**4. Gabazine, a competitive antagonist of GABAA receptors**

action of glycine on spinal neurones [15]. Curtis et al. [16] reported antagonist action against both glycine and GABA, but the results were inconsistent due to the lack of ionization using

These technical difficulties were eventually overcome using recombinant receptors with bath application of picrotoxin showing it to be a mixed/noncompetitive GABAA receptor antagonist [17]. But picrotoxin was also shown to be an antagonist of other CYS-loop receptors, including

as an experimental tool is mitigated by its lack of selectivity for GABAA receptors. It also has no

Many terpenoids related to picrotoxinin are convulsants acting on ionotropic receptors for GABA and glycine [19]. Of particular interest is tutin that occurs in the berries and flowers of the indigenous New Zealand tutu plant, *Coriaria arborea*, and has been credited with convulsing a circus elephant that consumed the berries [20]. Toxic honey was also produced from

The discovery of bicuculline as a selective antagonist of what became known as GABAA receptors arose out of a systematic study of convulsant alkaloids [3]. It was well known that the most widely understood convulsant alkaloid, strychnine, antagonized the inhibitory action of glycine without influencing the inhibitory action of GABA [21]. Indeed, most convulsant alkaloids turned out to be glycine receptor antagonists with the important exception of bicu-

While bicuculline is selective for GABAA receptors, having little effect on GABAB, GABAC,

[17, 23]. Selectivity for GABAA receptors makes bicuculline a powerful experimental tool but without any therapeutic potential owing to the nonselective nature of binding to all GABA<sup>A</sup> receptor subtypes, causing profound convulsive effects. Bicuculline binds at the orthosteric site to stabilize the receptor in a closed state. It is three times the size of GABA and thus is able to bind to sites on the receptor that GABA cannot reach [3]. Bicuculline acts as a competitive antagonist in which it competitively inhibits GABA agonist binding to GABAA receptors, and GABA competitively inhibits bicuculline binding [24]. Single channel studies show that by competing with GABA for its binding site, bicuculline acts to reduce both chloride channel

At physiological pH, bicuculline is slowly converted to bicucine, a much less active convulsant [26]. This transformation is slowly reversed at acidic pH. Thus, bicuculline solutions should always be freshly prepared in order to preserve maximum convulsant potency. Quaternary salts of bicuculline, such as bicuculline methiodide ("N-methyl bicuculline") or methochloride, are much more stable than bicuculline, are more water soluble, and are of similar potency as GABA receptor antagonists, but they do not cross the blood-brain barrier

receptors, its action is largely independent of GABAA subunit composition

**3. Bicuculline, a competitive antagonist of GABAA receptors**

receptors [18]. Thus, the utility of picrotoxin (and also picrotoxinin)

the microiontophoresis method of drug administration.

94 GABA And Glutamate - New Developments In Neurotransmission Research

therapeutic potential owing to its potent convulsant effects.

culline (**Figure 1**), an alkaloid from *Dicentra cucullaria* [22].

bees that collected nectar from the flowers.

glycine, GABAC, and 5-HT<sup>3</sup>

glycine, and 5-HT<sup>3</sup>

open time and opening frequency [25].

Gabazine (also known as SR 95531, **Figure 1**) resulted from a study of arylaminopyridazine analogs of GABA. It was found to be a relatively specific, potent, and competitive antagonist of GABAA receptors [34]. Although both are functionally competitive inhibitors, gabazine and bicuculline also interact with other residues on GABAA receptors [35, 36]. Neither gabazine nor bicuculline compete for the binding at the barbiturate or neurosteroid binding sites on GABAA receptors. It is suggested that both antagonists act "as allosteric inhibitors of channel opening for the GABAA receptor after binding to the GABA-binding site" [36]. Gabazine has little activity at GABAC receptors [37]. At binary β3δ recombinant GABAA receptors, gabazine antagonized GABA currents, whereas bicuculline activated these receptors [38]. Thus, while functioning as competitive antagonists for GABAA receptors, gabazine and bicuculline clearly interact with different residues on GABAA receptors.

Structural analogs of gabazine have identified more potent agents [39]. Gabazine analogs incorporating photoactive groups, such as GZ-B1 (**Figure 1**), have been developed as photoactivated antagonists of GABAA receptors [40]. These antagonists provide dynamic tools for visualizing GABAA receptors, permitting a novel means of investigating receptor location, function, and trafficking [40].

## **5. TPMPA and related compounds, competitive antagonists of GABAC receptors**

GABAC receptors, also known as GABA-ρ and GABAA-ρ receptors, have distinctive distribution and pharmacological properties to GABAA receptors, making them particularly interesting [41]. They are CYS-loop ligand-gated ion channels with a similar pentameric structure to GABA<sup>A</sup> receptors but are not so widely distributed. They are homomeric rather than heteromeric and therefore much simpler receptors. These properties make them important drug targets [42].

form-deprivation myopia [51], while (3-aminopropyl)-n-butylphosphinic acid (CGP36742 or SGS742) was found to inhibit the development of myopia in chicks [50]. Thus, TPMPA and related GABAC receptor antagonists have been suggested for the treatment of myopia, administered intravitreally, orally, and ophthalmically [52]. Indeed, GABAC receptor antagonists

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GABAC receptor antagonists have also been useful experimental tools for demonstrating a role for these receptors in learning and memory [49, 50, 54]. TPMPA and the structural analog P4MPA ((piperidin-4-yl)methylphosphinic acid) were shown to enhance short-term memory in a bead discrimination task following injection into the multimodal association forebrain area in chicks [49]. Injection of bicuculline caused the opposite effect. TPMPA has also been used to demonstrate a role for GABAC receptors in fear learning and memory in rats [54]. Rats administered TPMPA via bilateral cannulae injections into the lateral amygdala showed reduced freezing in a foot shock conditioned fear task. This reduction in fear learning and memory is likely mediated by presynaptically located GABAC receptors in the

Arising out of studies on the orally active GABA(B/C) receptor antagonist (3-aminopropyl) n-butylphosphinic acid (CGP36742 or SGS742) [55], *cis*- and *trans*-(3-aminocyclopentanyl) butylphosphinic acid were found to be selective potent GABAC receptor antagonists that

Based on the structure of the selective GABAC receptor antagonist (*S*)-4-ACPBPA [(4-aminocyclopenten-1-yl)-butylphosphinic acid], a series of fluorescent ligands were produced linking fluorophores to the parent compound [56]. One of these fluorescent ligands, (*S*)-4-ACPBPA-C5-BODIPY, showed moderately potent antagonism for GABAC receptors with greater than 100 times selectivity for these receptors over GABAA receptors. (*S*)-4-ACPBPA-C5-BODIPY thus provides a valuable molecular probe for the role of GABAC receptors in physiological

Bilobalide (**Figure 2**) and a series of terpenoids known as ginkgolides isolated from *Ginkgo biloba* are structurally related to picrotoxinin and are relatively potent GABAA and GABA<sup>C</sup>

Unlike picrotoxin, bilobalide is an anticonvulsant. This may be due to its potent action on GABAC receptors [57]. Bilobalide also has differing effects to those of picrotoxin on the modulation of GABAA receptors by structurally different modulators [60], suggesting a different

Thus, there are GABAA receptor antagonists that act as channel blockers and negative modulators that do not produce convulsions in vivo. Explanation of this apparent paradox includes selective actions on GABAA receptor subtypes, reduction of glutamate release from presynaptic terminals via presynaptic receptors and effects on GABA metabolism, together with

receptors [58, 59].

enhanced learning and memory in rats in the Morris water maze task [50].

**6. Bilobalide, a nonconvulsant channel blocker**

receptor antagonists [57], but they also act on glycine and 5-HT<sup>3</sup>

binding profile to picrotoxin to negatively modulate the GABAA receptors.

have been patented for the treatment of myopia [52].

lateral amygdala [54].

and pathological processes [56].

actions on non-GABAergic systems.

TPMPA ((1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid, **Figure 2**) was the first selective GABAC receptor antagonist to be synthesized [43, 44]. Other GABAC receptor antagonists include the bicyclic GABA analog, THIP (Gaboxadol, 4,5,6,7-**t**etra**h**ydro**i**soxazolo(5,4-c)**p**yridin-3-ol, **Figure 2**), which is a moderately potent antagonist at the GABAC receptors, yet a potent agonist at GABAA receptor receptors [45]. Aza-THIP (1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridin-3-ol) is inactive at GABAA receptors but shows moderately potent antagonism at GABA<sup>C</sup> receptors. Phosphinic, phosphonic, and seleninic analogs of isonipecotic acid have also been shown to act as selective GABAC receptor antagonists [46], as have amide and hydroxamate analogs of 4-aminocyclopent-1-enecarboxylic acid [47].

