Accepted Manuscript

Glutathione in the human brain: Review of its roles and measurement by magnetic resonance spectroscopy

Caroline D. Rae, Stephen R. Williams

PII: S0003-2697(16)30434-1 DOI: 10.1016/j.ab.2016.12.022 Reference: YABIO 12589

To appear in: Analytical Biochemistry

Received Date: 18 July 2016 Revised Date: 21 December 2016 Accepted Date: 23 December 2016

Please cite this article as: C.D. Rae, S.R. Williams, Glutathione in the human brain: Review of its roles and measurement by magnetic resonance spectroscopy, Analytical Biochemistry (2017), doi: 10.1016/ j.ab.2016.12.022.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Glutathione in the human brain: review of its roles and measurement by magnetic resonance spectroscopy

Caroline D Rae a and Stephen R Williams b* aNeuroscience Research Australia, Barker St Randwick, NSW 2031 Australia and School of Medical Science, The University of New South Wales, NSW, 2052, Australia bDivision of Informatics, Imaging and Data Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK

*Address correspondence to: Professor S R Williams; Imaging Science; Stopford Building; Oxford Rd; Manchester M13 9PT [email protected]

A B S T R A C T

We review the transport, synthesis and catabolism of glutathione in the brain as well as its compartmentation and biochemistry in different brain cells. The major reactions involving glutathione are reviewed and the factors limiting its availability in brain cells are discussed. We also describe and critique current methods for measuring glutathione in the human brain using magnetic resonance spectroscopy, and review the literature on glutathione measurements in healthy brains and in neurological, psychiatric, neurodegenerative and neurodevelopmental conditions In summary: Healthy human brain glutathione concentration is ~1-2 mM, but it varies by brain region, with evidence of gender differences and age effects; in neurologicalMANUSCRIPT disease glutathione appears reduced in multiple sclerosis, motor neurone diseas e and epilepsy, while being increased in meningiomas; in psychiatric disease the picture is complex and confounded by methodological differences, regional effects, length of disease and drug-treatment. Both increases and decreases in glutathione have been reported in depression and schizophrenia. In Alzheimer’s disease and mild cognitive impairment there is evidence for a decrease in glutathione compared to age-matched healthy controls. Improved methods to measure glutathione in vivo will provide better precision in glutathione determination and help resolve the complex biochemistry of this molecule in health and disease. Keywords: Glutathione Magnetic resonance spectroscopy Brain Glyoxalase Psychiatric disease Neurological disease

ACCEPTED

1

ACCEPTED MANUSCRIPT

List of abbreviations : GSH, glutathione; GSSG, oxidized glutathione; NMDA, N-methyl-D-aspartate; GCL, glutamate-cysteine ligase; GCLC, glutamate-cysteine ligase subunit with catalytic activity; GCLM, glutamate- cysteine ligase subunit with modifier activity; MRS, magnetic resonance spectroscopy; PRESS, Point Resolved

SpectroScopy; MEGA-PRESS, Mescher-Garwood Point resolved spectroscopy; BSO, L-buthionine-S,R - sulfoximine; MSO, methionine sulfoximine; ASC1, neutral amino acid transporter 1; EAAT3, excitatory amino acid transporter 3; ROS, reactive oxygen species; MRP, multidrug resistance pump: GST, glutathione-S- transferase; NAA, N-acetyl aspartate; STEAM, stimulated echo acquisition mode: 2D-COSY, 2-dimensional chemical shift correlated spectroscopy; TR, repetition time; TE, echo time; CRLB, Cramer-Rao Lower Bounds;

BOLD, blood oxygenation level dependent; fMRI, functional magnetic resonance imaging; SPECIAL, Spin- echo full intensity acquired localized.

MANUSCRIPT

ACCEPTED

2

ACCEPTED MANUSCRIPT

1. Biology and Biochemistry of Glutathione

1.1. Introduction

Glutathione (GSH; γ-L-glutamyl-L-cysteinylglycine: (2 S)-2-amino-4-{[(1 R)-1-[(carboxymethyl)carbamoyl]-2-

sulfanylethyl]carbamoyl}butanoic acid) is a tripeptide thiol found in virtually all cells. Essential for cellular

function, it plays roles in oxidation-reduction reactions, acts as a cofactor in enzyme reactions, protects against

reactive oxygen species and potentially toxic xenobiotics, provides storage for cysteine and regulates cellular events including gene expression, DNA and protein synthesis, cell proliferation and apoptosis, signal transduction, cytokine production and immune response, and protein glutathionylation [1].

Two of the most challenging chemical species to deal with in biological systems are reactive oxygen species produced as a consequence of aerobic metabolism and an aerobic environment, and the production of oxoaldehydes, which are formed as a function of the oxidation of molecules such as glucose. The glutathione system was developed early in evolution as a solution to deal with both of these chemical challenges [2]. Since both oxygen and glucose are key ingredients in brain function, there is therefore considerable interest in this molecule and in the measurement of it in vivo . Here, we reviewMANUSCRIPT what is known about glutathione biosynthesis, metabolism and roles in the brain and also review best practice measurement of glutathione in vivo using 1H

magnetic resonance spectroscopy (MRS). There are a number of excellent, older reviews on glutathione in the

central nervous system which retain much relevance if further reading is sought [3, 4].

1.2. Where is Glutathione found in the Brain?

Studies using staining for sulfhydryl compounds have shown extensive brain staining for GSH with the common theme of relatively little reactivity in neuronal somata. Staining for GSH appears confined mostly to the neuropil

and white matter tracts with the exception of some neurons in the cerebellum, such as cerebellar granule cells and Purkinje cells [5,ACCEPTED 6]. A study using two-photon imaging of monochlorobimane fluorescence [7], a selective enzyme-mediated marker

for reduced GSH, reported high levels in lateral ventricle ependymal cells (2.73 ± 0.56 mM) and meningeal cells

(1.45 ± 0.09 mM), with lower levels in astroglial cells (0.9 ± 0.08 mM) and relatively low levels in cortical

3

ACCEPTED MANUSCRIPT

neurons (0.21 ±0.02 mM). Levels of GSH were up to threefold higher in developing neurons (e.g. in the subgranular zone of the dentate gyrus) than in older neurons in the neighbouring granular layer [8]. Earlier work, which measured GSH content then calculated GSH values for glia and neurons based on relative volumes, reported glial GSH as 3.8 mM vs 2.5 mM for neurons but these calculations could have been biased by higher concentrations of GSH in other cell types which were not estimated, such as ependymal cells [9]. Values in oligodendrocytes at various stages of development have been reported as less than half the levels found in astrocytes (i.e. ~12 vs 28 nmol/mg protein [10]. Total values reported in biopsies of fresh human cerebral cortex range from 0- 0.77 µmol/g wet weight for GSH and 0.24 – 1.22 µmol/g wet weight for glutathione disulfide (GSSG) [11]. This contrasts with values recorded from cells in culture with levels in human neurons reported at 93 ± 13 µmol/g protein and 177 ± 10 µmol/g protein in astrocytes [12].

Careful study of the relative amounts of reduced vs oxidised glutathione has shown levels of GSSG in rat and monkey brain to be 1.2% [13], or less [14], of total glutathione indicating that previous reports of levels as high

as 40% GSSG in brain are likely erroneous. Induction of ischemia in rat brain has been shown to decrease GSH without any concomitant increase in GSSG [15], while induction of oxidative stress by lipid peroxidation did result in accumulation of GSSG [14] showing different mechanismsMANUSCRIPT at work in these conditions. While it is plain that the ratio of GSH/GSSG is altered in various disease states (e.g. [16]) and that the levels of total glutathione may also be reduced under pathological conditions (Table 1) it is also apparent that care must be taken when measuring GSH and GSSG.

The concentration of glutathione in extracellular fluid is relatively low. It has been reported in the rat cortex via

microdialysis measurement at 2.10 ± 1.78 µM (N = 18; [17] and in the caudate nucleus (2.0 ± 0.1), rising in the

latter case under potassium depolarization to 3.0 ± 0.9 µM (N = 3; [18]. Glutathione efflux is also increased by

application of N-methyl-D-aspartate (NMDA) and kainate and by imposition of ischaemia or hypoxic conditions

[19].

In summary, the location of glutathione in the brain is highly tissue dependent and the majority of it is located in

non-neuronal cells.ACCEPTED

4

ACCEPTED MANUSCRIPT

1.3. Synthesis of glutathione

Glutamate, the major excitatory neurotransmitter in the brain, is a precursor for GSH synthesis. It is combined with cysteine, an amino acid with a free sulfhydryl group, in an ATP-dependent reaction catalyzed by γ- glutamylcysteine synthetase (more properly glutamate-cysteine ligase; GCL; E.C. 6.3.2.2) to form γ- glutamylcysteine (Fig. 1). This dipeptide is then further combined with glycine, another potential neurotransmitter, in another ATP-dependent reaction catalysed by glutathione synthase (E.C. 6.3.2.3).

MANUSCRIPT

Figure 1 Brain synthesis, compartmentation and metabolism of glutathione.

The enzyme GCL isACCEPTED often referred to as “rate limiting” although in neurons, for example, the enzyme has been shown to have ample capacity if given sufficient substrate, particularly cysteine, whose levels are highly controlled in neurons [7]. The enzyme is known to be composed of two dissociable subunits; a heavy (73 kDa)

5

ACCEPTED MANUSCRIPT

chain which possesses catalytic activity (GCLC) and a light (27.7 kDa) chain which is a modifier (GCLM) [20].

Although the catalytic subunit GCLC can produce γ-glutamylcysteine, the presence of the modifier increases enzyme activity; gclm -/- mice show tissue levels of GSH between 9 and 40% of those of the wild-type [21]. The presence of GCLM lowers the K M for glutamate and ATP and increases the catalytic rate of the enzyme

complex [22].

These two subunits are encoded in mice and men by separate genes located on different chromosomes [23]. The

ability of GCLM to modify GCL enzyme activity has made the gclm locus the subject of investigation across a

range of condition. Alterations in GCLM have been associated with disorders such as schizophrenia [24] but

others have indicated that the association does not hold in independent patient populations [25, 26] nor in self-

reported depression [27].

The apparent K M of GCL for glutamate (1.8 mM) is well below the cellular concentration of glutamate (~8-12

mM), as estimated from numerous 1H MRS studies in vivo . There are few reliable reports of glutamate concentrations within neurons in situ, with values reported from as high as 5.77 mM in rat cerebellar parallel fibre terminals [28]) while the apparent K M for cysteine is considerably lower at 0.1 - 0.3 mM, more proximate to the concentration of cysteine within the cell. The activityMANUSCRIPT of the enzyme is regulated by non-allosteric feedback competitive inhibition by GSH (Ki = 2.3 mM) thus controlling intracellular levels of both GSH and cysteine [29].

GLC can be irreversibly and rapidly inhibited by L-buthionine-S,R -sulfoximine (BSO) with limiting pseudo-first order rate constant of 3.7 min (t 1/2 ≈ 11 s) [30]as well as by methionine sulfoximine (MSO) [31], a commonly used inhibitor of glutamine synthetase [32]. Judicial use of lower concentrations of MSO and relatively short incubation times, however, mean that MSO can be used to inhibit glutamine synthetase without significant effects on glutathione concentrations [33]. Depletion of brain glutathione in rats with BSO administration for

10 days resulted in decreased complex IV and citrate synthase activity as well as decreased brain N- acetylaspartate levels, suggesting mitochondrial and neuronal damage [34], in agreement with an earlier study

showing gross mitochondrialACCEPTED enlargement and degeneration [35].

The final step in GSH synthesis, catalysed by glutathione synthetase, is generally considered to have less

metabolic control over the rate of glutathione synthesis. It is a homo-dimer and its absence results in the most

6

ACCEPTED MANUSCRIPT

common of the inborn errors of GSH metabolism with around 25% of sufferers dying early, such as during the neonatal period [36]. There is some suggestion that supplementation with vitamin C from an early age may improve long term outcomes [37]; this may boost neuronal antioxidant capacity in the absence of glutathione.

The time constant for GSH turnover in rat brain has been reported as 13 ± 2 h or about 0.05% of the overall metabolic rate of glucose oxidation [38], however others have pointed out that there are likely several pools of glutathione that have different turnover rates, some with a half-life as short as 30 min [35].

Some consideration of the nature of the GSH precursors is instructive. There is some evidence that GSH helps to modulate glutamate levels [39], and vice versa, as well as levels of cysteine, a compound which is neurotoxic unless concentrations are kept low [40]. Glutathione turnover has been shown to be rapid in astrocytes and to depend on the relative availability of glutamate and cysteine [41]. The redirection of label from 15 N-glutamate into glutathione synthesis following depletion of intracellular glutamate shows the relative importance of glutathione synthesis to these cells.

Neurons do not have an uptake mechanism for GSH and therefore, rely on their own synthesis of GSH to maintain adequate concentrations. Unlike astrocytes, they are not capable of using cysteine as a precursor and are therefore reliant on uptake of cysteine in order to synthesiseMANUSCRIPT the GSH precursor γ-glu-cys [42]. However, extracellular concentrations of cysteine, under normal circumstances, are relatively low (6 µM) [43]. Cysteine is neurotoxic with a killing threshold around 1 mM [44]. The mechanism of its neurotoxicity is not well understood , involving both pre- and post-synaptic interactions with glutamatergic neurotransmission [40].

Cysteine can enter neurons via the neutral amino acid transporter ASC1, a sodium dependent transporter that also carries alanine and serine. It is likely that this route of uptake plays only a minor role in neuronal cysteine uptake as competitive inhibition of ASC1 with ten-fold higher concentrations of serine had negligible effects on cysteine concentrations [45]. Cysteine is also taken up via the excitatory amino acid transporter EAAT family, particularly EAAT3 (SLC1A1), with inhibition of EAAT3 causing inhibition of GSH synthesis. Mice which do not have EAAT3 (Slc1a1 -/-) have lower neuronal GSH content and suffer from age-related neurodegeneration.

Reduced neuronal GSHACCEPTED content in these mice can be reversed by application of N-acetylcysteine, which crosses

cellular plasma membranes via transmembrane diffusion [46].

7

ACCEPTED MANUSCRIPT

Oligodendrocytes have relatively low levels of GSH; this has been reported to be due to a number of factors, including lower ability to take up the precursors cystine and glutamate due to relatively lower density of the

- glutamate cystine exchanger X c , and the relative ability to reduce cystine to cysteine which is dependent on the

intracellular redox potential, low in oligodendrocytes [10]. Oligodendrocytes have levels of iron which are

twenty fold higher than in astrocytes, contributing to the six-fold higher levels of oxidative stress seen in

oligodendrocytes compared to astrocytes. Coupled with the lower intrinsic GSH levels, this means that

oligodendrocytes are more vulnerable to oxidative stress ; indeed the relative vulnerability can be eliminated by

increasing GSH levels through provision of precursor substrates [47].

