Foregut duplication cysts are developmental anomalies of the bronchopulmonary foregut and are common cystic lesions of the mediastinum. We describe a case of mediastinal foregut duplication cyst with in vivo (1)H MR spectroscopy on a 1.5T magnet showing a large metabolite peak at 2.02 ppm, attributable to N-acetylated compounds, in addition to a smaller peak at 1.33 ppm, considered to represent lipids. In vitro NMR spectroscopy (7.05T) of cyst fluid confirmed the presence of these peaks. In addition, a broad multiplet centered at 3.7 ppm, possibly from various protons of the hexose ring system, was also noted. Chemical analysis of the cyst fluid demonstrated the presence of N-acetylhexosamines, proteins, and lipids. Again, in vitro spectra of pure samples of N-acetylglucosamine and N-acetylgalactosamine were obtained for comparison, which better resolved the N-acetyl peak and the peaks at 3.7 ppm. The mucus secreted by respiratory epithelium and the mucous glands of the foregut cysts contains glycoproteins that have N-acetylhexosamines as components and lipid breakdown products that are thought to contribute to the observed spectrum. This information might be useful in predicting the cyst content and, in turn, the lining of the epithelium and the glandular elements.     

Assignment of resonance in the 1H spectrum of rat brain by two-dimensional shift correlated and J-resolved NMR spectroscopy.

Two-dimensional shift-correlated (COSY) and J-resolved NMR spectroscopy was used to identify and assign resonance in the aliphatic region (0.8 to 4.5 ppm) of the 1H spectrum of acid extracts and tissue of rat brain. The chemical shift and spin-spin coupling constants of several resonances, which could not be resolved in one-dimensional spectra of tissue, were determined. These properties, together with the appropriate multiplet structure and scalar coupling patterns observed in 2D J-resolved and COSY spectra, were used to assign the resonances of lactate, threonine, alanine gamma-amino butyrate, N-acetyl aspartate, aspartate, glutamate, glutamine, taurine, and myo-inositol. The single peak observed at 1.33 ppm in the 1D 1H spectrum of excised brain tissue, which is generally assigned to lactate, was shown instead to consist of two overlapping peaks in 2D COSY contour plots. The intensity of both resonances increased considerably after death. The second resonance was assigned to threonine and the assignment was confirmed by ion-exchange chromatography. Several cross peaks were identified in 2D COSY spectra of intact brain tissue that were not present in similar spectra of the acid extract. The nonmetabolite resonances were assigned to macromolecules. Analysis of 2D COSY contour plots in conjunction with 1D spectra revealed that the total creatinine (creatinine + phosphocreatine) resonance at 3.0 ppm was overlapped by both GABA and macromolecules. This macromolecule resonance was present in a 2D COSY spectrum of the rat brain obtained in situ with a surface coil following cardiac arrest, indicating that its presence in the 2D spectrum of excised brain tissue did not arise from tissue disruption.     

Detection and assignment of the glucose signal in 1H NMR difference spectra of the human brain.

The difference between 1H NMR spectra obtained during eu- and hyperglycemia exhibited well-resolved glucose peaks between 3 and 4 ppm as demonstrated by comparison with solution spectra. Estimated increases were consistent with recent 13C NMR quantitations of intracerebral glucose. Difference spectra were measured in 36-ml volumes from the human brain every 3 min.     

1H and 31P NMR measurement of cerebral lactate, high-energy phosphate levels, and pH in humans during voluntary hyperventilation: associated EEG, capnographic, and Doppler findings

In order to explore the sensitivity of spatially resolved 1H and 31P NMR spectroscopy on a whole-body NMR instrument, cerebral metabolic changes in human volunteers were measured during hyperventilation provocation. During hyperventilation the flow velocity in the middle cerebral artery decreased significantly and the EEG showed a marked increase in slow activity. 1H NMR spectra revealed an increase in cerebral lactate concentration. 31P NMR spectra showed no changes in ATP or PCr peak heights, but a shift toward tissue alkalosis was derived from changes in Pi chemical shift. During subsequent recovery, lactate concentration decreased and a slight intracellular acidosis was detected. In three experiments broadening of the lactate resonance peak resulted in separation into two components at 1.32 and 1.48 ppm, in which the latter signal possibly arose from alanine.     