Unlike many GABAA receptor antagonists, TPMPA is not a convulsant, consistent with many instances that GABAA and GABAC receptors have been shown to mediate opposing functions, for example, on excitability [48] and in memory formation [49].

TPMPA and other GABAC receptor antagonists have been used to demonstrate the important role of GABAC receptors in many aspects of vision [50–53]. TPMPA was shown to inhibit

**Figure 2.** GABAA receptor antagonists that are not convulsants.

form-deprivation myopia [51], while (3-aminopropyl)-n-butylphosphinic acid (CGP36742 or SGS742) was found to inhibit the development of myopia in chicks [50]. Thus, TPMPA and related GABAC receptor antagonists have been suggested for the treatment of myopia, administered intravitreally, orally, and ophthalmically [52]. Indeed, GABAC receptor antagonists have been patented for the treatment of myopia [52].

They are CYS-loop ligand-gated ion channels with a similar pentameric structure to GABA<sup>A</sup> receptors but are not so widely distributed. They are homomeric rather than heteromeric and therefore much simpler receptors. These properties make them important drug targets [42].

TPMPA ((1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid, **Figure 2**) was the first selective GABAC receptor antagonist to be synthesized [43, 44]. Other GABAC receptor antagonists include the bicyclic GABA analog, THIP (Gaboxadol, 4,5,6,7-**t**etra**h**ydro**i**soxazolo(5,4-c)**p**yridin-3-ol, **Figure 2**), which is a moderately potent antagonist at the GABAC receptors, yet a potent agonist at GABAA receptor receptors [45]. Aza-THIP (1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridin-3-ol) is inactive at GABAA receptors but shows moderately potent antagonism at GABA<sup>C</sup> receptors. Phosphinic, phosphonic, and seleninic analogs of isonipecotic acid have also been shown to act as selective GABAC receptor antagonists [46], as have amide and hydroxamate

Unlike many GABAA receptor antagonists, TPMPA is not a convulsant, consistent with many instances that GABAA and GABAC receptors have been shown to mediate opposing functions,

TPMPA and other GABAC receptor antagonists have been used to demonstrate the important role of GABAC receptors in many aspects of vision [50–53]. TPMPA was shown to inhibit

analogs of 4-aminocyclopent-1-enecarboxylic acid [47].

96 GABA And Glutamate - New Developments In Neurotransmission Research

**Figure 2.** GABAA receptor antagonists that are not convulsants.

for example, on excitability [48] and in memory formation [49].

GABAC receptor antagonists have also been useful experimental tools for demonstrating a role for these receptors in learning and memory [49, 50, 54]. TPMPA and the structural analog P4MPA ((piperidin-4-yl)methylphosphinic acid) were shown to enhance short-term memory in a bead discrimination task following injection into the multimodal association forebrain area in chicks [49]. Injection of bicuculline caused the opposite effect. TPMPA has also been used to demonstrate a role for GABAC receptors in fear learning and memory in rats [54]. Rats administered TPMPA via bilateral cannulae injections into the lateral amygdala showed reduced freezing in a foot shock conditioned fear task. This reduction in fear learning and memory is likely mediated by presynaptically located GABAC receptors in the lateral amygdala [54].

Arising out of studies on the orally active GABA(B/C) receptor antagonist (3-aminopropyl) n-butylphosphinic acid (CGP36742 or SGS742) [55], *cis*- and *trans*-(3-aminocyclopentanyl) butylphosphinic acid were found to be selective potent GABAC receptor antagonists that enhanced learning and memory in rats in the Morris water maze task [50].

Based on the structure of the selective GABAC receptor antagonist (*S*)-4-ACPBPA [(4-aminocyclopenten-1-yl)-butylphosphinic acid], a series of fluorescent ligands were produced linking fluorophores to the parent compound [56]. One of these fluorescent ligands, (*S*)-4-ACPBPA-C5-BODIPY, showed moderately potent antagonism for GABAC receptors with greater than 100 times selectivity for these receptors over GABAA receptors. (*S*)-4-ACPBPA-C5-BODIPY thus provides a valuable molecular probe for the role of GABAC receptors in physiological and pathological processes [56].

#### **6. Bilobalide, a nonconvulsant channel blocker**

Bilobalide (**Figure 2**) and a series of terpenoids known as ginkgolides isolated from *Ginkgo biloba* are structurally related to picrotoxinin and are relatively potent GABAA and GABA<sup>C</sup> receptor antagonists [57], but they also act on glycine and 5-HT<sup>3</sup> receptors [58, 59].

Unlike picrotoxin, bilobalide is an anticonvulsant. This may be due to its potent action on GABAC receptors [57]. Bilobalide also has differing effects to those of picrotoxin on the modulation of GABAA receptors by structurally different modulators [60], suggesting a different binding profile to picrotoxin to negatively modulate the GABAA receptors.

Thus, there are GABAA receptor antagonists that act as channel blockers and negative modulators that do not produce convulsions in vivo. Explanation of this apparent paradox includes selective actions on GABAA receptor subtypes, reduction of glutamate release from presynaptic terminals via presynaptic receptors and effects on GABA metabolism, together with actions on non-GABAergic systems.

Owing to their unique characteristics, bilobalide and other natural terpenoids from *Gingko biloba* are being investigated as cognitive enhancers via their effects on the GABAergic system [61]. Bilobalide has been shown to improve cognition in cognitive- and memory-impaired animals in a variety of animal models [62–65]. As a result to its nonconvulsant effects, bilobalide is a superior candidate for therapeutic use in memory impairment related to dementia and other neurological disorders compared with other GABAA receptor antagonists like picrotoxin which are pro-convulsive. Natural terpenoids from *Gingko biloba*, such as bilobalide, are being investigated in the treatment of neurological disorders via their effects on the GABAergic system [61].

Salicylidene salicylhydrazide has been reported as a potent antagonist of GABAA receptors containing the β1 subunit using a high-throughput screen [77]. It was suggested that salicylidene salicylhydrazide is interacting at a previously unidentified site on the β1 subunit, but

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The most potent GABAA receptor antagonist is the convulsant steroid derivative RU5135, being some 500 times more potent than bicuculline [78]. It acts as a competitive antagonist, sharing a common site of action with bicuculline. However it lacks specificity, as it is also a

There is still widespread interest in GABA receptor antagonists after many years of investigation. Reflecting on the use of GABA receptor antagonists in the last 10 years, citation counts via the Web of Science for publications citing GABA together with a GABA antagonist in the title or abstract are as follows: bicuculline 1203, picrotoxin or picrotoxinin 564, gabazine or SR 95531 290, TPMPA 48, and bilobalide 14. Thus far, there are only four publications directly related to

Nonconvulsant antagonists of ionotropic GABA receptors have considerable therapeutic potential in the treatment of cognitive problems, myopia, and other CNS disorders. Such antagonists may be useful in the treatment of Down syndrome [62]. The myriad of possible subtypes of ionotropic GABA receptors in the CNS as a result of different combinations of protein subunits make the search for more subtype-specific agents highly desirable. The highthroughput analysis of ionotropic GABA receptor subtypes should result in the discovery of novel subtype-specific agonists, antagonists, and modulators that have therapeutic potential [80]. Clearly, we are going to hear a lot more about S44819 and other yet to be discovered ionotropic GABA receptor antagonists that act selectively on ionotropic GABA receptor subtypes.

this does not appear to have been followed up after the initial publication in 2004.

glycine receptor antagonist sharing a common site of action with strychnine [79].

**9. Conclusion**

the effects of S44819 on cognition.

**Acknowledgements**

Tina Hinton and Graham A. R. Johnston\*

**Author details**

Australia

The authors are grateful to Dr. Ken Mewett for helpful comments.

\*Address all correspondence to: graham.johnston@sydney.edu.au

School of Medical Sciences (Pharmacology), The University of Sydney, Sydney, NSW,

The plant-derived triterpenoids, asiatic, oleanolic, and ursolic acids (**Figure 2**), are negative modulators of GABAA receptor activation acting in vivo as anxiolytics, anticonvulsants, and antidepressants in animal models [66, 67].

