Noninvasive quantification of ascorbate and glutathione concentration in the elderly human brain

In this study, ascorbate (Asc) and glutathione (GSH) concentrations were quantified noninvasively using double-edited (1)H MRS at 4 T in the occipital cortex of healthy young [age (mean ± standard deviation) = 20.4 ± 1.4 years] and elderly (age = 76.6 ± 6.1 years) human subjects. Elderly subjects had a lower GSH concentration than younger subjects (p < 0.05). The Asc concentration was not significantly associated with age. Furthermore, the lactate (Lac) concentration was higher in elderly than young subjects. Lower GSH and higher Lac concentrations are indications of defective protection against oxidative damage and impaired mitochondrial respiration. The extent to which the observed concentration differences could be associated with physiological differences and methodological artifacts is discussed. In conclusion, GSH and Asc concentrations were compared noninvasively for the first time in young vs elderly subjects.     

Noninvasive quantification of human brain antioxidant concentrations after an intravenous bolus of vitamin C

Until now, the lack of a means to detect a deficiency or to measure the pharmacologic effect in the human brain in situ has been a hindrance to the development of antioxidant‐based prevention and treatment of dementia. In this study, a recently developed ¹H MRS approach was applied to quantify key human brain antioxidant concentrations throughout the course of an aggressive antioxidant‐based intervention. The concentrations of the two most abundant central nervous system chemical antioxidants, vitamin C and glutathione, were quantified noninvasively in the human occipital cortex prior to and throughout 24 h after bolus intravenous delivery of 3 g of vitamin C. Although the kinetics of the sodium‐dependent vitamin C transporter and physiologic blood vitamin C concentrations predict theoretically that brain vitamin C concentration will not increase above its homeostatically maintained level, this theory has never been tested experimentally in the living human brain. Therefore, human brain vitamin C and glutathione concentrations were quantified noninvasively using MEGA‐PRESS double‐edited ¹H MRS and LCModel. Healthy subjects (age, 19–63 years) with typical dietary consumption, who did not take vitamin supplements, fasted overnight and then reported for the measurement of baseline antioxidant concentrations. They then began controlled feeding which they adhered to until after vitamin C and glutathione concentrations had been measured at 2, 6, 10 and 24 h after receiving intravenous vitamin C. Two of the twelve studies were sham controls in which no vitamin C was administered. The main finding was that human brain vitamin C and glutathione concentrations remained constant throughout the protocol, even though blood serum vitamin C concentrations spanned from the low end of the normal range to very high. Copyright © 2010 John Wiley & Sons, Ltd.     

Noninvasive quantification of human brain ascorbate concentration using 1H NMR spectroscopy at 7 T

Ascorbate (Asc, vitamin C) was quantified in the human brain noninvasively using two different ¹H NMR spectroscopy methods: short‐echo time STEAM and MEGA‐PRESS homonuclear editing. Taking advantage of increased sensitivity and chemical shift dispersion at 7 T, Asc was quantified with increased reliability relative to our previous study accomplished at 4 T. Asc concentration quantified from short‐echo time spectra measured from the occipital lobe of eight healthy subjects ([Asc] = 1.1 ± 0.3 µmol/g, mean ± SD) was in excellent agreement with Asc concentration quantified from the same volume of interest using homonuclear editing ([Asc] = 1.2 ± 0.2 µmol/g). This agreement indicates that at 7 T, Asc can be reliably quantified in the human brain simultaneously with 15 other metabolites. Additional advantages of the short‐echo time approach were: shorter measurement time than homonuclear editing and minimal effect of T₂ relaxation on Asc quantification. High magnetic field was also beneficial for Asc quantification with MEGA‐PRESS because increased chemical shift dispersion enabled editing with full efficiency, which resulted in a supra‐linear gain in signal‐to‐noise ratio relative to 4 T. Copyright © 2009 John Wiley & Sons, Ltd.     