Quantitative and qualitative fat analysis in human leg muscle of neuromuscular diseases by 1H MR spectroscopy in vivo

1H MR spectra were recorded from human gastrocnemius muscle at 63.86 MHz using the body coil of the Signa scanner as transmitter and a 3-in. surface coil as receiver. The fat content of the muscle was quantified relative to that of water in a selected volume or slice. The fat/water ratio was 0.05-0.07 for normal muscle but increased to 0.5-6.0 in primary and secondary muscular disorders such as Duchenne and myotonic dystrophy, Charcot-Marie-Tooth and polio muscular atrophy, cerebral palsy, and spina bifida. In Werdnig-Hoffmann spinal atrophy the ratio was above 10. Water-suppressed and slice-selective 1H spectroscopy was used for qualitative analysis of fat. The 1H profile of gastrocnemius muscles between healthy individuals and patients with neuromuscular diseases showed two major differences. In the normal muscle spectra, the resonance from the -(CH2)n- protons at 1.6 ppm was the most pronounced, whereas in the diseased muscle spectra resonances also appeared between 1.1 and 1.4 ppm. Some diseased muscle spectra showed multiple resonances from -CH = CH- in polyunsaturated fatty acids between 5.5 and 7.0 ppm. The corresponding resonances from = CH-CH2-, 1.9-2.0 ppm, and = CH-CH2-CH =, 2.7-2.9 ppm, were also seen. These peaks are usually not detected in normal muscle.     

Localized 1H NMR spectra of glutamate in the human brain.

Localized 1H NMR spectra at TE = 12 ms were obtained from cerebral cortex of human subjects using ISIS with surface suppression. The 2.29-ppm resonance was assigned to C4 glutamate with contributions from C4 glutamine and GABA using in vivo spectral editing and comparison of chemical shift with pure compounds. The measured intensity ratio between the 2.29 resonance and the creatine resonance at 3.03 ppm was in good agreement with the ratio predicted from previously reported measurements of glutamate, glutamine, and GABA concentrations in biopsied human brain tissue.     

1H-MR波谱评价骨骼肌细胞内脂肪含量的在体和模型研究

目的 解释骨骼肌1H-MRS中的脂峰形态及其影响因素,建立利用骨骼肌1H-MRS评价肌细胞内脂肪的方法 .方法 取5名健康志愿者的小腿胫骨前肌和比目鱼肌区域行1H-MRS检查,改变小腿纵轴与主磁场(B0)之间的角度,观察0.80～1.80 ppm(×10-6)处脂峰形态的变化.体外模型研究采用毛细玻璃管束中灌注大豆油和脂肪乳液,分别模拟肌纤维细胞外、细胞内脂肪,比较脂峰随模璎与B0角度变化的特征.结果小腿骨骼肌在0.80～1.80 ppm处可以观察到3～4个峰,各峰相差约0.20～0.30 ppm;当小腿纵轴与B0所成角度增大时,胫骨前肌肌纤维间隙内的脂肪(EMCL)的亚甲基峰逐渐向右侧移位.体外模型很好地模拟了在体骨骼肌1H-MRS脂峰形态,在0.80～1.80 ppm处出现2组甘油三酸酯亚甲基峰和甲基峰,其中心频率相差0.20～0.30 ppm,分别代表肌细胞内、外脂肪.由于骨骼肌组织肌纤维走行的高度有序性及肌细胞内、外脂肪的分子分布状态不同,两者感应的化学位移不同而表现出波峰的分离.这种肌细胞内外脂肪峰的分离在肌束与B0一致时最大,在两者夹角接近魔角(54.7°)时无法分离.结论骨骼肌1H-MRS中肌细胞内、外脂肪发生分离,是一种无创性评价肌细胞内脂肪含量的有效方法 ;胫骨前肌是进行1H-MRS检查的理想部位.     

Selective suppression of lipid resonances by lipid-soluble nitroxides in NMR spectroscopy.