1.4. Catabolism of glutathione

The peptide bond linking glutamate with the cys-gly dipeptide is via the γ-carboxyl group rather than the more usual α-carboxyl. This has the outcome of rendering the bond impervious to hydrolysis by all but one enzyme,

γ-glutamyltransferase (GGT; E.C. 2.3.2.2). This enzyme is located in the cell membranes of a range of cells

throughout the body but its extracellular activity means that GSH is rendered resistant to intracellular

degradation [48]. GGT activity in the CNS is confined to non-neuronal cells, with ependymal cells staining intensely to GGT antibodies with moderate to intense reactivity MANUSCRIPT in Schwann and glial cells [49]. GGT activity is induced in blood brain barrier ependymal cells by a factor released by astrocytes [50]; it is not generally present in endothelial cells lining blood vessels in other parts of the body.

1.5. Glutathione oxidation and reduction (role of GSSH reductase, role of NADPH and PPP)

A major role for GSH is in the reduction of potentially toxic oxidizing reagents such as reactive oxygen species

(ROS), producing oxidized glutathione in the form of glutathione disulfide (GSSG). This compound is incapable of performing the functions that reduced glutathione performs and so must be itself reduced to reform GSH, a reaction catalyzed by glutathione reductase (Eqn 1: NADPH : oxidized glutathione oxidoreductase, EC 1.6.4.2:

[51]). In brain, this enzyme is reported to be most highly expressed in oligodendrocytes, microglia and neurons, with lower expressionACCEPTED levels in astrocytes [52].

GSSG + NADPH ↔ 2 x GSH + NADP + (Eqn 1).

8

ACCEPTED MANUSCRIPT

Reducing power in this case is provided by NADPH, a major source of which is generated via the metabolism

of the pentose phosphate pathway [51]. The enzyme glucose-6-phosphate dehydrogenase (G6PD) converts

glucose-6-phosphate to 6-phospho-D-glucono-1,5-lactone and in the process reduces NADP + to NADPH. It

has been proposed that in neurons, the primary metabolic role of glucose is to generate NADPH via the

pentose phosphate pathway in order maintain levels of reduced GSH [53, 54]

1.6. Glutathione detoxification – glutathione transferase reactions

A major role of glutathione is in the detoxification and removal of xenobiotics and other endogenous compounds, which can be conjugated with GSH by glutathione-S-transferase and subsequently exported from the cell via multi-drug resistance pumps (MRPs; recently reviewed in detail by [55]). MRPs are relatively large transmembrane proteins, which are ATP-driven export pumps, exporting GSSG as well as a multitude of glutathione-S-conjugates. Nine MRPs have so far been identified in humans; with MRP1,3,4 and 5 shown to be

expressed in astrocytes [56]. MRP1 is responsible for all of the GSSG export from astrocytes and ~60% of the

export of glutathione conjugates [57], while MRP4 appears to be the only MRP expressed at quantifiable levels

in the human blood brain barrier [58]. Glutathione-S-transferase activity is plainly of vital importanc MANUSCRIPTe because it has converged through evolution via at least four structurally distinct enzyme families [59]. The cytosolic GSTs (E.C. 2.5.1.18) are found in brain

and are best known for their ability to catalyse conjugation reactions between GSH and electrophilic substrates.

There are currently seven human cytosolic GSTs (known as alpha, mu pi, sigma, theta, zeta and omega) and

much remains to be determined about their distribution and role(s) in the brain. The Mu-form has been reported

in astrocytes, ependymal cells and in subventricular zone cells [60] with pi isoforms in oligodendrocytes [61].

Mitochondrial GST activity has also been reported in the brain. GST is stabilized by glutathione so that a GST-

deficiency can develop in persons with glutathione synthetase deficiency [36]. 1.7. Transport ofACCEPTED glutathione Glutathione is transported across the blood brain barrier in reduced form with an apparent K M = 5.84 mM [62],

while transport of GSSG is negligible (no different to sucrose). Transport across the blood brain barrier is likely

mediated by MRP4, which transports glutathione and conjugates [63] and is the only multidrug resistance pump

9

ACCEPTED MANUSCRIPT

expressed in the blood brain barrier in quantifiable levels [64]. Mean human blood GSH concentrations have been reported to be 1.02 mM [65] but uptake of intact GSH has been reported to be substantial [66-68] with some authors suggesting that GSH plays a role in maintaining blood brain barrier integrity [69].

GSH is released in significant amounts from astrocytes, but not other brain cell types, with MRP1 identified as a likely mediator of this release [70], accounting for around 60% of total GSH export and 100% of GSSG export

[57]. GSH is also likely released from astrocytes through open hemichannels [71]. Neurons and oligodendrocytes do not take up intact GSH, instead relying on uptake of precursors (Fig. 1); this has led to interest in supplying precursors such as cysteine in a less neurotoxic form, such as N-acetylcysteine (see 1.3 above).

Mitochondria, which are a major source of free radical production, contain GSH but are not capable of synthesizing it independently [72]. Indications are that GSH is able to exchange rapidly between mitochondrial and cytosolic compartments and that maintenance of mitochondrial GSH is important for cell viability and survival [73]. While GSH from the cytosol can cross into the outer mitochondrial space via porins, it cannot diffuse across the inner mitochondrial membrane and must instead use a carrier-mediated process. Two mitochondrial anion carriers are responsible for ~80% MANUSCRIPT of GSH uptake; the oxoglutarate carrier and the dicarboxylate carrier [74, 75], both of which are electroneutral. The activity of the oxoglutarate carrier is affected by mitochondrial lipid fluidity with increased cholesterol/phospholipids ratio resulting in lower mitochondrial GSH with subsequent increased vulnerability to apoptosis [76]. Much of the work on mitochondrial GSH uptake has been done in liver or kidney mitochondria.

1.8 Other glutathione-dependent reactions

GSH also acts as a cofactor in enzyme reactions. It plays a key role in the glyoxalase enzyme system which catalyses the detoxification of the ketoaldehyde methylglyoxal to D-lactate [77, 78] . Glutathione combines with methylglyoxal forming a hemithioacetal, both stereoisomers of which are substrates for glyoxalase II [79]. Glyoxalase II thenACCEPTED catalyses the cleavage of S-D-lactoylglutathione to form D-lactate and release of GSH. Methylglyoxal is a highly reactive molecule, capable of rapidly denaturing proteins by forming a Schiff base and engaging in a Maillard reaction with protein amines. Methylglyoxal can be formed by chemical breakdown

of glycolytic intermediates glyceraldehyde-3phosphate and dihydroxyacetone phosphate [80]. The rate of

10

ACCEPTED MANUSCRIPT

breakdown has been shown to be significantly accelerated by the actions of triose-phosphate isomerase [81], leading to methylglyoxal being branded as the “flaw in the glycolytic logic” [82]. In addition to its toxic actions on proteins, methylglyoxal has also been shown to act as a substrate for aldolase, substituting for glyceraldehyde-3-phosphate; this reaction has been shown to inactivate aldolase [83], possibly preventing glucose oxidation (Fig. 2).

MANUSCRIPT

Figure 2 ProductionACCEPTED and catabolism of methylglyoxal by the glutathione dependent glyoxalase enzyme system.

11

ACCEPTED MANUSCRIPT

It is therefore essential to detoxify methylglyoxal quickly and efficiently. The glyoxalase enzyme system is a highly reactive system and it is present in both neurons and glial cells in brain [84]. Work in cultured cells suggests that neurons have less glyoxalase activity than astrocytes and less capability to detoxify methylglyoxal but this was measured after “removal” of GSH with no control for lack of GSH, so it is difficult to know how much of the methylglyoxal toxicity was due to lack of GSH rather than the methylglyoxal itself [85]. Since the glyoxalase system is so reactive and needs little glutathione to function, in practice the effects of methylglyoxal may be mostly extinguished by an active glyoxalase system.

Glutathione is also a cofactor in the reaction catalyzed by the Class III glutathione-dependent alcohol

(formaldehyde) dehydrogenase, which is found in brain [86]. It is most abundant in neurons and in perivascular and subependymal astrocytes [86]. This enzyme has little affinity for ethanol, which is not significantly metabolized in brain [87], and its purpose is not well understood but possible roles include detoxification of formaldehyde and ώ-hydroxy-fatty acids [88]. Found in most cells in the brain [89] it is known to be actively involved in detoxifying formaldehyde in a range of brain cells [90] MANUSCRIPT 2. Measurement of Glutathione in the Brain by Magnetic Resonance Spectroscopy

Glutathione has three spin systems (Fig. 3, Table 2) resulting from each of the constituent amino acid residues,

γ-glutamyl, cysteinyl and glycine. In more details, these are the tightly coupled, second order γ-glutamyl αCH,

βCH2 and γCH2, the cysteinyl αCH and βCH2, and the glycine αCH2 [91]. The glycine αCH2 is best described as part of an A 2X system due to coupling with the Gly-NH proton which is itself in exchange with solvent water with a kinetic rate comparable to NMR time scales [92]. The Cys and Gly NH chemical shifts have been reported at pH 7.3 as 8.201 and 7.933 ppm, respectively [93] but it should be noted that they, and the associated

JNH coupling constants are highly susceptible to both pH and temperature. Despite these spin systemsACCEPTED having multiple resonance frequencies the overcrowded nature of the 1H spectrum of the brain means that, in this case, none of the resonances are properly resolved, including at very high fields such as 800 MHz (18.8 Tesla) and in high resolution tissue extracts. GSH resonances can be resolved through

use of 2D spectroscopic techniques, with the cross-peak between the cysteinyl αCH and βCH2 being particularly

12

ACCEPTED MANUSCRIPT

useful in this case, but in one-dimensional spectra at 3T or similar clinically feasible field strengths, GSH is not readily visible in the 1H MR spectrum (Fig. 3). Coupled with the low intrinsic concentration of GSH (see

Section 1.2) and the lower signal to noise caused by multiple spin couplings, this means that in practice GSH is difficult to detect in standard one-dimensional spectra.

Figure 3 Low field section of 1H magnetic resonance spectrum from the brain in vivo and from

GSH at 3T. MANUSCRIPT 2.1. Methods for detecting glutathione in vivo 2.1.1. Overview

Standard and widely available techniques for obtaining spectra from localized areas of human brain tissue in a single scan include Point RESolved Spectroscopy (PRESS, [94, 95] and STimulated Echo Acquisition Mode

(STEAM [96]). These methods rely on localizing signal from the volume of interest using three, frequency selective RF pulses accompanied by orthogonally applied field gradients. Maximal receiver gain is achieved by use of an appropriate frequency-selective water suppression routine to remove the dominant signal from water.

PRESS resolves signal from mobile molecules through the use of a double spin echo created by applying consecutive 90°, 180° and 180° pulses simultaneously with each field gradient (Fig. 4) while STEAM achieves this through three ACCEPTED consecutive 90° pulses, again applied simultaneously with each field gradient [97]. Each method has its own advantages. PRESS yields better signal to noise and is less sensitive to diffusion, motion and multiple quantum effects. STEAM allows use of a shorter echo time, which is advantageous for measurement of

13

ACCEPTED MANUSCRIPT

highly coupled metabolites with short T2 relaxation times, and the orientation along the z-axis of the magnetization during the mixing time (delay between the second and third 90° pulses) allows a useful period in which to implement further water suppression or “crusher” pulses to disperse unwanted signals.

Here, we discuss use of these localization techniques to measure GSH in brain, as well as less widely available editing sequences that have been employed to date in the literature.

2.1.1.2 The Glutathione 1H spectrum

As indicated above, the glutathione spectrum consists of a series of resonances from the peptide components which are shifted to a greater or lesser degree from the isolated peptide resonances. All of the signals from glutathione overlap with resonances from other neurochemicals present at higher concentrations, most importantly N-acetylaspartate (NAA) at 2.02 ppm and 2.45 ppm, glutamate multiplets centred at 2.05 ppm and

2.35 ppm, creatine plus phosphocreatine at 3.03 and 3.96 ppm and amino acids at 3.75-3.8 ppm such as glutamate, glutamine and aspartate. This is illustrated in Fig. 3 in which the GSH spectrum is plotted below a human brain spectrum. The first approaches to measure GSH in human brain used spectroscopic editing methods to selectively retain GSH while suppressing other resonances [98]. At the available field strength of 1.5T for human spectroscopy at that time this was really theMANUSCRIPT only feasible approach to measuring such a low concentration metabolite in normal brain, though GSH measurement from short echo time STEAM spectra has been reported at 1.5T for a paediatric sample [99] and in a study of brain tumours [100]. The double quantum filter method described by Trabesinger has been developed further for use at 3T [101, 102], but most spectral editing methods for GSH detection have used variants of spin-echo J-modulation methods, such as MEGA-

PRESS [103]. These methods were developed for animal studies in the 1980s to detect compounds such as lactate and GABA in vivo , in the presence of overlapping lipid (lactate) or glutamate/NAA signals (GABA)

[104, 105], but have proved robust and useful for detection of GSH in human brain (see Table 1) [106, 107].

Some researchers have taken advantage of the superior spectroscopic resolution available from 2-dimensional chemical shift correlated spectroscopy (2D-COSY) and developed gradient-localized versions of this technique from high-resolutionACCEPTED NMR [108], but the technical challenges and relatively long acquisition time of such methods seem to have prevented follow up studies. With increasing availability of 3T scanners for human use, there have been numerous studies in which short echo spectra, without editing, acquired with either STEAM or

14

ACCEPTED MANUSCRIPT

PRESS localization, have been used to estimate GSH. Without exception, these studies have used the popular method, LCModel [109], to extract GSH concentration from the spectra (see Table 1 for numerous examples).

Some studies have used phantom data to provide evidence that LCModel can return a correct value for GSH under conditions in which there are no visually detectable signals from GSH in the spectrum [110-112], but as noted in Section 2.1.2.2 these do not provide completely compelling evidence that the standard PRESS acquisition with TE 30-35 ms, 8 ml voxels and 6-8 min acquisitions is adequate for measuring a low concentration metabolite such as GSH reliably. At 7T, with higher signal to noise and improved spectroscopic dispersion this problem is mitigated, though even at 7T a number of researchers prudently adopt techniques to simplify the spectrum and remove overlap [113-115]. One study has directly compared MEGA-PRESS to short echo STEAM (TR/TE 4500/5 ms) at 4T [107], showing there is no significant difference in GSH quantification using the two methods. This study is often quoted as a justification for the use of LCModel combined with short echo (30-35 ms) PRESS at 3T to measure GSH in clinical populations, but there are objections to this contention as noted below in Section 2.1.2.2 .