Sensitivity and specificity of human brain glutathione concentrations measured using short‐TE 1H MRS at 7 T

Although the MR editing techniques that have traditionally been used for the measurement of glutathione (GSH) concentrations in vivo address the problem of spectral overlap, they suffer detriments associated with inherently long TEs. The purpose of this study was to characterize the sensitivity and specificity for the quantification of GSH concentrations without editing at short TE. The approach was to measure synthetically generated changes in GSH concentrations from in vivo stimulated echo acquisition mode (STEAM) spectra after in vitro GSH spectra had been added to or subtracted from them. Spectra from five test subjects were synthetically altered to mimic changes in the GSH signal. To account for different background noise between measurements, retest spectra (from the same individuals as used to generate the altered data) and spectra from five other individuals were compared with the synthetically altered spectra to investigate the reliability of the quantification of GSH concentration. Using STEAM spectroscopy at 7 T, GSH concentration differences on the order of 20% were detected between test and retest studies, as well as between differing populations in a small sample (n = 5) with high accuracy (R² > 0.99) and certainty (p ≤ 0.01). Both increases and decreases in GSH concentration were reliably quantified with small impact on the quantification of ascorbate and γ‐aminobutyric acid. These results show the feasibility of using short‐TE ¹H MRS to measure biologically relevant changes and differences in human brain GSH concentration. Although these outcomes are specific to the experimental approach used and the spectral quality achieved, this study serves as a template for the analogous scrutiny of quantification reliability for other compounds, methodologies and spectral qualities. Copyright © 2016 John Wiley & Sons, Ltd.     

Proton T2 relaxation of cerebral metabolites of normal human brain over large TE range

T2 of NAA, creatine and choline-containing compounds were measured in posterior frontal white matter and occipital grey matter in 10 healthy human volunteers. Decay curves comprised signals from eight TE times ranging from 30 to 800 ms with TR 2000 ms acquired with a PRESS sequence on a 1.5 T clinical scanner. Simulations were conducted to assess the precision of T2 estimates from decay curves comprising varying numbers and ranges of TE points. Mean and standard errors for T2s of NAA, creatine and choline-containing compounds were 300(8), 169(3) and 239(4) ms in posterior frontal white matter and 256(6), 159(8) and 249(8) ms in occipital grey matter. In vivo T2s found for choline and NAA were shorter than the T2s in the literature. The elevation of literature T2s is accounted for by the simulation results, which demonstrated that there is a bias towards lengthened T2s when T2 is measured with a maximum TE approximately T2. Concentration estimates are at risk of being underestimated if previously reported T2 corrections are used.     

T2 measurement of J-coupled metabolites in the human brain at 3T

Proton T₂ relaxation times of metabolites in the human brain were measured using point resolved spectroscopy at 3T in vivo. Four echo times (54, 112, 246 and 374 ms) were selected from numerical and phantom analyses for effective detection of the glutamate multiplet at ~ 2.35 ppm. In vivo data were obtained from medial and left occipital cortices of five healthy volunteers. The cortices contained predominantly gray and white matter, respectively. Spectra were analyzed with LCModel software using volume‐localized calculated spectra of brain metabolites. The estimate of the signal strength vs. TE was fitted to a monoexponential function for estimation of apparent T₂ (T₂^?). T₂^? was estimated to be similar between the brain regions for creatine, choline, glutamate and myo‐inositol, but significantly different for N‐acetylaspartate singlet and multiplet. T₂^?s of glutamate and myo‐inositol were measured as 181 ± 16 and 197 ± 14 ms (mean ± SD, N = 5) for medial occipital cortices, and 180 ± 12 and 196 ± 17 ms for left occipital cortices, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Proton T1 relaxation times of cerebral metabolites differ within and between regions of normal human brain

