A Multi-Center Study on Human Brain Glutathione Conformation using Magnetic Resonance Spectroscopy

In vitro NMR studies

Aqueous solutions of GSH (Sigma-Aldrich, USA) and GSH with N-acetyl aspartate (NAA) (Sigma-Aldrich, USA) were prepared in PBS. One dimensional (1D) and two dimensional (2D) total correlation (TOCSY) spectroscopic data were acquired using a 500 MHz Bruker Avance III spectrometer equipped with a “broad band observe” probe (Department of Chemistry, Case Western Reserve University, Cleveland, OH, USA). Proton NMR data were collected at 25°C and 37°C for GSH and, at 25°C, 37°C, and 47°C for the mixed GSH and NAA samples. For internal chemical shift reference, TSP (3-(trimethylsilyl)-propionate-d4) was used. Two dimensional TOCSY data on GSH PBS solution were performed for establishing through bond connectivity at 25°C using a mixing time of 80 ms.

NMR data processing

One dimensional NMR data were processed using MestReNova (version 11.0.4) software. Data were apodized with mixed exponential (2.50 Hz) and Gaussian (4.70 Hz) windows, and zero-order phase correction was applied. Multipoint spline algorithm was used for baseline correction. Both 1D data and 2D NMR TOCSY data were referenced to TSP. In 2D TOCSY data processing, T₁-noise suppression was applied in both the indirect (f1) and acquisition (f2) directions of the 2D spectra. Data apodization was performed with mixed exponential (6.10 Hz) and Gaussian (2.40 Hz) windows.

In vivo human brain studies

In vitro NMR study on GSH conformations

We present the NMR derived results to show the variability in chemical shift positions of GSH peaks (specifically Cys-H _α and Cys-H _β ) indicating the importance of the local environment on GSH conformations. Figure 2 shows the 1D ¹H spectra of GSH and its correlation with COSY/TOCSY spectra in different sample conditions such as dimethyl sulfoxide (DMSO), the aqueous medium with degassing condition as well as in PBS. The chemical shifts of GSH peaks were referenced with respect to TSP in both the degassed aqueous medium condition [19] and in PBS solution. For the NMR studies of GSH solution in PBS, the external reference TSP (taken in a 2 mm NMR tube) inserted co-axially in the 5 mm NMR tube containing GSH solution in PBS was used. Figure 2A and 2B show the COSY and the TOCSY spectra of GSH in DMSO and degassed aqueous solutions, respectively, highlighting the through bond spin-spin connectivity between the Cys-H _α peak (at 4.40 ppm) with Cys-H _β (2.80 ppm) in DMSO and in degassed environment [19] representing the stable (or closed) form of GSH conformation. In the non-degassed PBS solution, the TOCSY spectrum of GSH (Fig. 2C) shows the through bond spin-spin connectivity between Cys-H _α peak (at 4.56 ppm) with Cys-H _β (at 2.95 ppm) representing the extended GSH conformation. Fig. 2D-F show the respective 1D-NMR spectra highlighting the Cys-H _α and Cys-H _β peak positions for GSH sample in the DMSO, degassed aqueous, and PBS buffer solutions.

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Fig.2

In vitro GSH sample study using NMR under different sample conditions. 2D NMR showing J-coupled GSH Cysteine peaks under different sample conditions: (A) COSY spectra of GSH in DMSO [18], (B) TOCSY spectra of GSH in aqueous D₂O (degassed with nitrogen) [19], (C) TOCSY spectra of GSH in phosphate buffer saline (PBS) solutions; 1D NMR sample study for GSH in (D) DMSO [18], (E) aqueous D₂O (degassed) [19], (F) PBS solutions (no degassing) (copyright permission obtained from the respective publishers).

Temperature dependent NMR studies have been carried out to show that ¹H chemical shift positions of GSH remains unaltered irrespective of the sample temperature, or pH 6.4, 6.9, 7.0, and 7.2 [19]. Figure 3A and 3B illustrate the comparative temperature based analysis of 1D ¹H spectra of GSH in PBS (27°C and 37°C), and degassed aqueous mediums (7°C (pH 7.0) and 17°C (pH 6.4, 6.9, and 7.2)) [19].

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Fig.3

Effect on Cysteine GSH peak positions under varying environmental conditions. (A) 1D NMR spectra of GSH in PBS solution using 500 MHz NMR at different temperatures (i) 25°C, (ii) 37°C, (B) 1D NMR of GSH in nitrogen degassed aqueous solution with different (i) pH 5.6, 6.4, 7.2 at temperature 290 K (17°C), [19] (ii) pH 7.0 at temperature 280 K (7°C). GSH peak positions remain consistent irrespective of pH and/or temperature (copyright permission obtained from the publisher).