2.1.2. Description and evaluation of methods

2.1.2.1. Metabolite-selective methods MANUSCRIPT

Metabolite-selective methods (double quantum filter, MEGA-PRESS, 2D chemical shift correlated, RF and echo time optimized PRESS) all rely on exploiting the fact that J-coupling leads to phase-modulation of coupled

signals during evolution periods (usually during TE), which can be manipulated with extra RF pulses to fully or partially isolate GSH signals from others. A full explanation of the principles of these methods requires an understanding of the generation of spin echoes and the evolution of coupled and uncoupled signals which is beyond the scope of this review. Readers are referred to the primary literature cited here, to [116, 117] and to

other reviews contained within this special issue [118-121].

MEGA-PRESS is a specific variant of spin-echo editing methods which has been adapted to be compatible with PRESS localization.ACCEPTED PRESS is the commonest technique used for clinical spectroscopy and is available on all the major clinical imaging platforms. As shown in Fig. 4, localization is achieved by the intersection of 3

selective pulses in the presence of orthogonal slice selective gradients. The spin-echo time, TE, is chosen so the

15

ACCEPTED MANUSCRIPT

cysteinyl residue of GSH at 2.98 ppm is inverted relative to the overlapping creatine + phosphocreatine residue.

In MEGA-PRESS, the interval between the 90 o and first 180 o pulse is minimized, and a pair of frequency

selective 180 o pulses are inserted around the second slice-selective 180 o pulse. The timings are set so that the interval between the centres of the two frequency selective pulses is equal to half the total spin-echo time

(TE/2). The extra pair of pulses are selective for the frequency of the cysteinyl αCH residue at 4.56 ppm in one sub-spectrum and their application inhibits the phase modulation and hence inversion of the signal at 2.98 ppm, while leaving the creatine residue unperturbed. The frequency of the editing pulses is set to a control value in the second sub-spectrum, not affecting phase modulation. Thus, subtraction of the two sub-spectra cancels out the creatine and retains the GSH resonance (Fig. 5). The aspartyl resonances of N-acetylaspartate co-edit with

GSH and also appear in the edited spectrum. The choice of the optimum echo time for GSH MEGA-PRESS has been a source of debate, with published work using TE values around 70 ms or 120 ms. Despite the loss of signal through T 2 decay it is now clear from simulations, phantoms and measurements in vivo , that TE~120 ms

is optimal [122].

MANUSCRIPT

ACCEPTED

16

ACCEPTED MANUSCRIPT

Figure 4 PRESS and MEGA-PRESS MANUSCRIPT sequences

One disadvantage of methods such as MEGA-PRESS is that they rely on subtraction of two sub-spectra and are thus potentially prone to movement artefacts. However, it has been shown that correcting for phase and frequency shifts throughout an acquisition by collecting data in short blocks of scans and, if necessary, rejecting pairs of sub-spectra which are badly affected results in good spectra free of subtraction errors (Mullins et al.

2014, Waddell et al. 2007). Double quantum filter methods, which achieve editing in a single shot, can also be adapted into a PRESS localization sequence, but despite some refinements of the method this approach has not gained much traction in clinical studies. The other metabolite selective methods, including some developed specifically for useACCEPTED at 7T, have also been used sparingly (Choi et al. 2010, Choi & Lee 2013, An et al. 2015b, Srinivasan et al. 2010, Thomas et al. 2001b).

17

ACCEPTED MANUSCRIPT

MANUSCRIPT Figure 5 Edited GSH spectrum

2.1.2.2. Non-selective Methods with Model based Fitting

The other main approach to detecting GSH is to acquire a conventional short echo localized spectrum, such as

PRESS or STEAM and extract GSH by fitting to a metabolite model. All the papers listed in Table 1 which adopt this methodology have used LCModel [123] to fit the data. This method is well established, with over

1500 citations since its invention in 1993. The user provides a basis set of metabolite spectra either acquired or simulated under the acquisition conditions of the experimental data and the program computes the best linear combination of the basis set that fits the data. The output provides metabolite concentrations and shows the fit, the residual and calculatesACCEPTED an estimate of the goodness of fit using Cramer-Rao lower bounds (CRLB). There are options to fit the baseline to a model and macromolecules and lipids can be included in the basis set alongside expected metabolites. It is common to include in the basis set metabolites which cannot be visually

18

ACCEPTED MANUSCRIPT

identified in the spectra, such as N-acetylaspartylglutamate, GABA and GSH, all of which overlap with other

resonances present at higher concentration. Confidence is given that estimates of these hidden metabolites are

correct by inspection of the CRLB, which may be 20% or less of the amplitude. Often researchers will use a

threshold value of CRLB (usually 20%, e.g. [124, 125]) to accept or reject a metabolite estimate. This approach

is flawed for two reasons: firstly it is dependent on signal-to-noise, so if there is a decrease in the signal from a

metabolite, the % CRLB will increase, thus data are more likely to be rejected if there is a real decrease in

concentration, such as in an experimental group [126]; secondly, the CRLB is only strictly applicable if the

model is correct and fully parametrized, but as explained in van Ormondt et al [127] inclusion of non-parametric

elements such as baseline fitting removes the conditions under which the CRLB is a good estimator.

Attempts to validate the short echo / LCModel approach have been carried out by a number of researchers, by

comparing results to MEGA-PRESS [128], or by conducting measurements in phantoms that mimic brain

spectra with GSH added over a range of concentrations [124, 129, 130]. The paper by Terpstra [128] is key to

the adoption of the short echo / LCModel method as it was the only work published comparing edited to non-

edited spectra, until one of us (SW) led a study comparing PRESS and MEGA-PRESS, published in 2016 [131]. Terpstra et al. [128] show that quantification of GSH is notMANUSCRIPT significantly different between the two methods. However, this does not fully test that the non-edited quantification is reliable, since it effectively only shows

equivalence at a single average GSH concentration. The evidence would be stronger if there was a comparison

of PRESS and MEGA-PRESS in a group of subjects in which the average GSH concentration is significantly

different to normal, with both techniques showing the same GSH concentration. It also seems unwarranted to

conclude that this result from a very short echo (TE=5 ms), relatively long (20 min) acquisition at 4T means that

longer echo PRESS at 3T is equally reliable for measuring GSH.

It is instructive to inspect the data presented in the phantom studies. As noted in Table 1, good linear

correlations are reported between true and LCModel-estimated GSH concentration, but this is driven by non-

physiological concentrations of GSH (5-20 mM) which give visually detectable resonances in the spectra. In the

three papers quotedACCEPTED [124, 129, 130], a significantly non-zero concentration of GSH (1-2 mM) is returned from

phantoms which do not contain the metabolite. As this concentration is in the expected range for GSH in brain,

the reliability of the LCModel approach has to be questioned.

19

ACCEPTED MANUSCRIPT

Our study [131] addressed these issues directly by acquiring PRESS (TE=35 ms) and MEGA-PRESS (TE=120 ms) spectra in a series of brain-mimicking phantoms with GSH concentrations from 0-24 mM, with a number of measurements below 4 mM GSH. We also acquired spectra from human volunteers in two voxels located in the anterior cingulate and occipital cortex. The spectra were analyzed with jMRUI rather than LCModel, but the conceptual approach of fitting to a linear combination of basis sets was the same. In the phantoms, GSH was not quantified reliably at concentrations below 3 mM from PRESS spectra though there was a strong linear correlation from 4 - 24 mM, whereas quantification from MEGA-PRESS was linear over the full range from 0 -

24 mM. Furthermore a significant difference in GSH was detected between anterior cingulate and occipital cortex GSH (3.2 ± 0.6 mM and 1.4 ± 0.4 mM, respectively) from MEGA-PRESS spectra, whereas no significant difference was evident from PRESS spectra (2.8 ± 0.3 mM; 2.5 ± 0.7 mM).

3. Glutathione Concentration in Health and Disease

Table 1 presents a summary of papers which describe GSH measurements in human brain. The table is arranged

with normal volunteers first, including studies of development, ageing, physiological and pharmacological challenge, followed by neurological then psychiatric diseases. MANUSCRIPT

3.1. Normal volunteers

In normal adult volunteers, there is general consensus that the concentration of GSH is around 1-2 mM, with eight of the papers in Table 1 reporting values between 0.8 and 1.5 mM. Six of the papers report higher values in the range 1.5-3.5 mM. There is limited information on regional comparisons of cerebral GSH or on white matter / grey matter differences, but An et al. reported 30% higher GSH in white matter dominated frontal

cortex compared to grey matter dominated prefrontal cortex [114]. As these data were presented as ratios to creatine, without quantificationACCEPTED of the % of grey and white matter they cannot be regarded as definitive. The study by Srinivasan et al. [113] carried out at 7T using spectral editing, reported higher GSH in grey matter (3.3

mM) than white matter (2.1 mM). Wijtenburg et al [130] reported GSH in ‘Institutional Units’ in posterior and

20

ACCEPTED MANUSCRIPT

anterior cingulate, but did not report a significance test of regional differences. Inspection of their data suggests a small but probably significant increase in GSH in the anterior as opposed to posterior cingulate (2.7 units as opposed to 2.1, with S.D. ~0.25 units). As noted in Section 2.1.2.2 , a significantly higher GSH concentration was reported in anterior cingulate (~3.2 mM) compared to occipital cortex (~1.4 mM) in the study by Sanaei-

Nezhad et al [131] using MEGA-PRESS, despite the lower grey matter in the anterior cingulate (42%) compared to the occipital cortex (59% grey matter). The most conservative explanation of these four studies is

that GSH varies across brain regions, in a manner which does not simply reflect differences in grey and white

matter content.

One study examined the effect of age on GSH [130], reporting significant changes in young adults (~20 yo), 1.5 times higher than the elderly group (~75-80 yo). At the start of the lifespan, GSH was one of the metabolites measured across gestational ages from 32-43 weeks [99]. Although GSH was reported, at relatively high levels compared to adult studies (average ~ 2.5 mM) no significant changes were shown and the metabolite was not

discussed. Given the low field strength (1.5 T) of this early study performed without editing in very small voxels

it is likely the estimates of GSH are unreliable. We have identified three studies in which GSH was measuredMANUSCRIPT following a challenge in normal volunteers – visual stimulation [132, 133] and ascorbic acid administration [134]. Another study investigated the effect of N-

acetylcysteine administration in Parkinson’s and Gaucher’s disease patients and normal volunteers [135], while

the influence of diet on brain GSH in older volunteers has also been investigated [136].

The visual stimulation studies were carried out at 7T using a functional paradigm, with the MRS voxel

positioned based on the location of the BOLD (Blood oxygenation level-dependent) response from fMRI. One

study [133] detected increases during stimulation in lactate, glutamate and GSH, but despite the high signal-to-

noise of 7T spectra it is difficult to believe that a change of less than 10% can be detected in such a low

concentration metabolite and it is also difficult to see what biochemistry might underlie such a large increase in

a relatively short period of time. Others [132] reported increases in lactate and glutamate, decreases in glucose

and aspartate but noACCEPTED significant change in GSH.

The ascorbic acid challenge produced large changes in the blood concentration of ascorbate without any effect

on either ascorbate or GSH in the brain, indicating that acute anti-oxidant administration has no direct effects on

21

ACCEPTED MANUSCRIPT

levels in the brain, either immediately or over 24h after injection. In contrast, a single injection of N-

acetylcysteine in both patients and healthy volunteers produced increases in brain GSH of 35-55%, peaking at

90-110 min. Gender differences in GSH were investigated by Mandal et al. [137] in several brain regions in

young volunteers (20-30 yo) using MEGA-PRESS at 3T. Significantly increased GSH levels in females

compared to males were reported in cortical and hippocampal volumes.

Choi et al used their double quantum spectroscopic imaging method to investigate the relationship between

brain GSH and diet in a large group of older volunteers (60 subjects, 69±6 years old) [136]. They investigated

diet in general (including calories, protein, carbohydrate and dairy intake) with a specific intention to test for

effects of dairy consumption by recruiting 3 groups with low, normal and high consumption of milk and dairy

products. They observed a highly significant positive correlation between dairy consumption and brain

glutathione independently in frontal, fronto-parietal and parietal areas. Milk may be a good source of cysteine

and superior to other sources of dietary protein, but this study did not address that question directly. It is

possible that the observation of Emir et al. that older subjects have lower brain GSH than young people could

reflect a difference in consumption of dairy products [138]. 3.2. Neurological disease MANUSCRIPT GSH has been measured by MRS in humans in a number of neurological diseases: stroke, cancer, Parkinson’s,

Gaucher’s, epilepsy, motor neurone disease and multiple sclerosis. It is only in multiple sclerosis that we have been able to identify more than one study [101, 113]. These studies were consistent in reporting lower GSH, possibly implicating oxidative stress in the pathology of multiple sclerosis. Decreases were also reported in motor neurone disease [139] and epilepsy [140]. In each of these four disease studies, versions of spectroscopic editing were used. The investigation of Parkinson’s and Gaucher’s disease [135] did not have sufficient numbers to detect the effect of disease on GSH but was instead focused on the effects of N-acetylcysteine infusion (see

Section 3.1, describing physiological and pharmacological interventions). There is one study in each of stroke [122] and cancer [100],ACCEPTED with the stroke study (J-editing) not identifying changes in GSH between stroke patients and controls, whereas the early study of cancer identified increased GSH in meningiomas. Despite the low field

(1.5 T) and lack of editing in the meningioma study, the 150% increase in GSH (1.2 to 3.3 mM) is convincing,

22

ACCEPTED MANUSCRIPT

especially as creatine is very low in meningioma [141] such that a plausible GSH signal can be visually identified.