Saturation recovery spectra (STEAM) were acquired at 1.5 T with 7 TRs ranging from 530 to 5000 ms and a constant TE of 30 ms in voxels (7.2 ml) located in occipital grey, parietal white and frontal white matter (10 subjects each location). Spectra were also acquired at 7, 21 and 37 degrees C from separate 100 mm solutions of inositol (Ins), choline-containing compounds (Cho), N-acetyl-aspartate (NAA) and creatine. Simulations of T(1) fits with 2, 3 and 7 TRs demonstrated that at typical SNR there is potential for both inaccurate and biased results. In vivo, different metabolites had significantly different T(1)s within the same brain volume. The same order from shortest to longest T(1) (Ins, Cho, NAA, creatine) was found for all three brain regions. The order (Ins, NAA, creatine, Cho) was found in the metabolite solutions and was consistent with a simple model in which T(1) is inversely proportional to molecular weight. For all individual metabolites, T(1) increased from occipital grey to parietal white to frontal white matter. This study demonstrates that, in spectra acquired with TR near 1 s, T(1) weightings are substantially different for metabolites within a single tissue and also for the same metabolites in different tissues.     

Linear discriminant analysis of brain tumour (1)H MR spectra: a comparison of classification using whole spectra versus metabolite quantification

(1)H MRS is an attractive choice for non-invasively diagnosing brain tumours. Many studies have been performed to create an objective decision support system, but there is not yet a consensus as to the best techniques of MRS acquisition or data processing to be used for optimum classification. In this study, we investigate whether LCModel analysis of short-TE (30 ms), single-voxel tumour spectra provide a better input for classification than the use of the original spectra. A total of 145 histologically diagnosed brain tumour spectra were acquired [14 astrocytoma grade II (AS2), 15 astrocytoma grade III (AS3), 42 glioblastoma (GBM), 41 metastases (MET) and 33 meningioma (MNG)], and linear discriminant analyses (LDA) were performed on the LCModel analysis of the spectra and the original spectra. The results consistently suggest improvement in classification when the LCModel concentrations are used. LDA of AS2, MNG and high-grade tumours (HG, comprising GBM and MET) correctly classified 94% using the LCModel dataset compared with 93% using the spectral dataset. The inclusion of AS3 reduced the accuracy to 82% and 78% for LCModel analysis and the original spectra, respectively, and further separating HG into GBM and MET gave 70% compared with 60%. Generally MNG spectra have profiles that are visually distinct from those of the other tumour types, but the classification accuracy was typically about 80%, with MNG with substantial lipid/macromolecule signals being classified as HG. Omission of the lipid/macromolecule concentrations in the LCModel dataset provided an improvement in classification of MNG (91% compared with 76%). In conclusion, there appears to be an advantage to performing pattern recognition on the quantitative analysis of tumour spectra rather than using the whole spectra. However, the results suggest that a two-step LDA process may help in classifying the five tumour groups to provide optimum classification of MNG with high lipid/macromolecule contributions which maybe misclassified as HG.     

Doubly selective multiple quantum chemical shift imaging and T1 relaxation time measurement of glutathione (GSH) in the human brain in vivo

Mapping of a major antioxidant, glutathione (GSH), was achieved in the human brain in vivo using a doubly‐selective multiple quantum filtering based chemical shift imaging (CSI) of GSH at 3 T. Both in vivo and phantom tests in CSI and single voxel measurements were consistent with excellent suppression of overlapping signals from creatine, γ‐Amino butyric acid (GABA) and macromolecules. GSH concentration in the fronto‐parietal region was 1.20 ± 0.16 µmol/g (mean ± SD, n = 7). The longitudinal relaxation time (T₁) of GSH in the human brain was 397 ± 44 ms (mean ± SD, n = 5), which was substantially shorter than that of other metabolites. This GSH‐CSI method permits us to address regional differences of GSH in the human brain under conditions where oxidative stress has been implicated, including multiple sclerosis, aging and neurodegenerative diseases. Copyright © 2012 John Wiley & Sons, Ltd.     