Since N-acetyl aspartate (NAA) is available in high concentration (9–14 mM) [28] in human brain and the aspartyl CH₂ peaks also appear in MEGA-PRESS pulse sequence during in vivo GSH detection [2, 30], we have also performed NMR studies of NAA and NAA+GSH samples at various temperatures. Figure 4 illustrates the temperature dependent NMR studies for GSH and GSH+NAA samples at various temperatures (25°C, 37°C, and 47°C). High resolution NMR spectra of GSH+NAA show that the aspartate-CH₂ peaks of NAA at 2.67 ppm and 2.49 ppm [8, 28] are well separated from the GSH Cys-H _β peak at 2.95 ppm. The motivation for this comparative temperature study as presented in Fig. 4, is to establish that only the water peak shifts with temperature, while the chemical shift positions of NAA and GSH are unaffected.

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Fig.4

Comparison of relative peak positions of GSH and GSH+NAA samples in phosphate buffer saline (PBS) using NMR study over different temperatures. (A) 1D NMR for GSH in PBS solution at 25°C, (B) 1D NMR for GSH+NAA PBS solution at 25°C, (C) GSH+NAA in PBS solution 37°C and, (D) GSH+NAA in PBS solution at 47°C using 500 MHz NMR.

In vivo human brain GSH and its conformations

Synthesis of information from the existing NMR studies [6] are extremely helpful for selecting the excitation pulse position (e.g. 4.40 ppm or 4.56 ppm) to identify GSH conformation in vivo. Figures 5–7 demonstrate the existence of the two GSH conformers in vivo irrespective of the selected parameters under the effect of variable TE and inversion (OFF) pulse positions, scanners, and population, respectively. In vivo MRS data have been acquired from NBRC and Bergen using the same experimental protocol. Further details of our MEGA-PRESS experiments are as follows:

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Fig.5

In vivo GSH conformer detection using MEGA-PRESS sequence using 3T Philips and GE scanners with the two MEGA-ON pulse positions, i.e., 4.40 ppm (A) and 4.56 ppm (B) and analysis of relative peak positions and phase of the two GSH conformation using same experimental parameters. Effect of different echo times on the two MEGA-ON pulse positions using both the Philips and GE scanners has been demonstrated for variable TE (120 ms, 125 ms, 130 ms and 140 ms) using MEGA-ON pulse at 4.40 ppm (A) and at 4.56 ppm (B). The selective excitation 180° pulse is applied at 4.40 ppm (for closed conformer) and 4.56 ppm (for extended conformer) (TE = 120 ms and TE = 125 ms give the two out of phase conformer peaks while the same two peaks appear in-phase for TE = 130 ms and TE = 140 ms. Excitation with ON-pulse at 4.40 ppm gives a sharp and heightened peak at ∼2.80 ppm and a broad peak at ∼2.95 ppm; however, ON-pulse at 4.56 ppm results in nearly equal peak shapes and area at two different positions) as the two GSH conformal peaks). The peaks are labeled as: GSH_ex refers to Cys-H _β extended form; GSH_cl refers to Cys-H _β closed form; NAA-CH₂ refers to NAA-Aspartate CH₂ group.

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Fig.6

Effect of inversion pulse position on the in vivo GSH conformer peak detection using MEGA-PRESS sequence. Validation of the two GSH conformal peaks in different published protocols for MEGA-ON, MEGA-OFF (ON/OFF = 4.40 ppm/5.0 ppm; 4.40 ppm/7.22 ppm; 4.56 ppm/5.0 ppm, 4.56 ppm/7.22 ppm; TE = 120 ms; TR = 2500 ms, voxel size = 3.5×3.5×3.5 cm³ on left parietal cortex) using (A) Philips scanner (Indian site) (B) GE scanner (Norwegian site) (both the peaks are present irrespective of the chosen protocols).

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Fig.7

Identification of the two in vivo GSH conformer peaks in controls subjects from different continents. (Data from Indian subject, subject from African, Australian, and American origins are collected using Philips scanner at NBRC, India, and data from Norwegian subject is collected using GE scanner at Bergen, Norway). Same MRS protocol was used for data acquisition: TE = 130 ms, TR = 2500 ms, (A) MEGA-ON/OFF = 4.40 ppm/5.00 ppm and, (B) MEGA-ON/OFF = 4.56 ppm/5.00 ppm) (voxel size = 3.5×3.5×3.5 cm³ on left parietal cortex).