The ability of lipid-soluble nitroxides to suppress selectively the peaks of lipid resonances in 31P, 1H, and 13C NMR spectra was investigated in serum as part of studies aimed at using these contrast agents for magnetic resonance imaging and magnetic resonance spectroscopy in vivo. Nitroxides are especially interesting potential contrast agents because they can reversibly be converted in cells to diamagnetic hydroxylamines, with conversion rates that are dependent on the redox potential and the intracellular concentration of oxygen; the characterization of nitroxide-dependent changes in NMR spectra may therefore be a useful means to measure oxygen-dependent redox metabolism in vivo. The fatty acid analogs, doxyl stearates, suppressed the methyl resonance of choline and the methyl and methylene peaks of lipids in the 1H NMR spectra of serum samples. As a consequence, lactate peaks, which were not readily detected became clearly resolved and could be evaluated quantitatively. The 31P resonance of phosphatidylcholine in the 31P NMR spectrum was suppressed by 5-doxyl stearate and 4-(N,N-dimethyl-N-hexadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxy l,iodid e (Cat16). In the 13C NMR spectrum, the resonances of the methyl groups of choline and the lipids also were broadened significantly by addition of 5-doxyl stearate. Differential suppression of lipid resonances can be employed to facilitate quantitation of lactate.     

In vivo 1H NMR spectroscopy of an intracerebral glioma in the rat

High-resolution 1H surface coil NMR spectroscopy (MRS) was used to evaluate in vivo the cerebral metabolism changes in rat brain induced by a glial tumor growing in situ. Tumor cells (C6 glioma cells) were stereotaxically placed in the right hemisphere superficially. 1H MRS was performed using 5-mm surface coils implanted over the right hemisphere and the water was suppressed using a binomial sequence. As the intracerebral tumor size increased, there was a marked decrease in the N-acetyl aspartate level and an increase in the 1.3 ppm peak. Edition of this peak showed that lactate increased but lipids increased much more than lactate. Moreover the ratio between the choline-phosphocholine and creatine-phosphocreatine peaks changed. This study demonstrates that high-resolution surface coil 1H MRS can be used to monitor changes in metabolism associated with growth of an experimentally induced rat brain tumor in situ.     

Quantitative proton spectroscopy of canine brain: in Vivo and in Vitro correlations

Quantitative, single-voxel proton NMR spectroscopy of normal brain was performed in five adult beagle dogs using the cerebral water signal as an internal intensity reference. The same brain regions were then rapidly isolated and frozen using a pneumatic biopsy drill, perchloric acid extracted, and analyzed by biochemical assay and high-resolution NMR spectroscopy. The concentrations of the major resonances in the in vivo and in vitro spectra were compared, and good agreement was found between the different measurements. The in vivo spectra contained three peaks at 3.21, 3.04, and 2.02 ppm, which are usually assigned to trimethylamines (TMA), creatines, and N-acetyl derivatives (NAc), which corresponded to be the following metabolite concentration values: 1.7 ± 0.6, 7.7 ± 2.1, and 10.9 ± 2.7 μmol/g wet weight respectively. In vitro, the following metabolite concentrations were measured: glycerophosphocholine (GPC) 1.3 ± 0.2, phosphocholine (PC) 0.5 ± 0.1, phosphocreatine (PCr) 2.6 ± 0.4, creatine (Cr) 5.9 ± 1.4, and N-Acetyl aspartate (NAA) 8.9 ± 1.8 μmol/g wet weight. Therefore, the 3.21 ppm resonance observed in the in vivo spectrum is predominantly GPC and PC in a ratio of 2.6:1, the 3.04 ppm resonance is Cr and PCr in a ratio of 2.3:1, and the 2.02 ppm resonance is predominantly (≈?80%) NAA with small contributions from N-acetylaspartyl-glutamate (NAAG) and glutamate. The data presented here validate the technique of water referencing as a simple and convenient means of quantitating single-voxel in vivo proton NMR spectra of the brain.     

In vivo monitoring of hepatic glutathione in anesthetized rats by 13C NMR.

A method for in vivo (13)C NMR monitoring of hepatic glutathione (GSH) in intact, anesthetized rats has been developed. Studies were conducted using a triple-tuned, surgically implanted surface coil designed for this animal model. The coil permitted complete decoupling and sufficient resolution in the (13)C NMR spectrum to monitor the time course of hepatic (13)C-metabolites of intravenously administered 2-(13)C-glycine, particularly GSH at 44.2 ppm and serine signals at 61.1 and 57.2 ppm, respectively. It further allowed concomitant monitoring of high-energy phosphagens and intracellular pH by (31)P NMR. To confirm in vivo NMR peak assignments, we compared high-resolution 2D (1)H[(13)C] heteronuclear multiple quantum coherence and 1D (13)C spectra of hepatic perchloric acid extracts to those of authentic standards. The fractional isotopic enrichment of hepatic (13)C-glycine increased exponentially at a rate of 1.68 h(-1) and reached its plateau level of 81% in 2 h. The (13)C fractional isotopic enrichment of GSH increased exponentially at a rate of 0.316 h(-1) and reached 55% after 4 h of 2-(13)C-glycine infusion, but without achieving a plateau. To confirm that the resonance at 44.2 ppm resulted from GSH, a rat was given an intravenous dose of 2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine precursor that increases intracellular GSH. As expected, with OTC administration the hepatic (13)C GSH-to-glycine peak area increased more than sevenfold.     