#### **7. S44819, an α5-selective competitive antagonist**

The α-β subunit interface has been highlighted as a novel target for subtype-selective drugs [68]. An example of a novel drug that targets this binding site and that is attracting considerable current attention as a new therapeutic agent is S44819 (Egis-13,529, 8-Methyl-5-[4-(trifluoromethyl)- 1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one, **Figure 2**), a novel oxazolo-2,3-benzodiazepine derivative, which selectively inhibits GABAA receptors that contain the α5-subunit [69, 70].

S44819 appears to act as a competitive antagonist at the orthosteric site at the α-β subunit interface of GABAA receptors containing only α5 subunits. Thus, S44819 is a competitive antagonist, unlike other α5-subunit selective drugs that act as negative allosteric modulators by binding to the benzodiazepine recognition site between at the α5-γ2 subunits [71, 72]. Agents that are selective for α5 subunit-containing GABAA receptors enhance cognitive performance in a variety of animal models without sedative or pro-convulsive effects [73]. SR44819 has been shown in healthy young humans to be orally active, reaching the cerebral cortex on oral administration where it increases cortical excitability [74], acting on extrasynaptic receptors to reduce tonic inhibition. Consequently, clinical trials are now underway.

#### **8. Other GABA receptor antagonists**

It has not been possible to cover all known GABA receptor antagonists in this perspective. Other important classes of antagonists include sulfated neurosteroids [75] and agents derived from 4-PIOL (5-(4-piperidyl)isoxazol-3-ol) [5]. Of particular interest is DPP-4-PIOL (4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide) that selectively antagonizes tonic over phasic GABAergic currents in the hippocampus, suggesting a degree of substrate specificity [76].

Salicylidene salicylhydrazide has been reported as a potent antagonist of GABAA receptors containing the β1 subunit using a high-throughput screen [77]. It was suggested that salicylidene salicylhydrazide is interacting at a previously unidentified site on the β1 subunit, but this does not appear to have been followed up after the initial publication in 2004.

The most potent GABAA receptor antagonist is the convulsant steroid derivative RU5135, being some 500 times more potent than bicuculline [78]. It acts as a competitive antagonist, sharing a common site of action with bicuculline. However it lacks specificity, as it is also a glycine receptor antagonist sharing a common site of action with strychnine [79].

#### **9. Conclusion**

Owing to their unique characteristics, bilobalide and other natural terpenoids from *Gingko biloba* are being investigated as cognitive enhancers via their effects on the GABAergic system [61]. Bilobalide has been shown to improve cognition in cognitive- and memory-impaired animals in a variety of animal models [62–65]. As a result to its nonconvulsant effects, bilobalide is a superior candidate for therapeutic use in memory impairment related to dementia and other neurological disorders compared with other GABAA receptor antagonists like picrotoxin which are pro-convulsive. Natural terpenoids from *Gingko biloba*, such as bilobalide, are being investigated in the treatment of neurological disorders via their effects on the GABAergic

The plant-derived triterpenoids, asiatic, oleanolic, and ursolic acids (**Figure 2**), are negative modulators of GABAA receptor activation acting in vivo as anxiolytics, anticonvulsants, and

The α-β subunit interface has been highlighted as a novel target for subtype-selective drugs [68]. An example of a novel drug that targets this binding site and that is attracting considerable current attention as a new therapeutic agent is S44819 (Egis-13,529, 8-Methyl-5-[4-(trifluoromethyl)- 1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one, **Figure 2**), a novel oxazolo-2,3-benzodiazepine derivative, which selectively inhibits GABAA receptors that

S44819 appears to act as a competitive antagonist at the orthosteric site at the α-β subunit interface of GABAA receptors containing only α5 subunits. Thus, S44819 is a competitive antagonist, unlike other α5-subunit selective drugs that act as negative allosteric modulators by binding to the benzodiazepine recognition site between at the α5-γ2 subunits [71, 72]. Agents that are selective for α5 subunit-containing GABAA receptors enhance cognitive performance in a variety of animal models without sedative or pro-convulsive effects [73]. SR44819 has been shown in healthy young humans to be orally active, reaching the cerebral cortex on oral administration where it increases cortical excitability [74], acting on extrasynaptic receptors to reduce

It has not been possible to cover all known GABA receptor antagonists in this perspective. Other important classes of antagonists include sulfated neurosteroids [75] and agents derived from 4-PIOL (5-(4-piperidyl)isoxazol-3-ol) [5]. Of particular interest is DPP-4-PIOL (4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide) that selectively antagonizes tonic over phasic GABAergic currents in the hippocampus, suggesting a degree of substrate

system [61].

antidepressants in animal models [66, 67].

contain the α5-subunit [69, 70].

**7. S44819, an α5-selective competitive antagonist**

98 GABA And Glutamate - New Developments In Neurotransmission Research

tonic inhibition. Consequently, clinical trials are now underway.

**8. Other GABA receptor antagonists**

specificity [76].

There is still widespread interest in GABA receptor antagonists after many years of investigation. Reflecting on the use of GABA receptor antagonists in the last 10 years, citation counts via the Web of Science for publications citing GABA together with a GABA antagonist in the title or abstract are as follows: bicuculline 1203, picrotoxin or picrotoxinin 564, gabazine or SR 95531 290, TPMPA 48, and bilobalide 14. Thus far, there are only four publications directly related to the effects of S44819 on cognition.

Nonconvulsant antagonists of ionotropic GABA receptors have considerable therapeutic potential in the treatment of cognitive problems, myopia, and other CNS disorders. Such antagonists may be useful in the treatment of Down syndrome [62]. The myriad of possible subtypes of ionotropic GABA receptors in the CNS as a result of different combinations of protein subunits make the search for more subtype-specific agents highly desirable. The highthroughput analysis of ionotropic GABA receptor subtypes should result in the discovery of novel subtype-specific agonists, antagonists, and modulators that have therapeutic potential [80]. Clearly, we are going to hear a lot more about S44819 and other yet to be discovered ionotropic GABA receptor antagonists that act selectively on ionotropic GABA receptor subtypes.

#### **Acknowledgements**

The authors are grateful to Dr. Ken Mewett for helpful comments.

#### **Author details**

Tina Hinton and Graham A. R. Johnston\*

\*Address all correspondence to: graham.johnston@sydney.edu.au

School of Medical Sciences (Pharmacology), The University of Sydney, Sydney, NSW, Australia

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**Chapter 7**

**Provisional chapter**

**Clinical Application of MR Spectroscopy in Identifying**

Magnetic resonance spectroscopy (MRS) provides useful information regarding metabolic composition in the tissues, and advanced spectroscopic methods are used to quantify markers of tumor membrane turnover and proliferation (e.g., choline (Cho)), energy homoeostasis (e.g., creatine (Cr)), intact glioneuronal structures (e.g., N-acetylaspartate (NAA)), and necrosis (e.g., lactate (Lac) or lipids). Results are usually expressed as metabolite ratios rather than absolute metabolite concentrations. Because glial tumors have some specific metabolic characteristics that differ according to the grade of tumor, there is a potential for MR spectroscopy to increase the sensitivity of routinely used diagnostic imaging. MRS also has many diagnostic applications in neurosciences to support the diagnosis in conditions like demyelination, infections, and dementia and in postradiotherapy cases. Biochemical changes in the metabolism of tumor cells related to malignant transformation are reflected in changes of particular metabolite concentration in the tumor tissue. Our prospective study aimed to analyze the usefulness of proton MR spectroscopy in grading of glioma and to correlate various metabolite ratios like choline/creatine, choline/N-acetylaspartate, N-acetylaspartate/creatine, and lactate/creatine with the histopathological grades of glioma.

**Clinical Application of MR Spectroscopy in Identifying** 

DOI: 10.5772/intechopen.71728

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Magnetic resonance spectroscopy (MRS) is an analytical method used for the identification and quantification of metabolites. It differs from conventional magnetic resonance imaging (MRI) since it provides physiological and chemical information instead of only anatomy [1].