J-couplings of Cys-H _α peaks in the extended (4.56 ppm) and the closed (4.40 ppm) conformations of GSH are used in our in vivo MEGA-PRESS study. Figure 5 shows the in vivo results from two centers including data from Philips and GE scanners for analyzing the effects of editing pulse positions at 4.40 ppm (Fig. 5A) and 4.56 ppm (Fig. 5B). It has been found that, in both conditions, the two GSH conformer peaks, i.e., extended form at ∼2.95 ppm (marked by GSH_ex) and closed form at ∼2.80 ppm (marked by GSH_cl), as well as the aspartate (-CH₂) group of NAA at 2.49 ppm and 2.67 ppm (marked by NAA-CH₂) are present. Based on the observations from NMR sample study, in vivo MEGA-PRESS data was acquired with the editing ON-pulse applied at 4.40 ppm (GSH Cys-H _α ), and the J-coupled peak at 2.80 ppm (GSH Cys-H _β ) was observed. Figure 5A1 and 5B1 show the results for edited GSH MEGA-PRESS data acquired from 3T Philips scanner with a voxel size of 3.5×3.5×3.5 cm³ on left parietal cortex with ON-pulse set at 4.40 ppm and 4.56 ppm, respectively. The two figures are arranged as stacked edited spectra of the two ON-pulse positions acquired with different TE values, and it is observed that, while the Cys-H _β of the two GSH conformers appear out-of-phase for TE = 120 ms and 125 ms, they are in-phase for TE values of 130 ms and 140 ms. In the case of the ON-pulse at 4.40 ppm, the signal from the closed conformer at 2.80 ppm is sharp and more intense, while the other form of GSH at 2.95 ppm appears as a broad peak. In contrast, application of the ON-pulse at 4.56 ppm shows a higher proportion of the extended conformer peak at 2.95 ppm over the peak at 2.80 ppm. However, relative MRS signal observed for one of the two detected GSH conformer peaks depends on the selection of 180° refocusing (ON) pulse, which is clearly evident in Fig. 5A1(iv) and 5B1(iv). Conclusively then, as demonstrated in our in vitro GSH studies, the two GSH conformers also exist in vivo and the MRS signal intensity of the particular conformer depends on the selectivity of 180° excitation pulse at 4.40 ppm for the closed and 4.56 ppm for the extended form of GSH. In Fig. 5 (A2 and B2), MEGA-PRESS data recorded on the GE scanner (Norway) showed a similar GSH pattern for the data acquired using identical experimental conditions. Our multi-center GSH study with different populations and scanners therefore establishes that GSH exists in two different conformations irrespective of diversity of population and data acquisition methodology. To the best of our knowledge, the two GSH conformations have not been studied with respect to their relative in vivo changes.