Classification of single-voxel 1H spectra of brain tumours using LCModel

This study presents a novel method for the direct classification of ¹H single‐voxel MR brain tumour spectra using the widespread analysis tool LCModel. LCModel is designed to estimate individual metabolite proportions by fitting a linear combination of in vitro metabolite spectra to an in vivo MR spectrum. In this study, it is used to fit representations of complete tumour spectra and to perform a classification according to the highest estimated tissue proportion. Each tumour type is represented by two spectra, a mean component and a variability term, as calculated using a principal component analysis of a training dataset. In the same manner, a mean component and a variability term for normal white matter are also added into the analysis to allow a mixed tissue approach. An unbiased evaluation of the method is carried out through the automatic selection of training and test sets using the Kennard and Stone algorithm, and a comparison of LCModel classification results with those of the INTERPRET Decision Support System (IDSS) which incorporates an advanced pattern recognition method. In a test set of 46 spectra comprising glioblastoma multiforme, low‐grade gliomas and meningiomas, LCModel gives a classification accuracy of 90% compared with an accuracy of 95% by IDSS. Copyright © 2011 John Wiley & Sons, Ltd.     

Glutamine protects activated human T cells from apoptosis by up-regulating glutathione and Bcl-2 levels

Glutamine is the most abundant amino acid in the body. A decrease of plasma glutamine concentrations is found in catabolic stress and is related to susceptibility to infections. Glutamine is known to modulate lymphocyte activation; however, little is known about glutamine modulation of cell death of activated human T cells. Using Jurkat T cells, we investigated glutamine modulation of T-cell apoptosis activated by PMA plus ionomycin. We found that glutamine at various concentrations significantly enhanced IL-2 production, cell proliferation, and cell viability of Jurkat T cells. Glutamine also decreased the number of apoptotic cells stimulated with PMA plus ionomycin as demonstrated by flow cytometry. Meanwhile, glutamine down-regulated CD95 and CD95L expression, but up-regulated CD45RO and Bcl-2 expression in activated T cells. Further investigation of CD95-mediated caspase activities revealed that supplementation of glutamine significantly decreased caspase-3 and caspase-8 activities in activated T cells. Since oxidative stress is closely associated with induction of lymphocyte apoptosis, we found that glutamine significantly increased glutathione (GSH), but decreased reactive oxygen species levels in activated T cells. Blockade of intracellular GSH formation enhanced, but exogenous GSH supplementation decreased, activated T-cell apoptosis. Studying normal peripheral lymphoproliferation, we also found that the presence of glutamine increased lymphoproliferation as well as Bcl-2 and CD95 expression; but decreased CD95L and activation-induced T-cell death. Taken together, glutamine appeared to augment lymphoproliferation but suppressed activation-induced T-cell death in both Jurkat T cells and human peripheral T lymphocytes.     

The age dependence of T2 relaxation times of N‐acetyl aspartate,creatine and choline in the human brain at 3 and 4T

Knowledge of the T₂ age dependence is of importance for MRS clinical studies involving subject groups with a wide age range. A number of studies have focused on the age dependence of T₂ values in the human brain, with rather conflicting results. The aim of this study was to analyze the age dependence of T₂ values of N‐acetyl aspartate (NAA), creatine (Cr) and choline (Cho) in the human brain using data acquired at 3T and 4T and to assess the influence of the macromolecule (MM) baseline handling on the obtained results. Two distinct groups of young and elderly controls have been measured at 3T (T_E = 30–540 ms, 9 young and 11 elderly subjects) and 4T (T_E = 10–180 ms, 18 young and 14 elderly subjects) using single‐voxel spectroscopy. In addition, MM spectra were measured from two subjects using the inversion‐recovery technique at 4T. All spectra were processed with LCModel using basis sets with different MM signals (measured or simulated) and also with MM signals included for a different T_E range. Individual estimated T₂ values were statistically analyzed using the R programming language for the age dependence of T₂ values as well as the influence of the MM baseline handling. A significant decrease of T₂ values of NAA and Cr in elderly subjects compared with young subjects was confirmed. The same trend was observed for Cho. Significantly higher T₂ values calculated using the measured MM baseline for all studied metabolites at 4T were observed for both young and elderly subjects. To conclude, while the handling of MM and lipid signals may have a significant effect on estimated T₂ values, we confirmed the age dependence of T₂ values of NAA and Cr and the same trend for Cho in the human brain. Copyright © 2016 John Wiley & Sons, Ltd.     