**Biochemical Composition of the Intracranial**

Sankar Neelakantan and Sunitha Palasamudram Kumaran

**Keywords:** MRS, MRI, grade, glioma, NAA, choline, creatine, ratio

**Biochemical Composition of the Intracranial** 

B C Hamsini, Bhavana Nagabhushana Reddy,

B C Hamsini, Bhavana Nagabhushana Reddy,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Pathologies**

**Pathologies**

Sankar Neelakantan and

**Abstract**

**1. Introduction**

Sunitha Palasamudram Kumaran

http://dx.doi.org/10.5772/intechopen.71728

**Provisional chapter**

#### **Clinical Application of MR Spectroscopy in Identifying Biochemical Composition of the Intracranial Pathologies Clinical Application of MR Spectroscopy in Identifying Biochemical Composition of the Intracranial Pathologies**

DOI: 10.5772/intechopen.71728

B C Hamsini, Bhavana Nagabhushana Reddy, Sankar Neelakantan and Sunitha Palasamudram Kumaran B C Hamsini, Bhavana Nagabhushana Reddy, Sankar Neelakantan and Sunitha Palasamudram Kumaran Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71728

#### **Abstract**

Magnetic resonance spectroscopy (MRS) provides useful information regarding metabolic composition in the tissues, and advanced spectroscopic methods are used to quantify markers of tumor membrane turnover and proliferation (e.g., choline (Cho)), energy homoeostasis (e.g., creatine (Cr)), intact glioneuronal structures (e.g., N-acetylaspartate (NAA)), and necrosis (e.g., lactate (Lac) or lipids). Results are usually expressed as metabolite ratios rather than absolute metabolite concentrations. Because glial tumors have some specific metabolic characteristics that differ according to the grade of tumor, there is a potential for MR spectroscopy to increase the sensitivity of routinely used diagnostic imaging. MRS also has many diagnostic applications in neurosciences to support the diagnosis in conditions like demyelination, infections, and dementia and in postradiotherapy cases. Biochemical changes in the metabolism of tumor cells related to malignant transformation are reflected in changes of particular metabolite concentration in the tumor tissue. Our prospective study aimed to analyze the usefulness of proton MR spectroscopy in grading of glioma and to correlate various metabolite ratios like choline/creatine, choline/N-acetylaspartate, N-acetylaspartate/creatine, and lactate/creatine with the histopathological grades of glioma.

**Keywords:** MRS, MRI, grade, glioma, NAA, choline, creatine, ratio

#### **1. Introduction**

Magnetic resonance spectroscopy (MRS) is an analytical method used for the identification and quantification of metabolites. It differs from conventional magnetic resonance imaging (MRI) since it provides physiological and chemical information instead of only anatomy [1].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Many of nuclei have been used to obtain MR spectra; few of them include proton (1 H), phosphorus (31P), fluorine (19F), carbon (13C), and sodium (23Na).

Proton (1 H) MR spectroscopy is most commonly used since hydrogen nucleus is abundant in human tissues [1].

Magnetic field strength clinically used for conventional MRI ranges from 0.2 to 3 T.

For MRS, higher-field strength (1.5 T or more) is required since the main aim of MRS is to detect weaker signals from metabolites. Higher-field strength units have the advantage of higher signal-to-noise ratio (SNR), better resolution, and shorter acquisition times.

#### **2. Metabolites in MRS**

#### **2.1. N-Acetylaspartate (NAA)**

N-Acetylaspartate (NAA) peak is the most prominent peak in normal adult brain proton MRS which resonates at 2.0 ppm [2] (**Figure 1**).

NAA is a derivative of aspartic acid. It is synthesized and stored primarily in the neurons and hence it is called a neuronal marker or a marker of neuronal density and viability [2].

As a neuronal marker, NAA concentration declines with the destruction of neurons in highvolume lesions, dementia, hypoxia, or multiple sclerosis.

Due to the relationship between the decline in NAA concentration and increasing glioma grade, it is possible to use NAA as a substantial marker [13]. A high level of NAA is associated with a good prognosis [3].

NAA is also helpful for the differentiation of primary brain tumors from metastasis and nonneuronal tumors where the metabolite is lacking in the spectra [4].

#### **2.2. Creatine (Cr)**

Creatine (Cr) is synthesized from amino acids primarily in the kidneys and liver and transported to the peripheral organs by blood and is called as energy metabolism marker [3].

Total Cr indicates the quantity of phosphocreatine (PCr) and Cr contained in neurons and glial cells and visualized as a prominent peak in MR spectra at 3.0 ppm; an additional peak for creatine may be visible at 3.94 ppm [3].

A decrease in the Cr level in high-grade gliomas (HGGs) is due to increased metabolic demands of the tumorous tissue in the brain. Cr is a relatively constant element of cellular energetic metabolism of the brain and it is frequently used as a reference metabolite for in vivo MRS, for example, for mainly calculating the metabolite ratios such as choline (Cho)/Cr, NAA/Cr, lipid (Lip)-lactate (Lac)/Cr, or myoinositol/Cr [3].

**2.3. Choline (Cho)**

Choline (Cho) is a metabolic marker of cellular proliferation and cell membrane integrity, that is, phospholipids synthesis and degradation. Choline peak is seen at 3.2 ppm and is the most

**Figure 1.** Normal spectra obtained with short TE sequence (TE = 30 ms). Ins dd1, myoinositol; Cho, choline; Cr, creatine;

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109

important metabolic peak for the diagnosis of a glioma [3].

Glx, glutamate glutamine; NAA, N-acetylaspartate [9].

**Figure 1.** Normal spectra obtained with short TE sequence (TE = 30 ms). Ins dd1, myoinositol; Cho, choline; Cr, creatine; Glx, glutamate glutamine; NAA, N-acetylaspartate [9].

#### **2.3. Choline (Cho)**

Many of nuclei have been used to obtain MR spectra; few of them include proton (1

Magnetic field strength clinically used for conventional MRI ranges from 0.2 to 3 T.

higher signal-to-noise ratio (SNR), better resolution, and shorter acquisition times.

For MRS, higher-field strength (1.5 T or more) is required since the main aim of MRS is to detect weaker signals from metabolites. Higher-field strength units have the advantage of

N-Acetylaspartate (NAA) peak is the most prominent peak in normal adult brain proton MRS

NAA is a derivative of aspartic acid. It is synthesized and stored primarily in the neurons and

As a neuronal marker, NAA concentration declines with the destruction of neurons in high-

Due to the relationship between the decline in NAA concentration and increasing glioma grade, it is possible to use NAA as a substantial marker [13]. A high level of NAA is associated

NAA is also helpful for the differentiation of primary brain tumors from metastasis and non-

Creatine (Cr) is synthesized from amino acids primarily in the kidneys and liver and transported to the peripheral organs by blood and is called as energy metabolism marker [3].

Total Cr indicates the quantity of phosphocreatine (PCr) and Cr contained in neurons and glial cells and visualized as a prominent peak in MR spectra at 3.0 ppm; an additional peak

A decrease in the Cr level in high-grade gliomas (HGGs) is due to increased metabolic demands of the tumorous tissue in the brain. Cr is a relatively constant element of cellular energetic metabolism of the brain and it is frequently used as a reference metabolite for in vivo MRS, for example, for mainly calculating the metabolite ratios such as choline (Cho)/Cr,

hence it is called a neuronal marker or a marker of neuronal density and viability [2].

H) MR spectroscopy is most commonly used since hydrogen nucleus is abundant in

phorus (31P), fluorine (19F), carbon (13C), and sodium (23Na).

108 GABA And Glutamate - New Developments In Neurotransmission Research

Proton (1

human tissues [1].

**2. Metabolites in MRS**

**2.1. N-Acetylaspartate (NAA)**

with a good prognosis [3].

**2.2. Creatine (Cr)**

which resonates at 2.0 ppm [2] (**Figure 1**).

for creatine may be visible at 3.94 ppm [3].

NAA/Cr, lipid (Lip)-lactate (Lac)/Cr, or myoinositol/Cr [3].

volume lesions, dementia, hypoxia, or multiple sclerosis.

neuronal tumors where the metabolite is lacking in the spectra [4].

H), phos-

Choline (Cho) is a metabolic marker of cellular proliferation and cell membrane integrity, that is, phospholipids synthesis and degradation. Choline peak is seen at 3.2 ppm and is the most important metabolic peak for the diagnosis of a glioma [3].

Increased choline reflects increased cell membrane synthesis and degradation. Thus, all processes resulting in increased membrane turnover like primary brain neoplasm and myelin breakdown like demyelinating lesions lead to increased choline concentration [5].

An increased concentration of Ala may occur in oxidative metabolism defects [9].