GSH conformation in vitro

To understand the conformational changes of GSH, molecular dynamics [11–14], Raman spectroscopy [15, 16], and sample studies with NMR [17, 18] have been published. Molecular dynamics studies have shown that the structural conformation of GSH is very flexible in aqueous solution and an inter-conversion occurs between the extended and the folded forms [12]. The various chemical shifts of Cys-H _α and Cys-H _β resonance peaks are indicative of the conformational status of GSH. In our earlier report [6], we correlated Cys-H _α and Cys-H _β peak positions to the corresponding closed (4.40 ppm and 2.80 ppm) and extended (4.56 ppm and 2.95 ppm) conformations of GSH. The COSY spectra of aqueous GSH sample (Supplementary Figure 2) clearly show a J-coupling between Cys-H _α and Cys-H _β at 4.40 ppm and 2.80 ppm [18], which is additionally validated by results from the spin-connectivity between Cys-H _α and Cys-H _β at 4.40 ppm and 2.80 ppm in total correlation spectroscopy (TOCSY) of GSH in aqueous medium under degassed condition (Fig. 3B) [19]. The stable GSH form is reported in DMSO solvent [12] and two dimensional COSY spectrum of GSH in DMSO shows off-diagonal cross-peak connection of Cys-H _α , Cys-H _β at 4.40 ppm and 2.80 ppm [18]. In the aqueous medium, the closed conformation of GSH, the chemical shift of Cys-H _α and Cys-H _β [18] from COSY spectra as well as the TOCSY spectra of GSH shows cross-peak connectivity between 4.40 ppm and 2.80 ppm [19] resembling NMR spin connectivity of Cys-H _α and Cys-H _β (cross peak between 4.40 ppm and 2.80 ppm) of GSH in DMSO [18]. On the contrary, in the case of extended GSH Cys-H _α and Cys-H _β TOCSY spectra, cross peak connectivity is observed between 4.56 ppm and 2.95 ppm. The NMR chemical shift positions of Cys-H _α and Cys-H _β of GSH are reflective of conformational states (extended or closed conformer) or completely oxidized (GSSG) form. Published NMR studies are analyzed to demonstrate the oxidation sensitivity of GSH [19]. It is found that cysteine peaks (Cys-H _α and Cys-H _β ) are more susceptible to the surrounding environment, and the effect of oxygen tension in local environment is reflected as the transitional shift in both the Cys-H _α and Cys-H _β peak positions (GSH in degassed sample (Cys-H _α 4.40 ppm, -H _β : 2.80 ppm) [18, 19]; aqueous non-degassed (Cys-H _α 4.56 ppm,-H _β ppm) [8]; oxidized GSH (GSSG) (Cys-H _α : 4.65 ppm, -H _β (3.26 and 2.96 ppm) [38]). It is important to note that the chemical shift information of GSH conformers from phantom studies may not lead to sufficient accuracy, firstly because of the difficulty in reproducing the local cellular and anaerobic or aerobic physiological conditions in the brain and secondly due to the uncontrolled local environment within an MR scanner.

GSH conformation in vivo

GSH is sensitive to its surrounding environment due to the presence of thiol group and its sensitivity to biological activities. Low concentration of GSH in the brain and overlap of its spectral patterns with other metabolites make it difficult to detect it using commonly used PRESS sequences: thus a special MEGA-PRESS editing sequence based on GSH-Cysteine peak J-coupling is used for GSH detection. Information derived from the GSH NMR studies has confirmed two conformational states of GSH, which implies suitable experimental parameter selection in MEGA-PRESS MRS editing sequence for in vivo GSH detection. However, this is not the case for other popular in vivo metabolites such as lipid, lactate, NAA, glutamate, creatine, choline, and myo-inositol due to the absence of the thiol group in these metabolites. These metabolites do not have any known structural conformational states, and hence chemical shift information of these in vitro metabolites (except GSH) can be considered as gold standard for detection in vivo studies.

We overrule the interference from other metabolite peak positions in our GSH MEGA-PRESS data. The possible appearance of other brain metabolites at 2.80 ppm in terms of the peak positions of their protonic moieties may be rationalized further. As the human brain contains a variety of metabolites, the peak at 2.80 ppm should not be confused with NAA or aspartate (Asp). Selective editing pulse at 4.40 ppm being close to NAA-CH Asp moiety (i.e., 4.38 ppm), excites NAA–Asp CH₂ peaks, which appear in the range of 2.49 ppm to 2.67 ppm of the edited GSH MEGA-PRESS spectrum [8]. Comparative NMR sample study of the GSH and GSH+NAA samples presented in Fig. 4, clearly shows the NAA-Asp CH₂ peak at 2.67 ppm, which is absent in the 1D NMR spectrum of GSH sample. Neither any spectral shift could be reasonably envisaged as being due to a temperature difference between the high resolution NMR and in vivo experiments in the MRI scanner, nor could the peak at 2.80 ppm be considered as a spillover NAA peak. NAA peak under no condition can appear at 2.80 ppm giving a shift of 0.13 ppm (shift from 2.67 ppm). Similarly, for Asp, J-coupling between CH (3.89 ppm) and CH₂ (2.80 ppm) cancels out the possible excitation of Asp at 2.80 ppm by exciting the coupled moiety at 4.40 ppm or 4.56 ppm [8]. Therefore, the excitation at 4.40 ppm or 4.56 ppm is by no means linked to any NAA or Asp peaks. The peak at 2.80 ppm using MEGA-PRESS sequence therefore leads to an unambiguous assignment of the GSH Cys-H _β peak.