Improvement of resolution for brain coupled metabolites by optimized (1)H MRS at 7T

Resolution enhancement for glutamate (Glu), glutamine (Gln) and glutathione (GSH) in the human brain by TE‐optimized point‐resolved spectroscopy (PRESS) at 7 T is reported. Sub‐TE dependences of the multiplets of Glu, Gln, GSH, γ‐aminobutyric acid (GABA) and N‐acetylaspartate (NAA) at 2.2–2.6 ppm were investigated with density matrix simulations, incorporating three‐dimensional volume localization. The numerical simulations indicated that the C4‐proton multiplets can be completely separated with (TE₁, TE₂) = (37, 63) ms, as a result of a narrowing of the multiplets and suppression of the NAA 2.5 ppm signal. Phantom experiments reproduced the signal yield and lineshape from simulations within experimental errors. In vivo tests of optimized PRESS were conducted on the prefrontal cortex of six healthy volunteers. In spectral fitting by LCModel, Cramér–Rao lower bounds (CRLBs) of Glu, Gln and GSH were 2 ± 1, 5 ± 1 and 6 ± 2 (mean ± SD), respectively. To evaluate the performance of the optimized PRESS method under identical experimental conditions, stimulated‐echo spectra were acquired with (TE, TM) = (14, 37) and (74, 68) ms. The CRLB of Glu was similar between PRESS and short‐TE stimulated‐echo acquisition mode (STEAM), but the CRLBs of Gln and GSH were lower in PRESS than in both STEAM acquisitions. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of in vivo 31P NMR brain spectra using LCModel

Quantification of ³¹P NMR spectra is commonly performed using line‐fitting techniques with prior knowledge. Currently available time‐ and frequency‐domain analysis software includes AMARES (in jMRUI) and CFIT respectively. Another popular frequency‐domain approach is LCModel, which has been successfully used to fit both ¹H and ¹³C in vivo NMR spectra. To the best of our knowledge LCModel has not been used to fit ³¹P spectra. This study demonstrates the feasibility of using LCModel to quantify in vivo ³¹P MR spectra, provided that adequate prior knowledge and LCModel control parameters are used. Both single‐voxel and MRSI data are presented, and similar results are obtained with LCModel and with AMARES. This provides a new method for automated, operator‐independent analysis of ³¹P NMR spectra. Copyright © 2015 John Wiley & Sons, Ltd.     

High-field MRS of the human brain at short TE and TR

In vivo MRS of the human brain at 7 tesla allows identification of a large number of metabolites at higher spatial resolutions than currently possible at lower field strengths. However, several challenges complicate in vivo localization and artifact suppression in MRS at high spatial resolution within a clinically feasible scan time at 7 tesla. Published MRS sequences at 7 tesla suffer from long echo times, inherent signal‐to‐noise ratio (SNR) loss, large chemical shift displacement artifacts or long repetition times because of excessive radiofrequency (RF) power deposition. In the present study a pulse‐acquire sequence was used that does not suffer from these high field drawbacks. A slice selective excitation combined with high resolution chemical shift imaging for in‐plane localization was used to limit chemical shift displacement artifacts. The pulse‐acquire approach resulted in a very short echo time of 1.4 ms. A cost function guided shimming algorithm was developed to constrain frequency offsets in the excited slice, therefore adiabatic frequency selective suppression could be employed to minimize artifacts from high intensity lipids and water signals in the excited slice. The high sensitivity at a TR of 1 s was demonstrated both on a supraventricular slice as well as in an area very close to the skull in the frontal cortex at a nominal spatial resolution of 0.25 cc within a feasible scan time. Copyright © 2011 John Wiley & Sons, Ltd.     