In addition to alanine, other amino acids may be seen in various pathologies. Peaks of various amino acids may be seen at many levels, 0.9—valine and 3.6—leucine. Amino acids are also

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This is mainly used for glioma grading. Differentiation between low-grade and high-grade gliomas is important for estimating the prognosis and for therapeutic planning. Proton MRS may indicate the tumor grade with more accuracy because it assesses a larger amount of tis-

Gliomas are heterogeneous and their spectra vary depending on the region sampled by MRS. Hence, the region of interest chosen for analysis has a higher influence on the results. Multivoxel spectroscopy is generally considered preferable because it allows metabolic het-

Statistically significant higher values of Cho/Cr and Cho/NAA are observed in high-grade

Although the cutoff values of metabolite ratios for grading of gliomas are not well established, Cho/Cr is the most frequently used ratio. Some institutions use a threshold value of 2.0 for Cho/Cr to differentiate low-grade from high-grade gliomas; others use a cutoff value

Although increased Cho is related to tumor grade, few of grade IV gliomas have lower levels of Cho than grade II or grade III gliomas [11]. This may be due to the presence of necrosis in high-grade tumors, because necrosis is associated with a prominent lipid peak along with the

The presence of lipids and lactates correlates with the necrosis in high-grade gliomas. High-grade and low-grade tumors and their margins could be differentiated based on the

In tumors, an elevated level of Ala is considered specific for meningiomas [1].

**2.8. Other amino acids**

*3.1.1. Choline*

of 2.5.

than in low-grade gliomas.

*3.1.2. Lipids and lactates*

lactate/lipid peaks.

reduction of rest other metabolites [12].

**3. MRS in tumor grading**

seen in abscesses and neurocysticercosis [10].

sue than what is usually excised during the biopsy.

erogeneity to be evaluated in different components of the tumor.

**3.1. Useful metabolites for suggesting the tumor grade**

#### **2.4. Myoinositol (Myo)**

Myoinositol (Myo) is a simple sugar assigned at 3.56 ppm.

It is considered as a glial marker because it is primarily synthesized in glial cells, almost only in astrocytes. It is also the most important osmolyte in astrocytes [3].

The elevation in Myo detected can be found in cerebral diseases associated with marked gliosis. Myo concentration is higher in low-grade glioma (LGG) when compared to high-grade glioma. Thus, a higher Myo/Cr ratio is seen in low-grade glioma [6].

#### **2.5. Lactate (lac) and lipids (lip)**

Lactate (Lac) and lipids (Lip) are known as anaerobic metabolism markers. They are represented as a doublet peak in the MR spectra at 1.31 ppm. It is important to note that lactate is not detected in healthy adult brain tissue. There is direct correlation between Lac level and glioma grade [3].

Malignant transformation of the glial tumors is usually accompanied by an increase in cell density with their relative ischemia resulting in a higher level of Lac. When ischemia of tumorous tissue progresses further, the peaks of Lip increase, indicating the presence of necrosis and the destruction of myelin sheaths [7].

Thus, the level of Lip detected by MRS appears to reflect the severity of tissue damage.

#### **2.6. Glutamate (Glu)-glutamine (Gln) and gamma-amino butyric acid**

Glx is a complex peak from glutamate (Glu), glutamine (Gln), and gamma-amino butyric acid (GABA) assigned at 2.05–2.50 ppm [1].

These metabolite peaks are difficult to separate at 1.5 T. Glu is an important excitatory neurotransmitter and plays a role in the redox cycle [8, 9].

An elevated concentration of Gln is found in a few conditions such as hepatic encephalopathy [9].

#### **2.7. Alanine (Ala)**

Alanine (Ala) is an amino acid and is a doublet peak assigned at 1.48 ppm.

This peak is located above the baseline in spectra obtained with short or long TE and inverts below the baseline at intermediate TE = 135–144 ms [1].

The function of Ala is uncertain; however, it plays a role in the citric acid cycle. Alanine peak may be obscured by lactate peak [8].

An increased concentration of Ala may occur in oxidative metabolism defects [9].

In tumors, an elevated level of Ala is considered specific for meningiomas [1].

#### **2.8. Other amino acids**

Increased choline reflects increased cell membrane synthesis and degradation. Thus, all processes resulting in increased membrane turnover like primary brain neoplasm and myelin

It is considered as a glial marker because it is primarily synthesized in glial cells, almost only

The elevation in Myo detected can be found in cerebral diseases associated with marked gliosis. Myo concentration is higher in low-grade glioma (LGG) when compared to high-grade

Lactate (Lac) and lipids (Lip) are known as anaerobic metabolism markers. They are represented as a doublet peak in the MR spectra at 1.31 ppm. It is important to note that lactate is not detected in healthy adult brain tissue. There is direct correlation between Lac level and

Malignant transformation of the glial tumors is usually accompanied by an increase in cell density with their relative ischemia resulting in a higher level of Lac. When ischemia of tumorous tissue progresses further, the peaks of Lip increase, indicating the presence of necrosis

Glx is a complex peak from glutamate (Glu), glutamine (Gln), and gamma-amino butyric acid

These metabolite peaks are difficult to separate at 1.5 T. Glu is an important excitatory neu-

An elevated concentration of Gln is found in a few conditions such as hepatic encephalopathy [9].

This peak is located above the baseline in spectra obtained with short or long TE and inverts

The function of Ala is uncertain; however, it plays a role in the citric acid cycle. Alanine peak

Thus, the level of Lip detected by MRS appears to reflect the severity of tissue damage.

**2.6. Glutamate (Glu)-glutamine (Gln) and gamma-amino butyric acid**

Alanine (Ala) is an amino acid and is a doublet peak assigned at 1.48 ppm.

breakdown like demyelinating lesions lead to increased choline concentration [5].

Myoinositol (Myo) is a simple sugar assigned at 3.56 ppm.

110 GABA And Glutamate - New Developments In Neurotransmission Research

in astrocytes. It is also the most important osmolyte in astrocytes [3].

glioma. Thus, a higher Myo/Cr ratio is seen in low-grade glioma [6].

**2.4. Myoinositol (Myo)**

**2.5. Lactate (lac) and lipids (lip)**

and the destruction of myelin sheaths [7].

(GABA) assigned at 2.05–2.50 ppm [1].

may be obscured by lactate peak [8].

rotransmitter and plays a role in the redox cycle [8, 9].

below the baseline at intermediate TE = 135–144 ms [1].

glioma grade [3].

**2.7. Alanine (Ala)**

In addition to alanine, other amino acids may be seen in various pathologies. Peaks of various amino acids may be seen at many levels, 0.9—valine and 3.6—leucine. Amino acids are also seen in abscesses and neurocysticercosis [10].

#### **3. MRS in tumor grading**

This is mainly used for glioma grading. Differentiation between low-grade and high-grade gliomas is important for estimating the prognosis and for therapeutic planning. Proton MRS may indicate the tumor grade with more accuracy because it assesses a larger amount of tissue than what is usually excised during the biopsy.

Gliomas are heterogeneous and their spectra vary depending on the region sampled by MRS. Hence, the region of interest chosen for analysis has a higher influence on the results. Multivoxel spectroscopy is generally considered preferable because it allows metabolic heterogeneity to be evaluated in different components of the tumor.

#### **3.1. Useful metabolites for suggesting the tumor grade**

#### *3.1.1. Choline*

Statistically significant higher values of Cho/Cr and Cho/NAA are observed in high-grade than in low-grade gliomas.

Although the cutoff values of metabolite ratios for grading of gliomas are not well established, Cho/Cr is the most frequently used ratio. Some institutions use a threshold value of 2.0 for Cho/Cr to differentiate low-grade from high-grade gliomas; others use a cutoff value of 2.5.

Although increased Cho is related to tumor grade, few of grade IV gliomas have lower levels of Cho than grade II or grade III gliomas [11]. This may be due to the presence of necrosis in high-grade tumors, because necrosis is associated with a prominent lipid peak along with the reduction of rest other metabolites [12].

#### *3.1.2. Lipids and lactates*

The presence of lipids and lactates correlates with the necrosis in high-grade gliomas. High-grade and low-grade tumors and their margins could be differentiated based on the lactate/lipid peaks.

Lipids peak is also found in metastasis. When lipids peak is observed in solid tumors, lymphoma should also be considered.