Effect of TE on relative phasing of the Cysteine β-CH₂ peak of the two GSH conformers are also investigated. Figures 5 and 6 demonstrate the presence of two conformations of in vivo GSH at ∼2.95 ppm and ∼2.80 ppm. However, selection of ON-pulse position controls the relative proportions of the observed peaks of the two conformers. Spectral patterns corresponding to the two conformers indicate flexible conformal states of GSH molecules in vivo, possibly indicating GSH at two different local cellular environments. An initial investigation in protocol standardization led to the observation of relative phase changes in the two GSH conformers. It is observed that the two GSH peaks appear out of phase for TE = 120 ms, while they are in-phase for TE = 130 ms. Modulation of the relative peak phases due to such small difference of TE values is still unclear; however, the results demonstrate the same observation with data from another center. To further validate the observation on the effectiveness of the TE selection in MEGA-PRESS MRS sequence, results with transitional TE values (i.e., 120, 125, 130, and 140 ms) have also been demonstrated in Fig. 5. TE = 120 ms and 125 ms result in opposite-phasing of the two conformers, while higher TEs of 130 ms and 140 ms give in-phase conformal peaks at their consistent positions. This is a possible indication of a slight variability in the spin-spin relaxation time (T2) of the two conformers. In any case, the above echo time variation substantiates the existence of in vivo GSH conformers, where the change of echo time should only affect the relative peak phasing, and not the resonating coupled peak positions.

Effect of inversion pulse position on in vivo GSH conformers are observed by following the earlier published studies which considered only one (extended (2.95 ppm) [23] or closed (2.80 ppm) [2]) form of GSH. In the present study, to validate the co-existence of the two conformal peaks both the excitation ON-pulse positions, i.e., 4.40 ppm (closed/stable) and 4.56 ppm (extended), are compared with different OFF-pulse positions (5.00 ppm and 7.22 ppm) with TE = 120 ms. Figure 6 shows the edited GSH spectra from the data reproduced in our center (Fig. 6A) and Bergen (Fig. 6B) using following MEGA-PRESS experimental protocols: 1) Editing ON-pulse at 4.40 ppm, OFF-pulse at 5.00 ppm, TE = 120 ms, and TR = 2500 ms; 2) Editing ON-pulse at 4.56 ppm, OFF-pulse at 7.22 ppm, TE = 120 ms, and TR = 2500 ms [30].

Figure 6A shows the edited difference spectra of GSH from 3T Philips (India) using MEGA-PRESS sequence in the brain of a single subject, where application of the selective 180° pulse (ON-pulse) at 4.40 ppm enables observation of the closed form of GSH, while the ON-pulse at 4.56 ppm allows observation of the extended conformation of GSH. In both the cases, the inversion OFF-pulse was applied at 5.00 ppm. To further validate the observation of the peaks from the two GSH conformers, Fig. 6 shows results for additional data acquired with the inversion (OFF) pulse set at 7.22 ppm. For both cases of ON-pulse setting at 4.40 ppm and 4.56 ppm, it is found that the appearance of the two peaks and their positions remain consistent irrespective of the different inversion pulse positions at 5.00 ppm and 7.22 ppm. The two sets of data from two different scanners provide the same results, emphasizing our correct assignment of the two conformational peaks of interest.

Investigation of GSH conformers in various ethnicity in our multi-center study following the same experimental protocol (TE = 130 ms; TR = 2500 ms; ON-pulse at 4.40 ppm and 4.56 ppm; OFF-pulse at 5.00 ppm; voxel size = 3.5×3.5×3.5 cm³) is performed to further investigate the existence of the two GSH conformal states in voluntary subjects from different continental origins. Accordingly, GSH profiling over different population from different continents and different scanners was performed (Fig. 7). However, no significant differences were observed. This validates the selection of parameters for GSH conformer selectivity in vivo as the two conformal peaks observed at ∼2.80 ppm (closed) and ∼2.95 ppm (extended) without any ambiguity. Small dispersion of chemical shift in the range of 2.90 to 2.95 ppm and 2.76 to 2.80 ppm due to subject variability is reflective of the local micro-environmental differences in individual brains.

In order to compare results from our processing scheme in KALPANA for two sets of MEGA-PRESS data (selective 180° refocusing pulse at 4.40 ppm as well as at 4.56 ppm, TE = 120 ms, TR = 2500 ms), we have also processed the same data using jMRUI [39] and compared their spectral pattern in resulted edited spectra (Fig. 8A, B). Both the edited spectra were phase corrected for peak at ∼2.80 ppm, and the opposite phase peak at ∼2.90 ppm is highlighted with aligned dashed marker line. This clearly shows reproducibility of our data using KALPANA [2] and another independent jMRUI package [39]. Data processing validated with different software shows no ambiguity in processing and positions of the two GSH peaks of interest.