Identification of amide protons of glutathione in MR spectra of tumour cells

Signals attributable to amide protons and used in previous studies to measure intracellular pH were observed in the low-field region of the (1)H-MR spectra of four tumour cell lines: T98G, MCF-7, A172 and HeLa. The signals were more intense in the spectra of the two cell lines (T98G and MCF-7) characterised by higher concentrations of glutathione (GSH). After comparison with (1)H-MR spectra of GSH in solution at different pH values, the peaks were attributed to NHs of the Cys and Gly residues of GSH. Modification of the intracellular concentration of GSH by treatment with buthionine sulfoximine produced comparable decreases in the intensity of aliphatic signals of GSH and NHs under examination. The assignment was therefore confirmed.     

Classification of brain tumours from MR spectra: the INTERPRET collaboration and its outcomes

The INTERPRET project was a multicentre European collaboration, carried out from 2000 to 2002, which developed a decision‐support system (DSS) for helping neuroradiologists with no experience of MRS to utilize spectroscopic data for the diagnosis and grading of human brain tumours. INTERPRET gathered a large collection of MR spectra of brain tumours and pseudo‐tumoural lesions from seven centres. Consensus acquisition protocols, a standard processing pipeline and strict methods for quality control of the aquired data were put in place. Particular emphasis was placed on ensuring the diagnostic certainty of each case, for which all cases were evaluated by a clinical data validation committee. One outcome of the project is a database of 304 fully validated spectra from brain tumours, pseudotumoural lesions and normal brains, along with their associated images and clinical data, which remains available to the scientific and medical community. The second is the INTERPRET DSS, which has continued to be developed and clinically evaluated since the project ended. We also review here the results of the post‐INTERPRET period. We evaluate the results of the studies with the INTERPRET database by other consortia or research groups. A summary of the clinical evaluations that have been performed on the post‐INTERPRET DSS versions is also presented. Several have shown that diagnostic certainty can be improved for certain tumour types when the INTERPRET DSS is used in conjunction with conventional radiological image interpretation. About 30 papers concerned with the INTERPRET single‐voxel dataset have so far been published. We discuss stengths and weaknesses of the DSS and the lessons learned. Finally we speculate on how the INTERPRET concept might be carried into the future. Copyright © 2015 John Wiley & Sons, Ltd.     

Feasibility and reproducibility of neurochemical profile quantification in the human hippocampus at 3 T

Hippocampal dysfunction is known to be associated with several neurological and neuropsychiatric disorders such as Alzheimer's disease, epilepsy, schizophrenia and depression; therefore, there has been significant clinical interest in studying hippocampal neurochemistry. However, the hippocampus is a challenging region to study using ¹H MRS, hence the use of MRS for clinical research in this region has been limited. Our goal was therefore to investigate the feasibility of obtaining high‐quality hippocampal spectra that allow reliable quantification of a neurochemical profile and to establish inter‐session reproducibility of hippocampal MRS, including reproducibility of voxel placement, spectral quality and neurochemical concentrations. Ten healthy volunteers were scanned in two consecutive sessions using a standard clinical 3 T MR scanner. Neurochemical profiles were obtained with a short‐echo (T_E = 28 ms) semi‐LASER localization sequence from a relatively small (~4 mL) voxel that covered about 62% of the hippocampal volume as calculated from segmentation of T₁‐weighted images. Voxel composition was highly reproducible between sessions, with test–retest coefficients of variation (CVs) of 3.5% and 7.5% for gray and white matter volume fraction, respectively. Excellent signal‐to‐noise ratio (~54 based on the N‐acetylaspartate (NAA) methyl peak in non‐apodized spectra) and linewidths (~9 Hz for water) were achieved reproducibly in all subjects. The spectral quality allowed quantification of NAA, total choline, total creatine, myo‐inositol and glutamate with high scan–rescan reproducibility (CV ≤ 6%) and quantification precision (Cramér–Rao lower bound, CRLB < 9%). Four other metabolites, including glutathione and glucose, were quantified with scan–rescan CV below 20%. Therefore, the highly optimized, short‐echo semi‐LASER sequence together with FASTMAP shimming substantially improved the reproducibility and number of quantifiable metabolites relative to prior reports. In addition, the between‐session variation in metabolite concentrations, as well as CRLB, was lower than the between‐subject variation of the concentrations for most metabolites, indicating that the method has the sensitivity to detect inter‐individual differences in the healthy brain. Copyright © 2015 John Wiley & Sons, Ltd.     