3.3. Psychiatric disease

Glutathione levels in depression, chronic fatigue, bipolar disease, schizophrenia and risky alcohol use have all been studied with magnetic resonance spectroscopy. A series of papers from one group have investigated depression [142], bipolar disorder with alcohol use [125, 129], mild cognitive impairment [143] and serious psychosis / mood disorders in young people [124]. These papers form the largest single body of work by a research team on GSH using MRS in humans with over 250 patients and 100 controls (there may be some double-counting in the controls). They report evidence for GSH disturbances in all these conditions, with risky alcohol use and bipolar disorder combining to reduce GSH in those patients compared to controls or to patients with lower alcohol use. In contrast, GSH is elevated in older adults at risk of depression and those with mild cognitive impairment. In young people with serious psychiatric disorders, statistical clustering of spectroscopic data revealed that the ratio of GSH to creatine was an influential discriminator of disease type. All these studies were carried out using PRESS (TR/TE 2000/35 ms) in small voxels (8 ml) and short acquisition times of 4.25 mins. LCModel was used to quantify GSH as a ratio to creatine. MANUSCRIPT The spectra shown in the articles are very good quality, nevertheless reliable quantification of GSH in such small voxels in just over 4 min must be called into question. The βCH2 cysteinyl resonances from GSH are close to the N-methyl creatine resonance at 3.03 ppm; that will make the ratio particularly sensitive to small shifts in the fit to this region. The control data from 2012

[124] show a GSH to creatine ratio of ~0.3, implying a GSH concentration of ~2.5 mM, which is at the upper range of normal volunteer data.

Studies from other researchers have also been reported in major depressive order and chronic fatigue [144, 145], using J-editing in occipital cortex; in bipolar disorder [146] in prefrontal and occipital cortex using SPECIAL

(Spin-echo full intensity acquired localized) / LCModel; and in mild cognitive impairment [137] using MEGA- PRESS in frontal ACCEPTED and parietal cortex, hippocampus and cerebellum. GSH was reported to be decreased in depression and chronic fatigue compared to healthy volunteers and negatively correlated with anhedonia ratings in depression (i.e lower GSH in patients with more severe symptoms) [144, 145]. These results are inconsistent

23

ACCEPTED MANUSCRIPT

with increased GSH in depression described in the preceding paragraph in this section [142, 143], but the discrepancies could be explained by the use of different brain regions (anterior and posterior cingulate) as well as the acquisition and analysis method. The other study in bipolar disorder [146] reported no significant difference in GSH, but again was from different cortical regions. Two studies by Mandal et al [137, 147] have investigated Alzheimer’s disease and mild cognitive impairment investigating relatively small numbers of patients (14 in total) in the first report [137], but then refining the methods and studying a larger cohort in the second study [147] (43 patients with 21 age-matched controls). GSH was significantly lower in the disease groups in both hippocampus and frontal cortex, which contradicts another study in mild cognitive impairment which reported an increase in GSH/creatine ratios in anterior and posterior cingulate cortex relative to healthy age-matched controls [126]. Though a regional effect cannot be ruled out, it is notable that the acquisition for the latter study [126] was in smaller voxels and for a much shorter acquisition time than the study reporting a

GSH increase [147]. In contrast to many MRS studies, the increase in metabolite level could also be used as a diagnostic biomarker to distinguish patients with disease from healthy age-matched controls In many published

studies of MRS in human disease, it is common to detect significant differences in average metabolite concentrations, but the overlap between groups is such thatMANUSCRIPT a given subject could not be classified on the basis of the MRS metabolite measurement.

The most studied psychiatric disorder is schizophrenia, though general conclusions are difficult to draw as seven

brain areas have been investigated (three medial prefrontal cortex, two in anterior cingulate, one dorsolateral

prefrontal cortex and one medial temporal lobe), three field strengths used (1.5, 3 and 4 T) with differing

acquisition strategies (J-editing, double quantum filter, PRESS and SPECIAL). Furthermore the disease status

of the patients varies across studies: early vs established; medicated vs drug-free. In summary, one study has

reported an increase in GSH [148] (medial temporal lobe, first episode); one a decrease [149] (prefrontal cortex,

mixed treatment and length of illness, from first episode to 14 y duration), and three studies have reported no

change [150] (posterior medial prefrontal cortex, established disease, all medicated); [128] (anterior cingulate

cortex, all medicatedACCEPTED with established disease); [151] (medial prefrontal lobe, early disease (2 ± 1.8 y) in young

patients, all medicated). A negative correlation between the severity of negative symptoms and GSH was

reported in one of the studies which showed no difference between patients and controls [150], while in another

24

ACCEPTED MANUSCRIPT

of these studies there was evidence for a difference in the relationship between GSH and connectivity measures between patients and controls [151]. Overall these disparate observations in schizophrenia support the contention that GSH is important in the pathogenesis of schizophrenia but its concentration is likely to be regionally heterogeneous and dependent on disease, medication and symptomatic status.

Finally amongst psychiatric disorders, we identified single publications on post-traumatic stress disorder [152] and autism spectrum disorder [153]. The investigation of post-traumatic stress used a MEGA-PRESS sequence optimized for GABA detection with relatively large voxels and long acquisitions in dorsolateral prefrontal cortex and anterior cingulate cortex (19 ml / 20 min; 30 ml / 10 min, respectively). Both GABA and GSH were

elevated in the patients, but the method used to measure GSH was not optimal. GSH was estimated using

LCModel analysis of the MEGA sub-spectrum without the GABA-selective pulse, thus it includes only half the acquired averages and, compared to conventional LCModel / PRESS, used a longer TE. The autism investigation used PRESS at 3T in small voxels in basal ganglia (6 ml, 6.5 min) and dorsomedial prefrontal cortex (7.7 ml, 4.8 min), reporting GSH ~ 3 mM with no effect of disease. Though the study was relatively large by the standards of MRS (21 patients, 29 controls) the intrinsic sensitivity of GSH detection using their method may be too low to detect differences. MANUSCRIPT In summary the interpretation of GSH in psychiatric disorders is confounded by differences in methodology - is

PRESS / LCModel appropriate for measuring GSH? It would be very instructive for a study of disease to be undertaken in which differences in GSH detected by one of the editing techniques could also be investigated using the non-editing approach. Nevertheless, the weight of evidence that GSH is altered in psychiatric disease is strong, but a lot of work is required to deal with the confounds of length of disease, medication, symptom status and regional effects.

4. Conclusions The biochemistry ofACCEPTED glutathione is varied and complicated, playing multiple roles in many different metabolic pathways. Levels of glutathione in brain are highest in non-neural cells such as ependymal cells and choroid plexus, lower in astrocytes and lower still in oligodendrocytes and neurons. Synthesis of glutathione is subject to strong metabolic regulation, mostly through control of the availability of the precursor cysteine and to a lesser

25

ACCEPTED MANUSCRIPT

extent, glutamate. Levels in human brain measured by magnetic resonance spectroscopy represent bulk tissue

glutathione, the vast majority of which is reduced glutathione, and are ~ 1-2 mM in healthy brain. Measurement

of glutathione at clinical field strengths (1.5 – 3 Tesla) is fraught with difficulty due to resonance overlap, the

high level of spin coupling and the low concentration of glutathione. It is best and most precisely accomplished

by using an editing sequence, such as MEGA-PRESS.

Acknowledgements

The authors are grateful to University of Manchester Ph.D. student Faezeh Sanaei Nezhad for providing simulations using jMRUI and for help in producing figures. We acknowledge the developers of jMRUI (Stefan et al. 2009) and the European Union for funding jMRUI (EC-HCM/ CHRX-CT94-0432, EC-TMR/

ERBFMRXCT970160, FAST (FP6-MC-RTN-035801), TRANSACT (FP7-PEOPLE-2012-ITN- 316679)).

MANUSCRIPT

ACCEPTED

26

ACCEPTED MANUSCRIPT

REFERENCES

[1] G. Wu, Y.-Z. Fang, S. Yang, J.R. Lupton, N.D. Turner, Glutathione Metabolism and Its Implications for Health, J Nutr, 134 (2004) 489- 492. [2] M. Deponte, Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes, Biochim Biophys Acta (BBA)- General Subjects, 1830 (2013) 3217-3266. [3] A.J.L. Cooper, B.S. Kristal, Multiple roles of glutathione in the central nervous system, Biol Chem, 378 (1997) 793-802. [4] R. Dringen, Metabolism and functions of glutathione in brain, Prog Neurobiol, 62 (2000) 649-671. [5] M.A. Philbert, C.M. Beiswanger, D.K. Waters, K.R. Reuhl, H.E. Lowndes, Cellular and regional distribution of reduced glutathione in the nervous system of the rat: Histochemical localization by mercury orange and o-phthaldialdehyde-induced histofluorescence, Toxicol Applied Pharmacol, 107 (1991) 215-227. [6] A. Slivka, C. Mytilineou, G. Cohen, Histochemical evaluation of glutathione in brain, Brain Res, 409 (1987) 275-284. [7] X. Sun, A.Y. Shih, H.C. Johannssen, H. Erb, P. Li, T.H. Murphy, Two-photon imaging of glutathione levels in intact brain indicates enhanced redox buffering in developing neurons and cells at the cerebrospinal fluid and blood-brain interface, J Biol Chem, 281 (2006) 17420-17431. [8] X. Sun, Two-photon imaging of glutathione levels in intact brain indicates sites of enhanced redox buffering, University of British Columbia, 2006. [9] M.E. Rice, I. Russo-Menna, Differential compartmentalization of brain ascorbate and glutathione between neurons and glia, Neurosci, 82 (1998) 1213-1223. [10] B.H.J. Juurlink, S.K. Thorburne, L. Hertz, Peroxide-scavenging deficit underlies oligodendrocyte susceptibility to oxidative stress, Glia, 22 (1998) 371-378. [11] T. Perry, S. Hansen, K. Berry, C. Mok, D. Lesk, Free amino acids and related compounds in biopsies of human brain, J Neurochem, 18 (1971) 521-528. MANUSCRIPT [12] V. Vitvitsky, M. Thomas, A. Ghorpade, H.E. Gendelman, R. Banerjee, A functional transsulfuration pathway in the brain links to glutathione homeostasis, J Biol Chem, 281 (2006) 35785-35793. [13] A. Slivka, M.B. Spina, G. Cohen, Reduced and oxidized glutathione in human and monkey brain, Neurosci Lett, 74 (1987) 112-118. [14] S. Rehncrona, J. Folbergrova, D.S. Smith, B.K. Siesjo, Influence of Complete and Pronounced Incomplete Cerebral Ischemia and Subsequent Recirculation on Cortical Concentrations of Oxidized and Reduced Glutathione in the Rat, J Neurochem, 34 (1980) 477-486. [15] A.J. Cooper, W.A. Pulsinelli, T.E. Duffy, Glutathione and ascorbate during ischemia and postischemic reperfusion in rat brain, J Neurochem, 35 (1980) 1242-1245. [16] A. Chauhan, T. Audhya, V. Chauhan, Brain region-specific glutathione redox imbalance in autism, Neurochem Res, 37 (2012) 1681- 1689. [17] C.-S. Yang, S.-T. Chou, N.-N. Lin, L. Liu, P.-J. Tsai, J.-S. Kuo, J.-S. Lai, Determination of extracellular glutathione in rat brain by microdialysis and high-performance liquid chromatography with fluorescence detection, J Chromat B: Biomed Sci Apps, 661 (1994) 231- 235. [18] M.W. Lada, R.T. Kennedy, In vivo monitoring of glutathione and cysteine in rat caudate nucleus using microdialysis on-line with capillary zone electrophoresis-laser induced fluorescence detection, J Neurosci Meth, 72 (1997) 153-159. [19] C. Wallin, S.G. Weber, M. Sandberg, Glutathione Efflux Induced by NMDA and Kainate, J Neurochem, 73 (1999) 1566-1572. [20] G.F. Seelig, R.P. ACCEPTED Simondsen, A. Meister, Reversible dissociation of gamma-glutamylcysteine synthetase into two subunits, J Biol Chem, 259 (1984) 9345-9347. [21] Y. Yang, M.Z. Dieter, Y. Chen, H.G. Shertzer, D.W. Nebert, T.P. Dalton, Initial Characterization of the Glutamate-Cysteine Ligase Modifier Subunit Gclm(−/−) Knockout Mouse: Novel mouse model system for a severely compromised oxidative stress response, J Biol Chem, 277 (2002) 49446-49452.

27

ACCEPTED MANUSCRIPT

[22] Y. Chen, H.G. Shertzer, S.N. Schneider, D.W. Nebert, T.P. Dalton, Glutamate Cysteine Ligase Catalysis: Dependence on ATP and modifier subunit for regulation of tissue glutathione levels. , J Biol Chem, 280 (2005) 33766-33774. [23] K. Tsuchiya, R.T. Mulcahy, L.L. Reid, C.M. Disteche, T.J. Kavanagh, Mapping of the glutamate-cysteine ligase catalytic subunit gene (GLCLC) to human chromosome 6p12 and mouse chromosome 9D-E and of the regulatory subunit gene (GLCLR) to human chromosome 1p21-p22 and mouse chromosome 3H1-3, Genomics, 30 (1995) 630-632. [24] M. Tosic, J. Ott, S. Barral, P. Bovet, P. Deppen, F. Gheorghita, M.-L. Matthey, J. Parnas, M. Preisig, M. Saraga, Schizophrenia and oxidative stress: glutamate cysteine ligase modifier as a susceptibility gene, Am J Hum Gen, 79 (2006) 586-592. [25] C. Butticaz, T. Werge, J.S. Beckmann, M. Cuénod, K.Q. Do, C. Rivolta, Mutation screening of the glutamate cysteine ligase modifier (GCLM) gene in patients with schizophrenia, Psychiatr Gen, 19 (2009) 201-208. [26] T. Kishi, M. Ikeda, T. Kitajima, Y. Yamanouchi, Y. Kinoshita, K. Kawashima, T. Inada, M. Harano, T. Komiyama, T. Hori, Glutamate Cysteine Ligase Modifier (GCLM) Subunit Gene Is Not Associated with Methamphetamine ‐Use Disorder or Schizophrenia in the Japanese Population, Proc Ann NY Acad Sci, 1139 (2008) 63-69. [27] M. Berk, S. Johansson, N.R. Wray, L. Williams, C. Olsson, J. Haavik, O. Bjerkeset, Glutamate cysteine ligase (GCL) and self reported depression: An association study from the HUNT, J Affect Disord, 131 (2011) 207-213. [28] O.P. Ottersen, N. Zhang, F. Walberg, Metabolic compartmentation of glutamate and glutamine: Morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum, Neurosci, 46 (1992) 519-534. [29] P. Richman, A. Meister, Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione, J Biol Chem, 250 (1975) 1422-1426. [30] O.W. Griffith, A. Meister, Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine), J Biol Chem, 254 (1979) 7558-7560. [31] P.G. Richman, M. Orlowski, A. Meister, Inhibition of γ-glutamylcysteine synthetase by L-methionine-S-sulfoximine, J Biol Chem, 248 (1973) 6684-6690. [32] R.A. Ronzio, W.B. Rowe, A. Meister, Mechanism of inhibition of glutamine synthetase by methionine sulfoximine, Biochem, 8 (1969) 1066-1075. [33] T.M. Jeitner, A.J. Cooper, Inhibition of human glutamine synthetase MANUSCRIPTby L-methionine-S, R-sulfoximine—relevance to the treatment of neurological diseases, Metabol Brain Dis, 29 (2014) 983-989. [34] R. Heales, S.E.C. Davies, T.E. Bates, J.B. Clark, Depletion of brain glutathione is accompanied by impaired micochondrial function and decreased N-acetyl aspartate concentration, Neurochem Res, 20 (1995) 31-38. [35] A. Jain, J. Mårtensson, E. Stole, P. Auld, A. Meister, Glutathione deficiency leads to mitochondrial damage in brain, Proc Natl Acad Sci USA, 88 (1991) 1913-1917. [36] R. Njälsson, S. Norgren, Physiological and pathological aspects of GSH metabolism, Acta Pædiatrica, 94 (2005) 132-137. [37] E. Ristoff, E. Mayatepek, A. Larsson, Long-term clinical outcome in patients with glutathione synthetase deficiency, J Pediatr, 139 (2001) 79-84. [38] I.-Y. Choi, R. Gruetter, Dynamic or inert metabolism? Turnover of N-acetyl aspartate and glutathione from d-[1-13C]glucose in the rat brain in vivo, J Neurochem, 91 (2004) 778-787. [39] M. Koga, A.V. Serritella, M.M. Messmer, A. Hayashi-Takagi, L.D. Hester, S.H. Snyder, A. Sawa, T.W. Sedlak, Glutathione is a physiologic reservoir of neuronal glutamate, Biochem Biophys Res Commun, 409 (2011) 596-602. [40] R. Janaky, V. Varga, A. Hermann, P. Saransaari, S.S. Oja, Mechanisms of L-cysteine neurotoxicity, Neurochem Res, 25 (2000) 1397- 1405. [41] M. Yudkoff, D. Pleasure,ACCEPTED L. Cregar, Z.-P. Lin, I. Nissim, J. Stern, I. Nissim, Glutathione Turnover in Cultured Astrocytes: Studies with [15N]Glutamate, J Neurochem, 55 (1990) 137-145. [42] R. Dringen, B. Hamprecht, N-acetylcysteine, but not methionine or 2-oxothiazolidine-4-carboxylate, serves as cysteine donor for the synthesis of glutathione in cultured neurons derived from embryonal rat brain, Neurosci Lett, 259 (1999) 79-82. [43] X.F. Wang, M.S. Cynader, Astrocytes provide cysteine to neurons by releasing glutathione, J Neurochem, 74 (2000) 1434-1442.