The water resonance as an alternative pH reference: relevance to in vivo 31P NMR localized spectroscopy studies.

pH measurements require a suitable pH reference within the 31P NMR spectrum with respect to which the chemical shift of Pi, and hence pH, may be calculated. In muscle spectra PCr is prominent and provides a reference frequency. However, recent localized tumor studies have reported the absence of PCr, for example, in breast tumors. The use of the alpha-, beta-, and gamma-ATP peaks as suitable references has been suggested, but the position of the beta- and gamma-ATP peaks is dependent upon the intracellular Mg2+ concentration. The alpha-ATP is not affected by ionic concentrations; however, it contains UDPG and NAD+, the presence of which can lead to peak-shape distortion. This paper considers the use of the H2O resonance from the proton spectrum used for shimming as a suitable pH reference, provided this is also localized to the same region of interest, using a sequence giving rise to eddy current effects comparable to those of the 31P NMR sequence. Localized in vivo measurements in the muscle and brain of volunteers indicate good agreement between the proton and phosphorus chemical shifts, allowing the PCr position to be predicted to within 0.01 ppm in all cases.     

In vitro and in vivo studies of 1H NMR visibility to detect deoxyhemoglobin and deoxymyoglobin signals in myocardium.

1H nuclear magnetic resonance (NMR) spectroscopy can be used noninvasively to detect the proximal histidyl N delta proton signals of deoxymyoglobin in the myocardium. However, the quantification of deoxymyoglobin is based on the assumption that the deoxymyoglobin signal detected is not contaminated by the deoxyhemoglobin signals contributed from the blood. The purpose of this study was to conduct in vitro and in vivo 1H NMR studies to examine the in vivo NMR visibility of deoxyhemoglobin in the myocardium. The results demonstrate that the NMR visibility of alpha and beta subunits of deoxyhemoglobin is sensitive to the pulse width for spin excitation because of short T2 relaxation times, and they are not NMR visible in the canine myocardium in vivo at 4.7 T when a 0.5-1.0 msec long Gaussian excitation pulse is used. Therefore, the resonance peak detected at approximately 72 ppm (relative to the water resonance) in the ischemic canine myocardium in vivo is dominated by deoxymyoglobin.     

beta-Ureidopropionase deficiency: a novel inborn error of metabolism discovered using NMR spectroscopy on urine.

In this work, NMR investigations that led to the discovery of a new inborn error of metabolism, beta-ureidopropionase (UP) deficiency, are reported. 1D (1)H-NMR experiments were performed using a patient's urine. 3-Ureidopropionic acid was observed in elevated concentrations in the urine spectrum. A 1D (1)H-(1)H total correlation spectroscopy (TOCSY) and two heteronuclear 2D NMR techniques (heteronuclear multiple bond correlation (HMBC) and heteronuclear single-quantum correlation (HSQC)) were used to identify the molecular structure of the compound that caused an unknown doublet resonance at 1.13 ppm. Combining the information from the various NMR spectra, this resonance could be assigned to 3-ureidoisobutyric acid. These observations suggested a deficiency of UP. With 1D (1)H-NMR spectroscopy, UP deficiency can be easily diagnosed. The (1)H-NMR spectrum can also be used to diagnose patients suffering from other inborn errors of metabolism in the pyrimidine degradation pathway.     

MR proton spectroscopy in multiple sclerosis.