**Fungal abscess:** A spectrum in fungal abscesses shows amino acids and lactate peak along with multiple peaks between 3.6 and 4.0 ppm. These peaks have been assigned to trehalose

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Proton MR spectroscopy is a useful tool in diagnosing metabolic brain disorders when used

In most of the inherited metabolic disorders, MRS findings are abnormal but are not specific

Few metabolic diseases have specific MRS findings, either abnormal elevation or reduction of

Specific MRS patterns are mainly found in Canavan's disease with prominently increased NAA peak and creatine deficiency with a characteristic reduced creatine peak. In nonketotic hyperglycemia, there is an appearance of glycine peak at 3.55 ppm and there is detection of

Neurodegenerative dementia is characterized by elevated myoinositol and decreased

An increase in myoinositol occurs before the reduction of NAA levels in Alzheimer's disease. An NAA/myo ratio in the posterior cingulate gyri decreases with an increasing burden of

In patients with mild cognitive impairment, 1H MRS is sensitive in the detection of the patho-

The characteristic spectroscopic finding in acute brain ischemia is the early appearance of a lactate peak, a decrease of NAA, and a slight increase of choline. Lactate is observed within few minutes following brain ischemia and its concentration reduces in sub-acute phase. The

The role of 1H–MRS in epilepsy is to help in characterizing and localizing the epileptogenic focus, especially in patients with refractory focal epilepsy without clear MR imaging findings. Temporal lobe epilepsy (TLE) is the most common cause of focal epilepsy; reduction in NAA concentration and NAA/Cho + Cr ratio is observed in TLE, which reflects the neuronal

sugar present in the fungal wall [15].

as an adjunct to conventional MRI.

for a single metabolic disease or syndrome.

a single normal peak or detection of abnormal metabolite peak.

branched-chain amino acids in maple syrup urine disease [16].

physiologic processes associated with the risk of dementia [17].

intensity of these peaks in the infracted area is related to the prognosis [18].

**4.3. Metabolic brain disorder**

**4.4. Dementia**

N-acetylaspartate levels.

Alzheimer's disease.

**4.5. Brain ischemia**

**4.6. Epilepsy**

damage [1].

An increase in the lipid peak is inversely correlated to prognosis [12].

#### *3.1.3. Myoinositol*

Useful information on tumor grade can be obtained by assessing the myo levels at short TE (30–35 ms).

Myo/Cr ratio is typically higher in low-grade than in high-grade gliomas. This may be due to a low mitotic index leading to lack of activation phosphatidylinositol metabolism resulting in the accumulation of myo in low-grade gliomas.

In gliomas without alteration in Cho/Cr ratio, increased levels of Myo have been reported to be useful in the identification of low-grade tumors [12].

#### *3.1.4. NAA and Cr*

A greatest reduction in NAA and Cr levels has been observed in higher-grade tumors. As the grade of the tumor increases, NAA/Cr ratio decreases and Cho/NAA ratio increases.

#### **4. Other applications of MRS in brain**

#### **4.1. Response to radiotherapy**

Differentiation between recurrent brain tumor and radiation change/injury is an important concern in postradiotherapy patients with brain tumors and particularly when fresh contrastenhancing lesions are seen in previously operated and/or irradiated regions.

Proton MRS is useful in differentiating radiation-induced tissue injury from tumor recurrence. Significantly reduced Cho and Cr levels suggest radiation necrosis; increased lipid and lactate signals can also be seen in necrotic areas.

Increased Cho levels relative to the normal tissue suggest recurrence of the tumor. Cho/Cr and/ or Cho/NAA ratios are significantly higher in recurrent tumor than in radiation injury [12].

#### **4.2. Infections**

**Pyogenic abscess:** Amino acid peak at 0.9 ppm is useful in differentiating pyogenic abscess from tumors. Abscess from anaerobic organism in addition has acetate and succinate peaks at 1.9 and 2.4 ppm, respectively [13] .

**Tuberculous abscess:** Spectra from tuberculous abscess shows lipid-lactate peak at 1.3 ppm. No amino acid peak is observed in tubercular abscess, which helps to differentiate it from pyogenic abscess [14].

**Fungal abscess:** A spectrum in fungal abscesses shows amino acids and lactate peak along with multiple peaks between 3.6 and 4.0 ppm. These peaks have been assigned to trehalose sugar present in the fungal wall [15].

#### **4.3. Metabolic brain disorder**

Lipids peak is also found in metastasis. When lipids peak is observed in solid tumors, lym-

Useful information on tumor grade can be obtained by assessing the myo levels at short TE

Myo/Cr ratio is typically higher in low-grade than in high-grade gliomas. This may be due to a low mitotic index leading to lack of activation phosphatidylinositol metabolism resulting in

In gliomas without alteration in Cho/Cr ratio, increased levels of Myo have been reported to

A greatest reduction in NAA and Cr levels has been observed in higher-grade tumors. As the

Differentiation between recurrent brain tumor and radiation change/injury is an important concern in postradiotherapy patients with brain tumors and particularly when fresh contrast-

Proton MRS is useful in differentiating radiation-induced tissue injury from tumor recurrence. Significantly reduced Cho and Cr levels suggest radiation necrosis; increased lipid and

Increased Cho levels relative to the normal tissue suggest recurrence of the tumor. Cho/Cr and/ or Cho/NAA ratios are significantly higher in recurrent tumor than in radiation injury [12].

**Pyogenic abscess:** Amino acid peak at 0.9 ppm is useful in differentiating pyogenic abscess from tumors. Abscess from anaerobic organism in addition has acetate and succinate peaks at

**Tuberculous abscess:** Spectra from tuberculous abscess shows lipid-lactate peak at 1.3 ppm. No amino acid peak is observed in tubercular abscess, which helps to differentiate it from

grade of the tumor increases, NAA/Cr ratio decreases and Cho/NAA ratio increases.

enhancing lesions are seen in previously operated and/or irradiated regions.

An increase in the lipid peak is inversely correlated to prognosis [12].

phoma should also be considered.

the accumulation of myo in low-grade gliomas.

**4. Other applications of MRS in brain**

lactate signals can also be seen in necrotic areas.

**4.1. Response to radiotherapy**

1.9 and 2.4 ppm, respectively [13] .

be useful in the identification of low-grade tumors [12].

112 GABA And Glutamate - New Developments In Neurotransmission Research

*3.1.3. Myoinositol*

*3.1.4. NAA and Cr*

**4.2. Infections**

pyogenic abscess [14].

(30–35 ms).

Proton MR spectroscopy is a useful tool in diagnosing metabolic brain disorders when used as an adjunct to conventional MRI.

In most of the inherited metabolic disorders, MRS findings are abnormal but are not specific for a single metabolic disease or syndrome.

Few metabolic diseases have specific MRS findings, either abnormal elevation or reduction of a single normal peak or detection of abnormal metabolite peak.

Specific MRS patterns are mainly found in Canavan's disease with prominently increased NAA peak and creatine deficiency with a characteristic reduced creatine peak. In nonketotic hyperglycemia, there is an appearance of glycine peak at 3.55 ppm and there is detection of branched-chain amino acids in maple syrup urine disease [16].

#### **4.4. Dementia**

Neurodegenerative dementia is characterized by elevated myoinositol and decreased N-acetylaspartate levels.

An increase in myoinositol occurs before the reduction of NAA levels in Alzheimer's disease. An NAA/myo ratio in the posterior cingulate gyri decreases with an increasing burden of Alzheimer's disease.

In patients with mild cognitive impairment, 1H MRS is sensitive in the detection of the pathophysiologic processes associated with the risk of dementia [17].

#### **4.5. Brain ischemia**

The characteristic spectroscopic finding in acute brain ischemia is the early appearance of a lactate peak, a decrease of NAA, and a slight increase of choline. Lactate is observed within few minutes following brain ischemia and its concentration reduces in sub-acute phase. The intensity of these peaks in the infracted area is related to the prognosis [18].

#### **4.6. Epilepsy**

The role of 1H–MRS in epilepsy is to help in characterizing and localizing the epileptogenic focus, especially in patients with refractory focal epilepsy without clear MR imaging findings.

Temporal lobe epilepsy (TLE) is the most common cause of focal epilepsy; reduction in NAA concentration and NAA/Cho + Cr ratio is observed in TLE, which reflects the neuronal damage [1].

#### **5. Materials and methods**

#### **5.1. Study site**

The study was conducted at the Department of Radio Diagnosis, Sri Sathya Sai Institute of Higher Medical Sciences, Whitefield, Bangalore 560066.

#### **5.2. Study population**

Initially, all patients suspicious for glioma and fulfilling the inclusion and exclusion criteria were imaged with conventional MRI and proton MR spectroscopy. All these patients underwent histopathological examination. Specimens were obtained via surgical resection/biopsy. Patients who had histopathological confirmation of glioma were included in the study. Histopathological grading was done according to WHO classification of brain tumors 2007 [19]. Grades I and II were graded as low-grade and grades III and IV were graded as high-grade tumors.