28

ACCEPTED MANUSCRIPT

[44] J.W. Olney, C. Zorumski, M.T. Price, J. Labruyere, L-cysteine, a bicarbonate-sensitive endogenous excitotoxin, Science, 248 (1990) 596-599. [45] Y. Chen, R.A. Swanson, The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures, J Neurochem, 84 (2003) 1332-1339. [46] K. Aoyama, S.W. Suh, A.M. Hamby, J. Liu, W.Y. Chan, Y. Chen, R.A. Swanson, Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse, Nat Neurosci, 9 (2006) 119-126. [47] S.K. Thorburne, B.H. Juurlink, Low glutathione and high iron govern the susceptibility of oligodendroglial precursors to oxidative stress, J Neurochem, 67 (1996) 1014-1022. [48] A. Meister, M.E. Anderson, Glutathione, Ann Rev Biochem, 52 (1983) 711-760. [49] M. Philbert, C. Beiswanger, M. Manson, J. Green, R. Novak, T. Primiano, K. Reuhl, H. Lowndes, Glutathione S-transferases and gamma-glutamyl transpeptidase in the rat nervous systems: a basis for differential susceptibility to neurotoxicants, Neurotoxicol, 16 (1994) 349-362. [50] K. Maxwell, J.A. Berliner, P.A. Cancilla, Induction of γ-glutamyl transpeptidase in cultured cerebral endothelial cells by a product released by astrocytes, Brain Res, 410 (1987) 309-314. [51] S.S. Hotta, Glucose metabolism in brain tissue: the hexose monophosphate shunt and its role in glutathione reduction, J Neurochem, 9 (1962) 43-51. [52] J.M. Gutterer, R. Dringen, J. Hirrlinger, B. Hamprecht, Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells, J Neurochem, 73 (1999) 1422-1430. [53] J.P. Bolanos, A. Almeida, S. Moncada, Glycolysis: a bioenergetic or a survival pathway?, Trends Biochem Sci, 35 (2010) 145-149. [54] J.P. Bolanos, A. Almeida, The pentose-phosphate pathway in neuronal survival against nitrosative stress, IUBMB Life, 62 (2010) 14- 18. [55] R. Dringen, M. Brandmann, M.C. Hohnholt, E.-M. Blumrich, Glutathione-dependent detoxification processes in astrocytes, Neurochem Res, 40 (2015) 2570-2582. [56] J. Hirrlinger, J. König, R. Dringen, Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes, oligodendrocytes, microglial cells and neurones, J Neurochem, 82 (2002) 716-719.MANUSCRIPT [57] T. Minich, J. Riemer, J.B. Schulz, P. Wielinga, J. Wijnholds, R. Dringen, The multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of glutathione and glutathione disulfide from brain astrocytes, J Neurochem, 97 (2006) 373-384. [58] R. Shawahna, Y. Uchida, X. Decleves, S. Ohtsuki, S. Yousif, S. Dauchy, A. Jacob, F. Chassoux, C. Daumas-Duport, P.-O. Couraud, Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels, Mol Pharmaceut, 8 (2011) 1332-1341. [59] P.G. Board, D. Menon, Glutathione transferases, regulators of cellular metabolism and physiology, Biochim Biophys Acta (BBA) - General Subjects, 1830 (2013) 3267-3288. [60] M. Abramovitz, H. Homma, S. Ishigaki, F. Tansey, W. Cammer, I. Listowsky, Characterization and Localization of Glutathione ‐S‐ Transferases in Rat Brain and Binding of Hormones, Neurotransmitters and Drugs, J Neurochem, 50 (1988) 50-57. [61] W. Cammer, F. Tansey, M. Abramovitz, S. Ishigaki, I. Listowsky, Differential localization of glutathione ‐S‐transferase Yp and Yb subunits in oligodendrocytes and astrocytes of rat brain, J Neurochem, 52 (1989) 876-883. [62] R. Kannan, J.F. Kuhlenkamp, E. Jeandidier, H. Trinh, M. Ookhtens, N. Kaplowitz, Evidence for carrier-mediated transport of glutathione across the blood-brain barrier in the rat, J Clin Invest, 85 (1990) 2009-2013. [63] A.T. Nies, G. Jedlitschky, J. König, C. Herold-Mende, H.H. Steiner, H.P. Schmitt, D. Keppler, Expression and immunolocalization of the multidrug resistanceACCEPTED proteins, MRP1–MRP6 (ABCC1–ABCC6), in human brain, Neurosci, 129 (2004) 349-360. [64] J.C. Kalvass, J.W. Polli, D.L. Bourdet, B. Feng, S.M. Huang, X. Liu, Q.R. Smith, L.K. Zhang, M.J. Zamek-Gliszczynski, C. International Transporter, Why Clinical Modulation of Efflux Transport at the Human Blood–Brain Barrier Is Unlikely: The ITC Evidence- Based Position, Clin Pharmacol Ther, 94 (2013) 80-94.

29

ACCEPTED MANUSCRIPT

[65] J.P. Richie, L. Skowronski, P. Abraham, Y. Leutzinger, Blood glutathione concentrations in a large-scale human study, Clin Chem, 42 (1996) 64-70. [66] B.V. Zlokovic, J.B. Mackic, J.G. McComb, M.H. Weiss, N. Kaplowitz, R. Kannan, Evidence for Transcapillary Transport of Reduced Glutathione in Vascular Perfused Guinea-Pig Brain, Biochem Biophys Res Commun, 201 (1994) 402-408. [67] R. Kannan, R. Chakrabarti, D. Tang, K. Kim, N. Kaplowitz, GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na+-dependent GSH transport in HCEC, Brain Res, 852 (2000) 374-382. [68] R. Kannan, R.Y. Jian, D. Tang, Y. Li, B.V. Zlokovic, N. Kaplowitz, Evidence for the Existence of a Sodium-dependent Glutathione (GSH) Transporter J Biol Chem, 271 (1996) 9754-9758. [69] R. Agarwal, G.S. Shukla, Potential role of cerebral glutathione in the maintenance of blood-brain barrier integrity in rat, Neurochem Res, 24 (1999) 1507-1514. [70] J. Hirrlinger, J.B. Schulz, R. Dringen, Glutathione release from cultured brain cells: multidrug resistance protein 1 mediates the release of GSH from rat astroglial cells, J Neuroscience Res, 69 (2002) 318-326. [71] S. Rana, R. Dringen, Gap junction hemichannel-mediated release of glutathione from cultured rat astrocytes, Neurosci Lett, 415 (2007) 45-48. [72] O.W. Griffith, A. Meister, Origin and turnover of mitochondrial glutathione, Proc Natl Acad Sci USA, 82 (1985) 4668. [73] J. Mårtensson, J. Lai, A. Meister, High-affinity transport of glutathione is part of a multicomponent system essential for mitochondrial function, Proc Natl Acad Sci USA, 87 (1990) 7185-7189. [74] T.B. McKernan, E.B. Woods, L.H. Lash, Uptake of glutathione by renal cortical mitochondria, Arch Biochem Biophys, 288 (1991) 653-663. [75] Z. Chen, L.H. Lash, Evidence for mitochondrial uptake of glutathione by dicarboxylate and 2-oxoglutarate carriers, J Pharmacol Exp Therap, 285 (1998) 608-618. [76] O. Coll, A. Colell, C. García ‐Ruiz, N. Kaplowitz, J.C. Fernández ‐Checa, Sensitivity of the 2 ‐oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletion, Hepatology, 38 (2003) 692-702. [77] E. Racker, Glutathione as a coenzyme in intermediary metabolism, Glutathione, a symposium, 1954, pp. 165-183. [78] K. Lohmann, A study of the enzymatic transformation of synthetic methylglyoxalMANUSCRIPT to lactic acid, Biochem Z, 254 (1932) 332-354. [79] C. Rae, S.I. O'Donoghue, W.A. Bubb, P.W. Kuchel, Stereospecificity of substrate usage by glyoxalase 1: nuclear magnetic resonance studies of kinetics and hemithioacetal substrate conformation, Biochem, 33 (1994) 3548-3559. [80] V. Riddle, F. Lorenz, Nonenzymic, polyvalent anion-catalyzed formation of methylglyoxal as an explanation of its presence in physiological systems, J Biol Chem, 243 (1968) 2718-2724. [81] J.P. Richard, Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance, Biochem, 30 (1991) 4581-4585. [82] C. Walsh, Enzymatic reaction mechanisms, W.H.Freeman, New York, 1979. [83] C. Rae, W.A. Bubb, P.W. Kuchel, Aldolase-catalyzed diketone phosphate formation from oxoaldehydes. NMR studies and metabolic significance, J Biol Chem, 267 (1992) 9713-9717. [84] F. Chen, M.A. Wollmer, F. Hoerndli, G. Münch, B. Kuhla, E.I. Rogaev, M. Tsolaki, A. Papassotiropoulos, J. Götz, Role for glyoxalase I in Alzheimer's disease, Proc Natl Acad Sci USA, 101 (2004) 7687-7692. [85] M. Belanger, J.Y. Yang, J.M. Petit, T. Laroche, P.J. Magistretti, I. Allaman, Role of the Glyoxalase System in Astrocyte-Mediated Neuroprotection, J Neurosci, 31 (2011) 18338-18352. [86] O. Mori, T. Haseba, K. Kameyama, H. Shimizu, M. Kudoh, Y. Ohaki, Y. Arai, M. Yamazaki, G. Asano, Histological distribution of class III alcohol dehydrogenaseACCEPTED in human brain, Brain Res, 852 (2000) 186-190. [87] C.D. Rae, J.E. Davidson, A.D. Maher, B.D. Rowlands, M.A. Kashem, F.A. Nasrallah, S.K. Rallipalli, J.M. Cook, V.J. Balcar, Ethanol, not detectably metabolized in brain, significantly reduces brain metabolism, probably via action at specific GABA(A) receptors and has measureable metabolic effects at very low concentrations, J Neurochem, 129 (2014) 304-314.