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Fig.8

Validation of in vivo GSH conformers in edited MEGA-PRESS spectra processed using different software. A) Edited MEGA-PRESS spectra with excitation ON-pulse at 4.40 ppm, analyzed with two different phase corrections with respect to conformal peak positions. B) Edited MEGA-PRESS spectra with excitation ON-pulse at 4.56 ppm, analyzed with two different phase corrections w.r.t. conformal peak positions.

Quantitation of in vivo GSH conformations

The results of in vitro NMR studies of GSH in aqueous medium confirmed the existence of two structural conformations of GSH at physiologically relevant temperatures and pH. To investigate the same in vivo, MEGA-PRESS sequence using the two sets of excitation pulses based on the J-coupling peaks for closed and extended conformations were chosen. MEGA-PRESS selective ON-pulse at 4.40 ppm relating closed conformation and ON-pulse at 4.56 ppm relating to extended GSH conformation were applied to get corresponding edited spectra. Figure 9 shows the fitted GSH edited spectra for the two set of MEGA-PRESS sequence parameters and the corresponding areas under the peaks of interest. It is found that both the conformational states of GSH are present in brain as characterized by the two Cys-H _β peaks at 2.80 ppm and 2.95 ppm. Applying editing pulse at 4.40 ppm shows relatively intense peak at ∼2.80 ppm (closed/stable form of GSH), compared to the peak at ∼2.95 ppm (extended form of GSH) (Fig. 9A). Editing pulse at 4.56 ppm for estimating the extended form of GSH also resulted the two conformer peaks at same positions, and their relative amounts obtained with this editing pulse sequence were comparable (Fig. 9B). It is found that excitation at 4.56 ppm (ON-pulse) leads to the relatively intense peak for extended GSH conformer, while the similar processing in case of 4.40 ppm excitation results relatively intense peak for stable form of GSH. This relative content of the GSH can be linked to the probable distribution of the two conformations and choosing editing ON-pulse position at 4.40 ppm actually measures the stable form of GSH (Cys H _β peak at 2.80 ppm) within the voxel.

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Fig.9

Quantitation of the two in vivo GSH peaks using the two sets of excitation pulse positions at 4.40 ppm and 4.56 ppm. The MEGA-ON and MEGA-OFF excitation pulse positions are (A) 4.40 ppm and 5.00 ppm, (B) 4.56 ppm and 5.00 ppm, respectively. Data acquired from healthy young subject (voxel size = 3.5×3.5×3.5 cm³ on left parietal cortex) was processed to get absolute concentration of individual interest peaks (peak at ∼2.90 links to extended conformation and peak at ∼2.78 ppm links to closed (stable) form of in vivo GSH). The two peaks appear in-phase for the selected parameter set of TE = 130 ms, TR = 2500 ms).

Impact of GSH conformation identification in clinical studies

Most of the other published in vivo studies reported only one form of GSH (extended form) [3, 40] on healthy subjects. The closed form of GSH was obtained with the application of MEGA-PRESS editing ON-pulse at 4.40 ppm on three healthy age groups (20–40Y, 41–60Y, 61Y and above) [1], which indicates decrease in the closed form of GSH in the frontal cortices with healthy aging.

Studies from AD, PD, and DLB autopsy brains have reported that the GSH level in the cingulate cortex region of AD patients is reduced by 49% as compared to age-matched control subjects; but this GSH depletion is not specific to PD and DLB patients [36].

In our earlier in vivo MRS studies with AD, MCI, and age-matched healthy control subjects, we have reported that GSH is reduced significantly [2, 29]. The GSH reported earlier is now identified as the closed form of GSH. However, the two GSH_cl and GSH_ex forms together need be detected simultaneously to investigate the role of the two GSH conformers with AD progression. Hence an extensive multi-center clinical investigation on AD patients using the same protocol for identification of relative modulation of the two GSH conformers in pathological conditions is required.

Conclusion

This is the first report providing important evidence for the co-existence of two i n vivo GSH conformations in the human brain, linked with the observation from the state-of-the-art in vitro NMR studies on GSH. Critical observation and analysis led to the conclusion for the presence of different GSH conformations and their identification with MEGA-PRESS pulse sequence for in vivo study. Our work is expected to have a huge impact on the understanding of GSH and its profound role in the individual’s cognitive reserve and disease affinity depending upon their lifestyle and upbringing in different geographical environments. Furthermore observational studies on GSH supplement and its impact on cognitive reserve for healthy old and MCI patients using the present protocol is planned at NBRC.