#### **5.3. Study design**

The study design was a prospective study.

#### **5.4. Sample size**

$$\text{Sample size} = \frac{Z\_{1\sim n2}^2 SD^2}{d^2} \tag{1}$$

• Both male and female patient population.

• Postoperative and postradiotherapy patients. • Unavailability of histopathological examination.

• MR spectroscopy with artifact, baseline noise, and uninterruptable spectra.

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• Concurrence was taken from the chairman, academic committee, scientific committee, and

• Written informed consent was obtained prior to subject enrolment into the study. • All the patients were subjected to conventional MRI and proton MR spectroscopy.

For all patients, initially MR imaging was performed with Siemens 1.5 T Magnetom Aera.

A localizing sagittal T1-weighted image was obtained followed by non-enhanced axial and coronal T2-weighted (4500/102 [TR/TE]), axial fluid-attenuated inversion-recovery (FLAIR, 8500/86/2500 [TR/TE/TI]), and T1-weighted axial (600/9 [TR/TE]) images, Susceptibility weighted imaging (50/40 [TR/TE]). Contrast material-enhanced axial T1-weighted imaging was performed.

After the conventional MRI volume of interest from the lesion (VOI) was selected from T1 post-contrast images, VOI was selected from the solid part of lesion with edges of the voxel well within the mass and in most of the cases perilesional edema was included within the VOI. VOI was carefully selected so that it will not include areas of hemorrhage or calcification

In our institution, we used Multivoxel MR spectroscopy technique (chemical shift method) at

**5.7. Exclusion criteria**

• MRI contraindicated patients:

○ Pregnant patients ○ Mentally ill patients.

**5.8. Methodology**

○ Patients with cardiac pace maker

○ Patients with severe claustrophobia

ethical committee for the study.

**5.9. Magnetic resonance imaging**

**5.10. Magnetic resonance spectroscopy**

intermediate TE of −135 ms.

and unintended areas like ventricles, calvarium, and so on.

○ Patients with aneurysm clips in the brain

○ Patients with a metallic foreign body in the eye

where *Z*1-*a*/2 is the standard normal variant; SD is the standard deviation of variable; *d* is the absolute error of precision.

The standard deviation of Cho/Cr for high-grade glioma is 1.92; Cho/Cr ratio is taken because for other ratios the sample size becomes very low. Absolute error of precision was 0.5.

$$\begin{aligned} \text{Sample size} &= (1.96)^2 \times (1.92)^2 / 0.5 \\ &= 3.8 \times 3.6 / 0.25 \\ &= 54.72 \text{(rounded to 55)} \end{aligned} \tag{2}$$

The minimum sample size was around 55; we have included 70 patients.

#### **5.5. Duration of the study**

The duration of study was over a period of 14 months from 01-10-2014, to 01-02-2016.

#### **5.6. Inclusion criteria**


• Both male and female patient population.

#### **5.7. Exclusion criteria**

**5. Materials and methods**

Higher Medical Sciences, Whitefield, Bangalore 560066.

114 GABA And Glutamate - New Developments In Neurotransmission Research

The study was conducted at the Department of Radio Diagnosis, Sri Sathya Sai Institute of

Initially, all patients suspicious for glioma and fulfilling the inclusion and exclusion criteria were imaged with conventional MRI and proton MR spectroscopy. All these patients underwent histopathological examination. Specimens were obtained via surgical resection/biopsy. Patients who had histopathological confirmation of glioma were included in the study. Histopathological grading was done according to WHO classification of brain tumors 2007 [19]. Grades I and II

where *Z*1-*a*/2 is the standard normal variant; SD is the standard deviation of variable; *d* is the

The standard deviation of Cho/Cr for high-grade glioma is 1.92; Cho/Cr ratio is taken because

Sample size = (1.96)2 × (1.92)2 /0.5

<sup>=</sup> 3.8 <sup>×</sup> 3.6/0.25

= 54.72(rounded to 55)

for other ratios the sample size becomes very low. Absolute error of precision was 0.5.

The duration of study was over a period of 14 months from 01-10-2014, to 01-02-2016.

• All cases with neuroparenchymal space occupying lesions suspicious for gliomas on MRI.

The minimum sample size was around 55; we have included 70 patients.

<sup>2</sup> *SD*<sup>2</sup> \_\_\_\_\_\_\_

*<sup>d</sup>*<sup>2</sup> (1)

(2)

were graded as low-grade and grades III and IV were graded as high-grade tumors.

**5.1. Study site**

**5.2. Study population**

**5.3. Study design**

**5.4. Sample size**

absolute error of precision.

**5.5. Duration of the study**

**5.6. Inclusion criteria**

• All age groups.

The study design was a prospective study.

Sample size <sup>=</sup> *<sup>Z</sup>*1−*a*/2

	- Patients with cardiac pace maker
	- Patients with aneurysm clips in the brain
	- Patients with a metallic foreign body in the eye
	- Patients with severe claustrophobia
	- Pregnant patients
	- Mentally ill patients.

#### **5.8. Methodology**


#### **5.9. Magnetic resonance imaging**

For all patients, initially MR imaging was performed with Siemens 1.5 T Magnetom Aera.

A localizing sagittal T1-weighted image was obtained followed by non-enhanced axial and coronal T2-weighted (4500/102 [TR/TE]), axial fluid-attenuated inversion-recovery (FLAIR, 8500/86/2500 [TR/TE/TI]), and T1-weighted axial (600/9 [TR/TE]) images, Susceptibility weighted imaging (50/40 [TR/TE]). Contrast material-enhanced axial T1-weighted imaging was performed.

#### **5.10. Magnetic resonance spectroscopy**

After the conventional MRI volume of interest from the lesion (VOI) was selected from T1 post-contrast images, VOI was selected from the solid part of lesion with edges of the voxel well within the mass and in most of the cases perilesional edema was included within the VOI. VOI was carefully selected so that it will not include areas of hemorrhage or calcification and unintended areas like ventricles, calvarium, and so on.

In our institution, we used Multivoxel MR spectroscopy technique (chemical shift method) at intermediate TE of −135 ms.

A typical VOI consisted of an 8 × 8 cm region placed within a 16 × 16 cm field of view on a 1.5-cm transverse section. A 16 × 16 phase-encoding matrix was used to obtain 8 × 8 arrays of spectra in the VOI, with an in-plane resolution of 1 × 1 cm and a voxel size of 1 × 1 × 1.5 cm<sup>3</sup> . The time taken to acquire spectra was around 8 min.

The sensitivity, specificity, PPV, and NPV of proton MR spectroscopy in differentiating the grades of glioma were high in comparison to conventional MRI indicating that proton MRS

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Lac/Cr ratio had a total diagnostic accuracy of 95.12%. Cho/NAA and Cho/Cr ratios had a

Metabolite ratio that had the highest diagnostic value was lactate/Cr followed by Cho/NAA

The presence of lipid peak was found to be suggestive of high-grade gliomas and was found

MRS had the added advantage in combination with conventional MRI with good diagnostic accuracy in differentiating grade II and grade III gliomas. Lactate/Cr had the highest diagnos-

Tumor grade is the most important key factor in determining treatment plan. Treatment plan for low-grade glioma is surgical resection; adjuvant radio chemotherapy is only recommended for patients with incompletely resected grade II tumors or for patients older than age of 40 years. Whereas high-grade glioma always require chemotherapy and/or radiotherapy following surgery; therefore, preoperative grading of glioma is necessary for good patient

The current "gold standard" for the determination of glioma grade is histopathological examination. It has few limitations; the most significant one is limited number of samples which creates potential errors in determining the glioma grade. When samples are not taken from the most malignant region of tumor during biopsy or when the tumor has not been completely resected, histopathological grading becomes inaccurate. This problem is particularly observed in glioma because of the infiltrative and heterogeneous nature of these tumors; the region of highest malignancy may then be within the peritumoral or perilesional-enhancing

Because histopathological examination requires biopsy/surgical resection which are invasive, it has morbidity of up to 3.6%, hemorrhage rate of up to 8%, and mortality of up to 1.7%, as

Alternatively, many noninvasive or minimally invasive imaging technologies have been used

In our study, grades I and II were graded as low grade and grades III and IV were graded as

NAA/Cr ratio had poor diagnostic significance in differentiating the grades of gliomas.