30

ACCEPTED MANUSCRIPT

[88] M.D. Boleda, N. Saubi, J. Farres, X. Pares, Physiological Substrates for Rat Alcohol Dehydrogenase Classes: Aldehydes of Lipid Peroxidation, ω-Hydroxyfatty Acids, and Retinoids, Arch Biochem Biophys, 307 (1993) 85-90. [89] D. Galter, A. Carmine, S. Buervenich, G. Duester, L. Olson, Distribution of class I, III and IV alcohol dehydrogenase mRNAs in the adult rat, mouse and human brain, Eur J Biochem, 270 (2003) 1316-1326. [90] K. Tulpule, R. Dringen, Formaldehyde in brain: an overlooked player in neurodegeneration?, J Neurochem, 127 (2013) 7-21. [91] V. Govindaraju, K. Young, A.A. Maudsley, Proton NMR chemical shifts and coupling constant for brain metabolites, NMR Biomed, 13 (2000) 129-153. [92] L.G. Kaiser, M. Marjanska, G.B. Matson, I. Iltis, S.D. Bush, B.J. Soher, S. Mueller, K. Young, (1)H MRS detection of glycine residue of reduced glutathione in vivo, J Magn Reson, 202 (2010) 259-266. [93] M.J. York, G.R. Beilharz, P.W. Kuchel, Conformation of reduced glutathione in aqueous solution by 1H and 13C nmr, Int J Peptide Prot Res, 29 (1987) 638-646. [94] R.J. Ordidge, R. Gordon, Methods and apparatus for obtaining localised NMR spectra, US Patent, 1985. [95] P.A. Bottomley, Spatial localization in NMR spectroscopy in vivo, Ann NY Acad Sci, 508 (1987) 333-348. [96] J. Frahm, K.D. Merboldt, W. Hanicke, Localized proton spectroscopy using stimulated echoes, J Magn Reson, 72 (1987) 502-508. [97] C.T.W. Moonen, M.V. Kienlin, P.C.M. Van Zijl, J. Cohen, J. Gillen, P. Daly, G. Wolf, Comparison of single-shot localization methods (steam and press) for In vivo proton NMR spectroscopy, NMR Biomed, 2 (1989) 201-208. [98] A.H. Trabesinger, O.M. Weber, C.O. Duc, P. Boesinger, Detection of glutathione in the human brain in vivo by means of double quantum coherence filtering., Magn Reson Med, 42 (1999) 283-289. [99] R. Kreis, L. Hofmann, B. Kuhlmann, C. Boesch, E. Bossi, P.S. Hüppi, Brain metabolite composition during early human brain development as measured by quantitative in vivo 1H magnetic resonance spectroscopy, Magn Reson Med, 48 (2002) 949-958. [100] K. Opstad, S. Provencher, B. Bell, J. Griffiths, F. Howe, Detection of elevated glutathione in meningiomas by quantitative in vivo 1H MRS, Magn Reson Med, 49 (2003) 632-637. [101] I.Y. Choi, S.P. Lee, D.R. Denney, S.G. Lynch, Lower Levels of Glutathione (GSH) in the Brains of Secondary Progressive Multiple Sclerosis Patients Measured by (1)H Magnetic Resonance Chemical Shift Imaging at 3 T, Multiple Sclerosis, 17 (2011) 289-296. [102] T. Zhao, K. Heberlein, C. Jonas, D.P. Jones, X. Hu, New double quantumMANUSCRIPT coherence filter for localized detection of glutathione in vivo, Magn Reson Med, 55 (2006) 676-680. [103] M. Mescher, H. Merkle, J. Kirsch, M. Garwood, R. Gruetter, Simultaneous in vivo spectral editing and water suppression, NMR Biomed, 11 (1998) 266-272. [104] D.L. Rothman, K.L. Behar, H.P. Hetherington, R.G. Shulman, Homonuclear H1 double resonance difference spectroscopy of the rat brain in vivo, Proc Natl Acad Sci USA - Biol Sci, 81 (1984) 6330-6334. [105] S. Williams, D. Gadian, E. Proctor, A method for lactate detection in vivo by spectral editing without the need for double irradiation, J Magn Reson, 66 (1986) 562-567. [106] T. Satoh, Y. Yoshioka, Contribution of reduced and oxidized glutathione to signals detected by magnetic resonance spectroscopy as indicators of local brain redox state, Neurosci Res, 55 (2006) 34-39. [107] M. Terpstra, P.G. Henry, R. Gruetter, Measurement of reduced glutathione (GSH) in human brain using LCmodel analysis of difference-edited spectra, Magn Reson Med, 50 (2003) 19-23. [108] M.A. Thomas, K. Yue, N. Binesh, P. Davanzo, A. Kumar, B. Siegel, M. Frye, J. Curran, R. Lufkin, P. Martin, Localized two ‐ dimensional shift correlated MR spectroscopy of human brain, Magn Reson Med, 46 (2001) 58-67. [109] S.W. Provencher, Estimation of metabolite concentrations from localized in vivo proton NMR spectra, Magn Reson Med, 30 (1993) 672-679. ACCEPTED [110] S.A. Wijtenburg, F.E. Gaston, E.A. Spieker, S.A. Korenic, P. Kochunov, L.E. Hong, L.M. Rowland, Reproducibility of phase rotation STEAM at 3T: focus on glutathione, Magn Reson Med, 72 (2014) 603-609. [111] K.M. Chitty, J. Lagopoulos, I.B. Hickie, D.F. Hermens, Risky alcohol use in young persons with emerging bipolar disorder is associated with increased oxidative stress, J Affect Disord, 150 (2013) 1238-1241.

31

ACCEPTED MANUSCRIPT

[112] K.M. Chitty, J. Lagopoulos, I.B. Hickie, D.F. Hermens, The impact of alcohol and tobacco use on in vivo glutathione in youth with bipolar disorder: an exploratory study, J Psychiatr Res, 55 (2014) 59-67. [113] R. Srinivasan, H. Ratiney, K.E. Hammond-Rosenbluth, D. Pelletier, S.J. Nelson, MR spectroscopic imaging of glutathione in the white and gray matter at 7 T with an application to multiple sclerosis, Magn Reson Imag, 28 (2010) 163-170. [114] L. An, S. Li, J.B. Murdoch, M.F. Araneta, C. Johnson, J. Shen, Detection of glutamate, glutamine, and glutathione by radiofrequency suppression and echo time optimization at 7 Tesla, Magn Reson Med, 73 (2015) 451-458. [115] C. Choi, I.E. Dimitrov, D. Douglas, A. Patel, L.G. Kaiser, C.A. Amezcua, E.A. Maher, Improvement of resolution for brain coupled metabolites by optimized 1H MRS at 7 T, NMR Biomed, 23 (2010) 1044-1052. [116] G.S. Payne, Single-Voxel MR Spectroscopy, EMagRes, John Wiley & Sons, Ltd2015. [117] R.A. de Graaf, D.L. Rothman, Spectral Editing, eMagRes, John Wiley and Sons, Ltd2015. [118] W. Bogner, G. Hangel, O.C. Andronesi, 1D-spectral editing and 2D multispectral in vivo 1H-MRS and 1HMRSI - methods and applications, Anal Biochem, In Press (2016). [119] Z. Star čuk Jr, J. Star čuková, Quantum-mechanical simulations for in vivo MR spectroscopy: Principles and possibilities demonstrated with the program NMRScopeB, Anal Biochem, In Press (2016). [120] G. Gambarota, Optimization of metabolite detection by quantum mechanics simulations in magnetic resonance spectroscopy, Anal Biochem, In Press (2016). [121] L. Xin, I. Tká č, A practical guide to in vivo proton magnetic resonance spectroscopy at high magnetic fields, Anal Biochem, (2016). [122] L. An, Y. Zhang, D.M. Thomasson, L.L. Latour, E.H. Baker, J. Shen, S. Warach, Measurement of glutathione in normal volunteers and stroke patients at 3T using J ‐difference spectroscopy with minimized subtraction errors, J Magn Reson Imag, 30 (2009) 263-270. [123] S.W. Provencher, Estimation of metabolite concentrations from localised in vivo proton NMR spectra., Magn Reson Med, 30 (1993) 672-679. [124] D. Hermens, J. Lagopoulos, S. Naismith, J. Tobias-Webb, I. Hickie, Distinct neurometabolic profiles are evident in the anterior cingulate of young people with major psychiatric disorders, Transl Psychiatry, 2 (2012) e110. [125] K.M. Chitty, J. Lagopoulos, I.B. Hickie, D.F. Hermens, Risky alcohol use in young persons with emerging bipolar disorder is associated with increased oxidative stress, J Affect Disord, 150 (2013) 1238-1241.MANUSCRIPT [126] R. Kreis, The trouble with quality filtering based on relative Cramér ‐Rao lower bounds, Magn Reson Med, 75 (2016) 15-18. [127] D. van Ormondt, D. Graveron-Demilly, D.M. Sima, S. Van Huffel, S.R. Williams, Time-Domain Methods for Quantifying MR Spectra, EMagRes, John Wiley & Sons, Ltd2015. [128] M. Terpstra, T.J. Vaughan, K. Ugurbil, K.O. Lim, S.C. Schulz, R. Gruetter, Validation of glutathione quantitation from STEAM spectra against edited H-1 NMR spectroscopy at 4T: application to schizophrenia, Magn Reson Mat Phys Biol Med, 18 (2005) 276-282. [129] K.M. Chitty, J. Lagopoulos, I.B. Hickie, D.F. Hermens, The impact of alcohol and tobacco use on in vivo glutathione in youth with bipolar disorder: An exploratory study, J Psychiatr Res., 55 (2014) 59-67. [130] S.A. Wijtenburg, F.E. Gaston, E.A. Spieker, S.A. Korenic, P. Kochunov, L.E. Hong, L.M. Rowland, Reproducibility of phase rotation STEAM at 3T: focus on glutathione, Magn Reson Med, 72 (2014) 603-609. [131] F. Sanaei Nezhad, A. Anton, L.M. Parkes, B. Deakin, S.R. Williams, Quantification of glutathione in the human brain by MR spectroscopy at 3 Tesla: Comparison of PRESS and MEGA ‐PRESS, Magn Reson Med, (2016). [132] P. Bedna řík, I. Tká č, F. Giove, M. DiNuzzo, D.K. Deelchand, U.E. Emir, L.E. Eberly, S. Mangia, Neurochemical and BOLD responses during neuronal activation measured in the human visual cortex at 7 Tesla, J Cereb Blood Flow Metab, 35 (2015) 601-610. [133] Y. Lin, M.C. Stephenson, L. Xin, A. Napolitano, P.G. Morris, Investigating the metabolic changes due to visual stimulation using functional proton magneticACCEPTED resonance spectroscopy at 7 T, J Cereb Blood Flow Metab, 32 (2012) 1484-1495. [134] M. Terpstra, C. Torkelson, U. Emir, J.S. Hodges, S. Raatz, Noninvasive quantification of human brain antioxidant concentrations after an intravenous bolus of vitamin C, NMR Biomed, 24 (2011) 521-528. [135] M.J. Holmay, M. Terpstra, L.D. Coles, U. Mishra, M. Ahlskog, G. Öz, J.C. Cloyd, P.J. Tuite, N-acetylcysteine Boosts Brain and Blood Glutathione in Gaucher and Parkinson’s Diseases, Clin Neuropharmacol, 36 (2013) 103.

32

ACCEPTED MANUSCRIPT

[136] I.-Y. Choi, P. Lee, D.R. Denney, K. Spaeth, O. Nast, L. Ptomey, A.K. Roth, J.A. Lierman, D.K. Sullivan, Dairy intake is associated with brain glutathione concentration in older adults, Am J Clin Nutr, 101 (2015) 287-293. [137] P.K. Mandal, M. Tripathi, S. Sugunan, Brain oxidative stress: Detection and mapping of anti-oxidant marker 'Glutathione' in different brain regions of healthy male/female, MCI and Alzheimer patients using non-invasive magnetic resonance spectroscopy, Biochem Biophys Res Commun, 417 (2012) 43-48. [138] U.E. Emir, S. Raatz, S. McPherson, J.S. Hodges, C. Torkelson, P. Tawfik, T. White, M. Terpstra, Noninvasive quantification of ascorbate and glutathione concentration in the elderly human brain, NMR Biomed, 24 (2011) 888-894. [139] N. Weiduschat, X. Mao, J. Hupf, N. Armstrong, G. Kang, D. Lange, H. Mitsumoto, D. Shungu, Motor cortex glutathione deficit in ALS measured in vivo with the J-editing technique, Neurosci Lett, 570 (2014) 102-107. [140] S. Mueller, A. Trabesinger, P. Boesiger, H. Wieser, Brain glutathione levels in patients with epilepsy measured by in vivo 1H-MRS, Neurol, 57 (2001) 1422-1427. [141] C.L. Florian, N.E. Preece, K.K. Bhakoo, S.R. Williams, M.D. Noble, Cell type-specific fingerprinting of meningioma and meningeal cells by proton nuclear magnetic resonance spectroscopy, Cancer Res, 55 (1995) 420-427. [142] S.L. Duffy, J. Lagopoulos, N. Cockayne, D.F. Hermens, I.B. Hickie, S.L. Naismith, Oxidative stress and depressive symptoms in older adults: A magnetic resonance spectroscopy study, J Affect Disord, 180 (2015) 29-35. [143] S.L. Duffy, J. Lagopoulos, I.B. Hickie, K. Diamond, M.B. Graeber, S.J. Lewis, S.L. Naismith, Glutathione relates to neuropsychological functioning in mild cognitive impairment, Alzheimers Demen, 10 (2014) 67-75. [144] K.A. Lapidus, V. Gabbay, X. Mao, A. Johnson, J.W. Murrough, S.J. Mathew, D.C. Shungu, In vivo 1H MRS study of potential associations between glutathione, oxidative stress and anhedonia in major depressive disorder, Neurosci Lett, 569 (2014) 74-79. [145] D.C. Shungu, N. Weiduschat, J.W. Murrough, X. Mao, S. Pillemer, J.P. Dyke, M.S. Medow, B.H. Natelson, J.M. Stewart, S.J. Mathew, Increased ventricular lactate in chronic fatigue syndrome. III. Relationships to cortical glutathione and clinical symptoms implicate oxidative stress in disorder pathophysiology, NMR Biomed, 25 (2012) 1073-1087. [146] B.R. Godlewska, S.W. Yip, J. Near, G.M. Goodwin, P.J. Cowen, Cortical glutathione levels in young people with bipolar disorder: a pilot study using magnetic resonance spectroscopy, Psychopharmacol, 231 (2014) 327-332. [147] P.K. Mandal, S. Saharan, M. Tripathi, G. Murari, Brain GlutathioneMANUSCRIPT Levels–A Novel Biomarker for Mild Cognitive Impairment and Alzheimer’s Disease, Biol Psychiatry, 78 (2015) 702-710. [148] S.J. Wood, G.E. Berger, R.M. Wellard, T.-M. Proffitt, M. McConchie, M. Berk, P.D. McGorry, C. Pantelis, Medial temporal lobe glutathione concentration in first episode psychosis: a 1 H-MRS investigation, Neurobiol Dis, 33 (2009) 354-357. [149] K. Do, A. Trabesinger, M. Kirsten ‐Krüger, C. Lauer, U. Dydak, D. Hell, F. Holsboer, P. Boesiger, M. Cuenod, Schizophrenia: glutathione deficit in cerebrospinal fluid and prefrontal cortex in vivo, Eur J Neurosci, 12 (2000) 3721-3728. [150] D. Matsuzawa, T. Obata, Y. Shirayama, H. Nonaka, Y. Kanazawa, E. Yoshitome, J. Takanashi, T. Matsuda, E. Shimizu, H. Ikehira, Negative correlation between brain glutathione level and negative symptoms in schizophrenia: a 3T 1 H-MRS study, PLoS One, 3 (2008) e1944. [151] A. Monin, P. Baumann, A. Griffa, L. Xin, R. Mekle, M. Fournier, C. Butticaz, M. Klaey, J.-H. Cabungcal, P. Steullet, Glutathione deficit impairs myelin maturation: relevance for white matter integrity in schizophrenia patients, Mol Psychiatry, 20 (2014) 827-838. [152] L. Michels, T. Schulte-Vels, M. Schick, R.L. O’Gorman, T. Zeffiro, G. Hasler, C. Mueller-Pfeiffer, Prefrontal GABA and glutathione imbalance in posttraumatic stress disorder: preliminary findings, Psychiatry Res, 224 (2014) 288-295. [153] A. Durieux, J. Horder, M.A. Mendez, A. Egerton, S.C. Williams, C.E. Wilson, D. Spain, C. Murphy, D. Robertson, G.J. Barker, Cortical and subcortical glutathione levels in adults with autism spectrum disorder, Autism Res, (2015). [154] M.A. Thomas, K.ACCEPTED Yue, N. Binesh, P. Davanzo, A. Kumar, B. Siegel, M. Frye, J. Curran, R. Lufkin, P. Martin, B. Guze, Localised two-dimensional shift correlated MR spectroscopy of human brain., Magn Reson Med, 46 (2001) 58-67. [155] M. Terpstra, M. Marjanska, P.G. Henry, I. Tkáč, R. Gruetter, Detection of an antioxidant profile in the human brain in vivo via double editing with MEGA ‐PRESS, Magn Reson Med, 56 (2006) 1192-1199.