Voltammetrically monitored brain ascorbate as an index of excitatory amino acid release in the unrestrained rat

To discover the significance of changes in the extracellular concentration of brain ascorbate, we used linear sweep voltammetry to monitor the ascorbate signal. Recordings were made with carbon paste electrodes implanted in the striatum and hippocampus of anaesthetised and unanaesthetised rats under a variety of conditions. Intraperitoneal administration of excitatory amino acid transmitters, but not tyrosine or glycine, increased extracellular striatal ascorbate; similarly, microinfusion of L-glutamate beside striatal electrodes enhanced the ascorbate signal. Electrical stimulation of the perforant path increased the extracellular concentration of dentate ascorbate in the unanaesthetised, but not in the anaesthetised, rat. These results support our hypothesis that changes in the extracellular concentration of brain ascorbate monitored by voltammetry reflect the release of excitatory amino acids.     

31P‐MRS of healthy human brain: ATP synthesis,metabolite concentrations,pH, and T1 relaxation times

The conventional method for measuring brain ATP synthesis is ³¹P saturation transfer (ST), a technique typically dependent on prolonged pre‐saturation with γ‐ATP. In this study, ATP synthesis rate in resting human brain is evaluated using EBIT (exchange kinetics by band inversion transfer), a technique based on slow recovery of γ‐ATP magnetization in the absence of B₁ field following co‐inversion of PCr and ATP resonances with a short adiabatic pulse. The unidirectional rate constant for the P_i → γ‐ATP reaction is 0.21 ± 0.04 s^?1 and the ATP synthesis rate is 9.9 ± 2.1 mmol min^?1 kg^?1 in human brain (n = 12 subjects), consistent with the results by ST. Therefore, EBIT could be a useful alternative to ST in studying brain energy metabolism in normal physiology and under pathological conditions. In addition to ATP synthesis, all detectable ³¹P signals are analyzed to determine the brain concentration of phosphorus metabolites, including UDPG at around 10 ppm, a previously reported resonance in liver tissues and now confirmed in human brain. Inversion recovery measurements indicate that UDPG, like its diphosphate analogue NAD, has apparent T₁ shorter than that of monophosphates (P_i, PMEs, and PDEs) but longer than that of triphosphate ATP, highlighting the significance of the ³¹P–³¹P dipolar mechanism in T₁ relaxation of polyphosphates. Another interesting finding is the observation of approximately 40% shorter T₁ for intracellular P_i relative to extracellular P_i, attributed to the modulation by the intracellular phosphoryl exchange reaction P_i ? γ‐ATP. The sufficiently separated intra‐ and extracellular P_i signals also permit the distinction of pH between intra‐ and extracellular environments (pH 7.0 versus pH 7.4). In summary, quantitative ³¹P MRS in combination with ATP synthesis, pH, and T₁ relaxation measurements may offer a promising tool to detect biochemical alterations at early stages of brain dysfunctions and diseases. Copyright © 2015 John Wiley & Sons, Ltd.