PURPOSE: To elucidate the natural history of visualized MR abnormalities in patients with multiple sclerosis using proton spectroscopy. METHODS: MR imaging and proton spectroscopy (1H spectroscopy) were performed on 16 patients with clinically definite multiple sclerosis. All patients received gadopentetate dimeglumine (Gd-DTPA). RESULTS: Decreased levels of N-acetylaspartate (NAA) were demonstrated in 17 out of 21 lesions. No correlation was found between decreased NAA and Gd-DTPA enhancement. In five out of seven enhancing lesions, abnormal 1H spectra with extra peaks (termed marker peaks) at 2.1-2.6 ppm (ranging in absolute concentration from 10-50 mM protons) were observed. In nine out of 14 unenhancing lesions, no elevated marker peaks were observed. In the five other unenhancing lesions, the levels of these marker peaks were generally lower than the enhancing group. No correlation was found between the NAA levels and the levels of the marker peaks. We suggest two distinct biochemical processes: 1) decreased NAA reflecting neuronal cell loss, and 2) elevated marker peaks reflecting ongoing demyelination. CONCLUSIONS: Based upon these observations we infer that 1) the majority of enhancing lesions are demyelinating with extra peaks at 2.1-2.6 ppm representing a marker of this process, 2) enhancing lesions without this marker most likely represent edematous regions without significant demyelination, and 3) demyelination may be long in duration compared with transient blood-brain barrier disruption manifested by Gd-DTPA enhancement. Our results suggest that 1H spectroscopy has the ability to further categorize MR-demonstrated enhancing and unenhancing lesions in patients with multiple sclerosis and that it may be more sensitive than contrast enhancement in revealing the true time course of demyelination.     

Natural abundance 13C NMR spectra of human muscle, normal and diseased

13C NMR spectra of human surgical muscle samples have been obtained at 50 and 118 MHz. Numerous sharp peaks in the 13C spectrum have been assigned to carbon atoms of soluble metabolites and fatty acyl chains of neutral fats and membrane-bound phospholipids. Comparisons have been made of 13C NMR spectra of normal and diseased muscles after removal of neutral fat by extraction with isopentane. Creatine, lactic acid, and phospholipids are not removed from muscles by isopentane. The most striking difference between 13C NMR spectra of isopentane-extracted normal and diseased muscle samples was the size of the residual 30.5 ppm methylene carbon resonance, that is, small in normal muscles and in nonspecific muscle diseases, but large in myogenic and neurogenic muscle diseases. In addition, differences were also found between normal and diseased muscles in their creatine content and their ability to produce lactic acid. By deoxycholate treatment of isopentane-extracted diseased muscle it is estimated that about one-fifth of the total phospholipids are highly mobile. T1 and NOE measurements indicated that the differences in peak height for the 30.5 ppm resonance between normal and diseased muscle are due only to difference in the amount of highly mobile fatty acyl chains in the muscle and not due to differences in relaxation parameters. 13C NMR of isopentane-extracted muscle appears to permit differentiation of normal from diseased muscle and, within diseased muscle, grading of the severity of the disease.     

In vivo 1H NMR spectroscopy of rat brain at 1 ms echo time.

Using optimized, asymmetric radiofrequency (RF) pulses for slice selection, the authors demonstrate that stimulated echo acquisition mode (STEAM) localization with ultra-short echo time (1 ms) is possible. Water suppression was designed to minimize sensitivity to B1 inhomogeneity using a combination of 7 variable power RF pulses with optimized relaxation delays (VAPOR). Residual water signal was well below the level of most observable metabolites. Contamination by the signals arising from outside the volume of interest was minimized by outer volume saturation using a series of hyperbolic secant RF pulses, resulting in a sharp volume definition. In conjunction with FASTMAP shimming (Gruetter Magn Reson Med 1993;29: 804-811), the short echo time of 1 msec resulted in highly resolved in vivo 1H nuclear magnetic resonance spectra. In rat brain the water linewidths of 11-13 Hz and metabolite singlet linewidths of 8-10 Hz were measured in 65 microl volumes. Very broad intense signals (delta v(1/2) > 1 kHz), as expected from membranes, for example, were not observed, suggesting that their proton T2 are well below 1 msec. The entire chemical shift range of 1H spectrum was observable, including resolved resonances from alanine, aspartate, choline group, creatine, GABA, glucose, glutamate, glutamine, myo-inositol, lactate, N-acetylaspartate, N-acetylaspartylglutamate, phosphocreatine, and taurine. At 9.4 T, peaks close to the water were observed, including the H-1 of alpha-D-glucose at 5.23 ppm and a tentative H-1 resonance of glycogen at 5.35 ppm.