33

ACCEPTED MANUSCRIPT

[156] U.E. Emir, E.J. Auerbach, P.F.V.D. Moortele, M. Marja ńska, K. U ğurbil, M. Terpstra, I. Tká č, G. Öz, Regional neurochemical profiles in the human brain measured by 1H MRS at 7 T using local B1 shimming, NMR Biomed, 25 (2012) 152-160. [157] I.Y. Choi, P. Lee, Doubly selective multiple quantum chemical shift imaging and T1 relaxation time measurement of glutathione (GSH) in the human brain in vivo, NMR Biomed, 26 (2013) 28-34. [158] L. An, S. Li, J.B. Murdoch, M.F. Araneta, C. Johnson, J. Shen, Detection of glutamate, glutamine, and glutathione by radiofrequency suppression and echo time optimization at 7 tesla, Magn Reson Med, 73 (2015) 451-458. [159] K.L. Chan, N.A. Puts, K. Snoussi, A.D. Harris, P.B. Barker, R.A. Edden, Echo time optimization for J ‐difference editing of glutathione at 3T, Magn Reson Med, (2016). [160] I.-Y. Choi, P. Lee, D.R. Denney, K. Spaeth, O. Nast, L. Ptomey, A.K. Roth, J.A. Lierman, D.K. Sullivan, Dairy intake is associated with brain glutathione concentration in older adults, Am J Clin Nutr, 101 (2015) 287-293. [161] N. Weiduschat, X. Mao, J. Hupf, N. Armstrong, G. Kang, D.J. Lange, H. Mitsumoto, D.C. Shungu, Motor cortex glutathione deficit in ALS measured in vivo with the J-editing technique, Neurosci Lett, 570 (2014) 102-107.

MANUSCRIPT

ACCEPTED

34

ACCEPTED MANUSCRIPT

TABLES

Table 1. Outcomes and commentary of studies measuring GSH in human brain.

Disease Subjects Method / voxel Anatomy Outcomes and Comments Reference A: HV 12 HV, 1.5T, PRESS localized DQF, TR/TE 2000/75, Midsagittal in First paper to report GSH measurement. GSH 2-5 mM in HV. [98] (Healthy phantoms 128 aves, ~4.5 min, 2.4x2.2x3.3 cm prefrontal cortex Successful detection of GSH in phantoms and HV . volunteer) A: HV 15 HV, 1.5T, Volume localized COSY, 3x3x3 cm, 11, 30 Various locations Only two HV spectra reported. Detection of GSH only mentioned [154] phantoms or 35 min acquisition in the abstract. No spectra shown with GSH identified A: HV 9 HV, 3 4T MEGA-PRESS TR/TE 4500/68, 256-512 Occipital lobe GSH ~ 1.3 mM [107] repeated, aves, 20-40 min. 2x2x2 cm. phantoms. A: HV Phantoms, 4 HV 3.0 T MEGA-PRESS, TR/TE 2000/80, 256-512 Parietal lobe Phantoms confirm GSH MEGA-PRESS signal and that GSSG [106] aves, 9-18 min, 3x3x3 cm would be undetectable at concentrations expected in vivo. GSH in HV ~ 2 mM A: HV 4 HV 4T double editing for ascorbate and GSH, based Midsagittal in occipital GSH and ascorbate both detected at ~0.8 and ~1 mM [155] on MEGA-PRESS. 3x3x3 cm. TR/TE 4500/112, lobe respectively with S.D. of 0.1 512 aves (~40 min) A: HV 5 HV, phantoms 3T volume-selective DQF, not requiring phase Left and right parietal GSH ~0.9 mM, sem or sd ? ~ 0.1 mM. Sequence shown to be [102] calibration scan. 3x3x3 cm TR/TE/TM lobe invariant to phase difference between excitation and DQF 2000/70/16, 256 aves, 8.3 min generating pulse. Don’t think use of the sequence has been reported again. A: HV Simulation, 7T. Asymmetric PRESS (TE1/TE2: 37/63), Medial prefrontal Optimization of the TE delays in asymmetric PRESS enables [115] phantoms and 6 STEAM (TE/TM: 14/37; 74/68). TR=2500. 64 cortex separation of Glu, Gln, GSH at 3T without editing. HV aves. 2.5x3x3cm. MANUSCRIPT A: HV Simulation, 4T and 9.4T phantoms. HV at 4T. MEGA- Occipital lobe. The commonly used spectroscopic parameters for the GSH [92] phantoms and 3 PRESS TR/TE 3500/68, 512 aves ~30 min; glycine shown to be wrong. It behaves as an uncoupled doublet, HV ‘PRESS +4’ for GSH glycine editing TR/TE with solvent exchange inhibiting J-modulation but not the 3500/72, 256 aves ~15 min. 3x3x3 cm splitting. Editing the GSH glycine is shown to be effective, but it is compared to non-optimal cysteinyl editing at TE68 . A: HV 14 HV 7T. Optimisation of short echo STEAM (TR/TE 7 SV regions: incl 9 or more metabolites (including GSH) quantified in all brain [156] 5000/8) with 16 channel transmit/receive arrays frontal white matter, regions. No discussion of GSH, its reproducibility or variation and local B1 shimming. Single voxels from limbic system, across the brain 0.6x0.6x1.3 to 2x2x2 cm, with 128-512 aves posterior cingulate, occipital cortex. A: HV Phantoms, 13 3T. CSI with DQF. T1 measurement by single Slice through fronto- GSH=1.2 mM in fronto-parietal region. GSH T1~400ms [157] HV voxel DQF. 3cm slice, 8x8 matrix 20 cm FOV. parietal regions 8-16 aves, up to 19min scan time. Absolute quantification A: HV 10 HV scanned 3T Phase-rotation STEAM TR/TE/TM Anterior and posterior Good reproducibility for GSH ~2.5 ‘Institutional Units’ cf [130] twice, phantoms 2000/6.5/10. 6 ml voxels, 256 aves,ACCEPTED 8.5 min cingulate NAA~11 IU. Phantom shows excellent linear correlation to GSH, but still the zero GSH sample is ≈1 I.U. Data are fitted to 19 metabolite basis sets, with baseline handling with undefined

35

ACCEPTED MANUSCRIPT

extra parameters. Still difficult to have confidence in low metabolite quantification. A: HV 8 HV 7T, J-selective RF suppression, PRESS Medial prefrontal Glu, Gln and GSH separated. Glu, Gln higher in GM. GSH [158] [114] assymetric optimized TE. Quantified by ratios to cortex GM, frontal higher in WM. GSH and Gln similar concentration (20-27% of creatine cortex WM Cr) A: HV Simulations, 3T MEGA-PRESS, TE 68, 120. 3.6x3.6x3.6 cm. Midline parietal cortex Consistent with An et al (2009) in showing TE120 is optimal in [159] phantoms, 5 HV Frequency modulated pulses compared to vivo. Improvements with better slice-selective pulses with amplitude modulated narrower BW at longer TE A:HV Simulations, 3T MEGA-PRESS TR/TE 2000/130 and PRESS Anterior cingulate PRESS not reliable in measuring GSH in phantoms below 4 [131] phantoms, 7 HV TR/TE 2000/35. ACC 4x2.5x2.5 cm; OC 3x3x3 cortex (ACC) and mM. Good linear correlation for MEGA-PRESS in phnatoms cm. 256 aves (8.5 min). Data analysed using occipital cortex (OC) from 0-24 mM. Significant difference between ACC GSH jMRUI between PRESS and MEGA-PRESS A: HV: ageing 22 young 4T double editing for ascorbate and GSH, based Midsagittal in occipital GSH lower, lactate higher in elderly. No effect for ascorbate. [138] (~20yo), 22 on MEGA-PRESS. 3x3x3 cm. TR/TE 4500/122, lobe. GSH concentration is low ~0.25 mM in institutional units (not elderly (~77yo) 512 aves (~40 min) corrected for T2 relative to water). HV. Two repeat measurements on 10 subjects. A: HV: 21 infants on 28 1.5T. PRESS TR/TE 3000/20, 128 aves, ~6 min. White matter – Good data on maturational changes in major metabolites. GSH [99] paediatrics occasions, Voxels vols from 2.1-4.3 ml, average 2.9. LC centrum semiovale; data presented (2-3.4 mM, average ~ 2.5) but not discussed with gestational age Model Grey matter – no significant effects found. Voxel size and acquisition time 32-43 wk. thalamus, occipital. rather short for reliable detection of GSH A: HV: visual 10 HV. Visual 7T STEAM TR/TE/TM 3000/15/17, 32 aves per Occipital cortex, based Lactate, Glu and GSH all increased during stimulation. Resting [133] stimulation stimulation block (99s), 40 min functional paradigm. 2x2x2 on BOLD signal from GSH 2.3 mM, increases by ~7% cm. LC Model, with rejection of high CRLB visual stimulation MANUSCRIPT measurements A: HV: visual 12 HV Visual 7T s-LASER TR/TE 5000/26, 32 aves per block, Occipital cortex, based Lactate and Glu increased, Asp and glucose decreased. No [132] stimulation Stimulation 2x2x2 cm. LC Model with rejection of high on BOLD signal from change in GSH CRLB measurements visual stimulation A: HV: 12 HV, fasted, 4T double editing for ascorbate and GSH, based Occipital lobe Neither ascorbate (~0.68 mM) nor GSH (~0.8 mM) varied after [134] vitamin C scanned then on MEGA-PRESS. 3x3x3 cm, with averaging of the infusion or between vit C and sham (2 subjects). Blood level challenge receive multiple TE values. TR/TE 4500/102-152, 6x96 of asc increase ~10-fold at 2h intravenous vit aves (~40 min) C, with repeat scans at 2, 6, 10 and 24h A: HV: dairy 60 older HV, 3T. CSI with DQF. T1 measurement by single Slice through fronto- GSH positively correlated with dairy (milk and cheese) intake in [136, 160] intake and ~68 yo, on their voxel DQF. 3cm slice, 8x8 matrix 20 cm FOV. parietal regions frontal, parietal and fronto-parietal regions. Other factors also brain GSH usual diet. 8-16 aves, up to 19min scan time. Absolute influenced GSH (age, sex, lean mass, other dietary factors) , but quantification dairy intake remained significant after partial correlation B: Neurology: 6 meningioma, 6 1.5T STEAM TR/TE 2020/30, 1.5x1.5x1.5ACCEPTED – Within tumour or in Fit to data stated to be better if GSH included in model. GSH [100]

36

ACCEPTED MANUSCRIPT

cancer astrocytoma and 2x2x2 cm. Fit with LC Model parietal WM of HV elevated in meningiomas (~3.3 mM) compared to HV (~1.2 6 HV. Phantoms mM). Voxels rather small and at low field, so SNR low. Meningiomas very low in creatine and there is an arguable but

small signal adjacent to creatine where GSH cys βCH2 is expected B: Neurology: 19 epilepsy 1.5T PRESS-DQF, TR/TE , TR/TE 2000/75, 128 Bilateral parieto- GSH/water lower in both hemispheres of epilepsy patients. No [140] epilepsy patients, 12/19 aves occipital regions, hemispheric difference and no effect of active disease with active structurally normal disease, 8 HV B: Neurology: 3 Parkinsons, 3 7T Semi-LASER TR/TE 5000/26, 2.2x2.2x2.2 Occipital cortex N-acetylcysteine infusion increased blood GSH/GSSG ratio and [135] Gaucher / Gaucher, 3 HV. cm, brain GSH in all subjects. Parkinsons Effect of N- acetylcysteine B: Neurology: 11 motor 3T J-edited PRESS TR/TE 1500/68, 2x2.5x2.5 Pre-central gyrus GSH lower by 31% or 36% relative to water or creatine [161] motor neurone neurone disease cm, 15 min. Effectively MEGA-PRESS (motor cortex) respectively in patients. NAA/creatine ratio decreased slightly. disease (ALS) (ALS) patients, GSH 11 matched HV B: Neurology: 7 multiple 7T 2D-PRESS-BASING Slice above ventricles GSH higher in GM (3.3 mM) than WM in HV (2.1 mM). GSH [113] multiple sclerosis TR/TE 2000/130, 1.2 cm in-plane, 2 cm slice, 11 in centre of brain in GM significantly lower in patients relative to HV. Good sclerosis patients, 6 HV aves, 17 min (20x20 cm) reproducibility, very convincing GSH detection. Selective pulse @ 4.90 ppm B: Neurology: 17 MS patients, 3T. CSI with DQF to select GSH. 3cm slice, 8x8 Fronto-parietal regions Lower GSH in patients, by up to 18.5% in frontal regions [101] multiple 17HV encoding over 20 cm, 12 aves, 19min scan time sclerosis B: Neurology: 5 ischaemic 3T J-difference, effectively MEGA-PRESS. Left or right parietalMANUSCRIPT Optimal TE for this method = 130 ms. [122] stroke stroke patients. TR/TE 2000/131, 256 aves, 8.5 min. 5x3x3 cm lobe GSH in HV ~1.20mM. Stroke patients not significantly different 11 HV, much and not different ipsilateral vs contralateral younger than the stroke group (30 ± 11 y cf 57 ± 15 y)

C: Psychiatry: 21 autistic 3T PRESS TR/TE 3000/30. 2x2x1.5 cm. BG: Basal ganglia (BG) GSH 2.8-3 mM in both regions, no effect of disease. These are [153] autism spectrum ganglia (128 aves); DMPFC: 1.6x2.4x2 (96 aves) and dorsomedial rather small volumes for measuring low concentration disorder, 29 HV prefrontal cortex metabolites (DMPFC) C: Psychiatry: 13 bipolar 3T, SPECIAL TR/TE 3200/8.5. 1.5x3x1.5 cm Prefrontal and No significant differences in GSH expressed relative to creatine [146] bipolar disease patients, 11 age- 128 aves prefrontal, 6 min; 2x2.5x2 cm 256 aves occipital cortex (ratio ~ 0.2). No evidence of GSH peak in spectra. matched HV occipital, 12 min. Ratios to creatine. CRLB threshold used to reject data. C: Psychiatry: 33 bipolar, 17 3T PRESS TR/TE 2000/35. 2x2x2cmACCEPTED 128 av. Anterior cingulate GSH lower in bipolar disease with risky alcohol use. Increased [125]

37

ACCEPTED MANUSCRIPT

bipolar HV, 19-30 yo. LCModel. CRLB threshold used to reject data cortex oxidative stress associated with high alcohol use in patients. disease/alcohol Risky alcohol use measured. Phantoms to test GSH quantification C: Psychiatry: Expansion of 3T PRESS TR/TE 2000/35. 128 av. LCModel. Anterior cingulate Reduced GSH in patients associated with high alcohol use in [129] bipolar 2014 paper. 64 CRLB threshold used to reject data cortex (2x2x2cm); both ACC and Hc, but ACC effect abolished when controlled for disease/alcohol bipolar, 49 HV. hippocampus (1.5x3x1 tobacco use. No effect of alcohol in controls. Phantom data Phantom tests of cm) shows linear relationship of LCModel fit with GSH GSH concentration, (but proportionality constant of 2.4, not 1), but quantification. LC model returns ~1.5 mM when there is no GSH in the Phantom phantom . supposedly contains creatine, but spectra don’t show any creatine! C: Psychiatry: 11 major 3T J-edited PRESS TR/TE 1500/68, 3x3x2 cm. Occipital cortex Anhedonia and GSH negatively correlated. No data shown to [144] depression depressive Effectively MEGA-PRESS. compare major depressive disorder and HV disorder, 10 matched HV C: Psychiatry: 58 at-risk-of- 3T PRESS TR/TE 2000/35, 2x2x2 cm 128 aves, Anterior cingulate Increased GSH/creatine in at risk group compared to controls [142] depression depression older 4.2 min. LC Model fit, ratios to creatine cortex MANUSCRIPTand in patient group correlation with depressive symptoms and adults 66-80 yo). poor verbal learning test performance Depression and anxiety ratings. 12 HV C: Psychiatry: 15 chronic 3T J-edited PRESS TR/TE 1500/68, 3x3x2 cm, Occipital cortex Decreased GSH in both patient groups cf HV [145] depression and fatigue, 15 major 15 min. Effectively MEGA-PRESS. chronic fatigue depressive disorder, 13 HV C: Psychiatry: 54 MCI, 41 HV. 3T PRESS TR/TE 2000/35, 2x2x2 cm 128 aves, Anterior and posterior Increase GSH/creatine in MCI in both regions. Poorer executive [143] mild cognitive Neuropsycholog 4.2 min. LC Model fit, ratios to creatine cingulate cortex function correlated with anterior and poorer memory impairment y and clinical consolidation with posterior GSH/creatine examinations. C: Psychiatry: 25 HV male 3T MEGA-PRESS L& R frontal cortex Statistically higher GSH in LFC, L&R PC in female HC than [137] mild cognitive (26.4 ± 3.0 y) TE 120 ms (FC), parietal cortex male HC. Higher GSH in young, gender matched HC vs older impairment and 19 HV VOI 2.5 cm 3 hippocampus (HC) MCI or AD subjects. ‘Arbitrary units’ used (not clear how (MCI) and female (23.6 Selective pulse @ 4.40 ppm ACCEPTEDand cerebellum quantitation was calculated) Alzheimer’s ±2.1 y)

38

ACCEPTED MANUSCRIPT

disease (AD) AD N = 1F, 5M MCI N = 3F,5M C: Psychiatry: HV: 21 age- 3T MEGA-PRESS TR/TE 2500/120. VOI 15.6 L& R frontal cortex, Significant reductions in GSH in both FC and HC in disease [147] mild cognitive matched to 21 cm3. 320 avs per voxel = ~13 min L&R hippocampus groups. (HV ~1 mM, AD and MCI 0.5-0.7 mM in HC: values impairment AD, 22 MCI about 20% higher in FC). Ratios to Cr and Cho also decreased, and showing that atrophy is not the cause of reduced GSH. Important Alzheimer’s study as it also shows reduced GSH is a predictive biomarker for disease identification of AD and MCI C: Psychiatry: 12 post- 3T MEGA-PRESS optimized for GABA. TR/TE Dorsolateral prefrontal Higher GABA and GSH in patients. Measurement of GSH in the [152] post-traumatic traumatic stress 1800/68. Glu and GSH quantified from MEGA- cortex (DLPFC). subspectrum really lacks credibility, especially in DLPFC for stress disorder disorder patients, off subspectra by LC Model. 2.5x4x3 cm, 320 Anterior cingulate which only 5 min data are available. 17HV aves, ~10 min (DLPFC); 2.8x3x2.25 cm, 640 cortex (ACC) aves, ~ 20 min (ACC) C: Psychiatry: Phantoms, 14 1.5T DQF with PRESS. 2.4x2.2x3.3 cm, TR/TE Medial prefrontal In CSF, drug-free schizophrenia patients GSH decreased by [149] schizophrenia schizophrenia 2000/75, 128 ave cortex 27%. GSH in brain decreased by 52% in a group with mixed patients, 14 HV. drug-status. CSF samples done on 26 patients and 14 HV C: Psychiatry: 3 HV scanned 3 4T MEGA-PRESS TR/TE 4500/68, 512 aves, 40 Anterior cingulate GSH from MEGA-PRESS and STEAM not significantly [128] schizophrenia times for min, 17 ml volume and STEAM TR/TE/TM cortex different (~1.5 mM). No difference between patients and HV (MEGA-PRESS 4500/5/42, 256 aves, 20min, 2x2x2 cm and 17 ml from STEAM. LC Model not completely convincing. This paper and STEAM); 9 volume (dimensions not specified) . LC Model is used to justify use of PRESS TE30 by other authors, but HV and 13 quantification MANUSCRIPTSTEAM spectra have excellent SNR and are long acquisitions, so schizophrenic the assumption that s 4-8 min, longer echo PRESS at 3T can also patients measure GSH is not necessarily correct. (STEAM). C: Psychiatry: 20 schizophrenia 3T MEGA-PRESS TR/TE 1500/94. 2.8x3x2.2 Posterior medial HV and patients not different, but GSH correlated negatively [150] schizophrenia patients, 16 cm, 512 aves, 12.8 min. prefrontal cortex with severity of negative symptoms. Phantom data show MEGA- matched HV. PRESS detects low GSH correctly. Psychiatric ratings and cognitive assessments. Phantom C: Psychiatry: 30 early 3T SPECIAL, TR/TE 4000/6, 148 aves Medial bilateral Relationships between generalized fractional anisotropy and [151] schizophrenia psychosis prefrontal lobe GSH levels (early) schizophrenia. 40 HV) C: Psychiatry: 30 first episode 3T PRESS TR/TE 3000/30,2x2x2ACCEPTED cm voxel, 128 Bilateral medial GSH significantly higher by 22% in first episode patients, no [148] schizophrenia schizophrenia aves, 6.4 min temporal lobe effect of hemispheres. Good SNR, but still short acquisition on

39

ACCEPTED MANUSCRIPT

(first episode) patients, 18 HV small volume for quantification of low concentration metabolites. C: Psychiatry: 88 young people 3T PRESS TR/TE 2000/35. 2x2x2cm 128 aves, Anterior cingulate Clustering revealed 3 groups differentiated on 4 metabolites: [124] schizophrenia with major mood 4.2 min. LCModel, relative to creatine. CRLB cortex NAA, mI, Glu, GSH. GSH appeared the most important. There (mood or psychotic threshold used to reject data were no clinical or demographic features to distinguish these disorder, symptoms. clusters. The spectra are excellent, but it is suspicious that ratios psychosis) Cluster analysis to creatine throw up GSH as significant, since the two peaks are to correlate indistinguishable in the spectra. The phantom data shows that biochemistry zero GSH is returned by LCModel as nearly 2mM. with symptoms

Table 1 Legend Comments by the review authors are given in italics, after a summary of the conclusions in normal text. Aves – number of acquisitions; CRLB – Cramer-Rao lower bound;

CSF – cerebrospinal fluid; CSI – chemical shift imaging; HV – normal healthy human volunteer; GM – grey matter; GSH – reduced glutathione; WM – white matter;

MEGA-PRESS, PRESS, STEAM, DQF, BASING, – spectroscopic acquisition techniques, refer to text for explanation

LCModel – procedure used to fit spectra for quantification

NAA, GABA, Gln, Glu, mI – metabolites detected by MRS: N-acetylaspartate; γ-amino butyric acid; glutamine; glutamate; myo -inositol Concentrations are expressed as mM – the cited papers use a mixture of mmol/kg wet weight MANUSCRIPT and mmol/L in vivo , as well as ‘Institutional Units’, which approximate to mM in most circumstances

ACCEPTED

40

ACCEPTED MANUSCRIPT

Table 2. Chemical shifts and coupling constants for GSH

Compound Group Shift in water Multiplicity J(Hz) Connectivity 10 Glycine moiety CH 2 3.779 s 3.758 s 9NH 7.154 t Cysteine moiety 7CH 4.560 dd 7.09 7-7’ 7’ ’’ CH 2 2.926 dd 4.71 7-7 ’ ’’ 2.974 dd -14.06 6.34 7 -7 6NH 8.177 d Glutamate moiety 2CH 3.769 t 6.34 2-3 3 ’ CH 2 2.159 m 6.36 2-3 2.146 m -15.48 3-3’ 4 CH 2 2.510 m 6.7 3-4 2.560 m 7.6 3-4’ 7.6 3’-4 6.7 3’-4’ -15.92 4-4’

Table 2 Legend

The data in the table are adapted from Kaiser et al. (2010) and Govindaraju et al . (2000). Kaiser et al. showed that the α-CH 2 resonance is best modelled as two singlets, separated by 3.8Hz. OtherwiseMANUSCRIPT the data follow the information presented by Govindaraju et al.

ACCEPTED

41

ACCEPTED MANUSCRIPT

Figure Legends

Figure 1 Brain synthesis, compartmentation and metabolism of glutathione.

Glutathione is synthesised from glycine, glutamate and cysteine within each type of brain cell in a two-step pathway, both steps of which require ATP. GSH is exported from astrocytes and is catabolised by the ectozyme γ-glutamyl transferase with the γ-glutamyl group transferred to a receiving thiol and generating Cys-Gly. The resulting amino acids, further catabolised by aminopeptidases, can then be taken up by other cell types, such as neurons and oligodendrocytes. Oligodendrocytes and astrocytes can take up cystine via the cystine/glutamate exchanger to supply cysteine for GSH synthesis, or in the case of astrocytes, synthesise it via the transsulfuration pathway, but neurons are reliant on exogenous cysteine. Most cysteine (80%) goes into neurons via the excitatory amino acid carrier 3 (EAAC3) with only a small fraction supplied via ASC or system L.

Glutathione can also leave cells via the multidrug resistance pump, mostly in the form of GSSG or conjugated glutathione.

GSH is converted to GSSH via redox reactions and is restored to GSH by GSSG reductase; this reaction needs the cofactor

NADPH which is generated via a number of dehydrogenase reactions using NADP +/NADPH as cofactor; the most important of which are in the pentose phosphate pathway. Figure 2 Production and catabolism of methylglyoxal by theMANUSCRIPT glutathione dependent glyoxalase enzyme system .

Methylglyoxal is produced by spontaneous breakdown of glycolysis intermediates DHAP (dihydroxyacetone phosphate) and

Gra-3P (glyceraldehyde-3-phosphate). This reaction is accelerated by triosephosphate isomerase. Methylglyoxal may combine with DHAP in a reaction catalysed by aldolase to form a diketone phosphate, with aldolase being inactivated by this reaction. Methylglyoxal is inactivated by combining with glutathione with subsequent conversion to D-lactate by the highly active glyoxalase enzyme system. Glutathione is regenerated by this pathway.

Figure 3 A 1H magnetic resonance spectrum from the brain in vivo and a spectrum of GSH at 3 Tesla.

A spectrum of glutathioneACCEPTED was simulated using NMRSCOPE B in jMRUI and is shown below a human brain spectrum acquired using the same PRESS localization sequence at 3T that was used for the simulation (TE/TR 35/2000 ms, 2x2x2 cm 3 voxel, 6.4 min) . It is not possible to resolve the peaks of GSH (labelled as cysteine: Cys; glycine: Gly; and glutamate, Glu)

42

ACCEPTED MANUSCRIPT

as they are co-resonant with peaks from glutamate and glutamine (Glu, Gln, Glx), N-acetylaspartate (NAA), GABA, creatine/phosphocreatine (Crn) and aspartate as well as macromolecules.

Figure 4 PRESS and MEGA-PRESS sequence

A sequence diagram is shown for the PRESS localization technique (upper rows) and the adaptation for MEGA-PRESS

(bottom row). A combination of one 90 o and two 180 o frequency-selective pulses with orthogonal slice selection gradients results in signal only being acquired from the volume at the intersection of the three slices. Additional crusher gradients and water suppression (not shown) are incorporated into the sequence to remove artefacts from outer volume signals and to reduce the amplitude of the water resonance respectively. As explained in Section 2.1.2.1 the addition of frequency selective

180 o pulses either side of the second slice-selective 180 o pulse enables J-editing. The sequence timings are not to scale and in practical implementations of MEGA-PRESS the extra gradients are combined with PRESS crusher gradients.

Figure 5 Edited GSH spectrum MANUSCRIPT An example of MEGA-PRESS editing to detect GSH. A MEGA-PRESS spectrum was acquired from the anterior cingulate cortex of a volunteer at 3T (4x2.5x2.5 cm 3 voxel; TR/TE 2000/130 ms; 64 blocks of 4 averages per block, interleaving

MEGA pulses centred at 4.56 ppm [ON] and 1.44 ppm [OFF]). The top two traces show the two MEGA sub-spectra, with the lower one inverted because of the way the acquisition is handled on the scanner. Resonances from N-acetylaspartate

(NAA), creatine plus phosphocreatine (Cr) and choline-containing compounds (Cho) are identified. The addition of the sub- spectra results in an edited spectrum in which the resonance of GSH cysteinyl at 2.95 ppm can be detected along with the co- edited aspartyl resonances of NAA. The data were acquired on a Philips Achieva 3T scanner at Salford Royal Hospitals’

Trust, Salford, UK. ACCEPTED

43