Other metabolite peaks like myoinositol and glutamate could not be evaluated.

tic value followed by Cho/NAA and Cho/Cr in differentiating the two grades.

spectroscopy is a useful tool in differentiating grades of glioma.

total diagnostic accuracy of 88.57 and 88.43%, respectively.

and Cho/Cr in differentiating low- and high-grade gliomas.

care and to reduce the morbidity and mortality [19].

assessed over a large number of studies [22].

to evaluate the malignancy of brain tumors [20, 21].

in about 46% of high-grade gliomas.

**7. Discussion**

region [20, 21].

high grade [19].

The metabolite peaks were assigned as follows: Cho, 3.22 ppm; Cr, 3.02 ppm; NAA, 2.02 ppm; lactate was identified at 1.33 ppm by its characteristic doublet and inversion at intermediate TE. Lipid peak was demonstrated at 0.9–1.3 ppm without inversion at intermediate TE. Metabolite ratios were obtained for Cho/Cr, Cho/NAA, NAA/Cr, and lactate/Cr. Maximal Cho/Cr, Cho/NAA and lactate/Cr and minimum NAA/Cr ratios were obtained from spectral maps. Lactate/Cr was used instead of lipid-lactate/Cr used by other studies in literature because lipid peak was not consistently seen in most of the cases at intermediate TE.

#### **5.11. Statistical methods**

The data collected were entered into a Microsoft excel spreadsheet and analyzed using IBM SPSS Statistics, Version 22 (Armonk, NY: IBM Corp). Descriptive data were presented in the form of frequency, percentage, mean, median, standard deviation, and quartiles. The metabolite ratios of Cho/Cr, Cho/NAA, NAA/Cr, and lactate/Cr were calculated. The metabolite ratios between the low-grade and high-grade gliomas were compared using Mann–Whitney *U*-test. Comparisons of the categorical variables between the two groups were performed using the chi-squared test. Receiver-operating characteristic (ROC) curve analysis was done to evaluate the performance of the metabolite ratios in differentiating high-grade and low-grade gliomas. The area under the ROC curve was calculated to summarize the performance of each metabolite in differentiating the two grades of glioma. The sensitivity, specificity, positive-predictive value (PPV), negativepredictive value (NPV), and diagnostic accuracy with each metabolite were assessed using the cutoff value obtained with minimum C1 error from the ROC analysis. A *P*-value of <0.05 was considered as statistically significant. In the same way, ROC curve analysis was done to evaluate the performance of metabolite ratios in differentiating grade II and grade III glioma. By similar way, sensitivity, specificity, positive-predictive value, negative-predictive value, and diagnostic accuracy with each metabolite were assessed using the cutoff value obtained with minimum C1 error from the ROC analysis in differentiating grade II and grade III gliomas.

#### **6. Results**

Seventy histopathologically proved cases of gliomas were included in the study. Grade I [14] and grade II [19] gliomas were classified as low-grade and grade III [22] and grade IV [15] were classified as high-grade tumors.

All patients underwent conventional MRI sequences and proton MR spectroscopy. Quantitative values were calculated for Cho/Cr, Cho/NAA, NAA/Cr, and lactate/Cr ratios. Grading by conventional MRI was also done to know the additional usefulness of MRS in grading of gliomas.

The sensitivity, specificity, PPV, and NPV of conventional MRI in grading of gliomas were 62.2, 78.8, 76.7, and 65%, respectively, with the total diagnostic accuracy of 70%.

The sensitivity, specificity, PPV, and NPV of proton MR spectroscopy in differentiating the grades of glioma were high in comparison to conventional MRI indicating that proton MRS spectroscopy is a useful tool in differentiating grades of glioma.

Lac/Cr ratio had a total diagnostic accuracy of 95.12%. Cho/NAA and Cho/Cr ratios had a total diagnostic accuracy of 88.57 and 88.43%, respectively.

Metabolite ratio that had the highest diagnostic value was lactate/Cr followed by Cho/NAA and Cho/Cr in differentiating low- and high-grade gliomas.

NAA/Cr ratio had poor diagnostic significance in differentiating the grades of gliomas.

The presence of lipid peak was found to be suggestive of high-grade gliomas and was found in about 46% of high-grade gliomas.

Other metabolite peaks like myoinositol and glutamate could not be evaluated.

MRS had the added advantage in combination with conventional MRI with good diagnostic accuracy in differentiating grade II and grade III gliomas. Lactate/Cr had the highest diagnostic value followed by Cho/NAA and Cho/Cr in differentiating the two grades.

#### **7. Discussion**

A typical VOI consisted of an 8 × 8 cm region placed within a 16 × 16 cm field of view on a 1.5-cm transverse section. A 16 × 16 phase-encoding matrix was used to obtain 8 × 8 arrays of spectra in the VOI, with an in-plane resolution of 1 × 1 cm and a voxel size of 1 × 1 × 1.5 cm<sup>3</sup>

The metabolite peaks were assigned as follows: Cho, 3.22 ppm; Cr, 3.02 ppm; NAA, 2.02 ppm; lactate was identified at 1.33 ppm by its characteristic doublet and inversion at intermediate TE. Lipid peak was demonstrated at 0.9–1.3 ppm without inversion at intermediate TE. Metabolite ratios were obtained for Cho/Cr, Cho/NAA, NAA/Cr, and lactate/Cr. Maximal Cho/Cr, Cho/NAA and lactate/Cr and minimum NAA/Cr ratios were obtained from spectral maps. Lactate/Cr was used instead of lipid-lactate/Cr used by other studies in literature

The data collected were entered into a Microsoft excel spreadsheet and analyzed using IBM SPSS Statistics, Version 22 (Armonk, NY: IBM Corp). Descriptive data were presented in the form of frequency, percentage, mean, median, standard deviation, and quartiles. The metabolite ratios of Cho/Cr, Cho/NAA, NAA/Cr, and lactate/Cr were calculated. The metabolite ratios between the low-grade and high-grade gliomas were compared using Mann–Whitney *U*-test. Comparisons of the categorical variables between the two groups were performed using the chi-squared test. Receiver-operating characteristic (ROC) curve analysis was done to evaluate the performance of the metabolite ratios in differentiating high-grade and low-grade gliomas. The area under the ROC curve was calculated to summarize the performance of each metabolite in differentiating the two grades of glioma. The sensitivity, specificity, positive-predictive value (PPV), negativepredictive value (NPV), and diagnostic accuracy with each metabolite were assessed using the cutoff value obtained with minimum C1 error from the ROC analysis. A *P*-value of <0.05 was considered as statistically significant. In the same way, ROC curve analysis was done to evaluate the performance of metabolite ratios in differentiating grade II and grade III glioma. By similar way, sensitivity, specificity, positive-predictive value, negative-predictive value, and diagnostic accuracy with each metabolite were assessed using the cutoff value obtained with minimum C1

because lipid peak was not consistently seen in most of the cases at intermediate TE.

error from the ROC analysis in differentiating grade II and grade III gliomas.

Seventy histopathologically proved cases of gliomas were included in the study. Grade I [14] and grade II [19] gliomas were classified as low-grade and grade III [22] and grade IV [15]

All patients underwent conventional MRI sequences and proton MR spectroscopy. Quantitative values were calculated for Cho/Cr, Cho/NAA, NAA/Cr, and lactate/Cr ratios. Grading by conventional MRI was also done to know the additional usefulness of MRS in grading of gliomas.

The sensitivity, specificity, PPV, and NPV of conventional MRI in grading of gliomas were

62.2, 78.8, 76.7, and 65%, respectively, with the total diagnostic accuracy of 70%.

The time taken to acquire spectra was around 8 min.

116 GABA And Glutamate - New Developments In Neurotransmission Research

**5.11. Statistical methods**

**6. Results**

were classified as high-grade tumors.

.

Tumor grade is the most important key factor in determining treatment plan. Treatment plan for low-grade glioma is surgical resection; adjuvant radio chemotherapy is only recommended for patients with incompletely resected grade II tumors or for patients older than age of 40 years. Whereas high-grade glioma always require chemotherapy and/or radiotherapy following surgery; therefore, preoperative grading of glioma is necessary for good patient care and to reduce the morbidity and mortality [19].

The current "gold standard" for the determination of glioma grade is histopathological examination. It has few limitations; the most significant one is limited number of samples which creates potential errors in determining the glioma grade. When samples are not taken from the most malignant region of tumor during biopsy or when the tumor has not been completely resected, histopathological grading becomes inaccurate. This problem is particularly observed in glioma because of the infiltrative and heterogeneous nature of these tumors; the region of highest malignancy may then be within the peritumoral or perilesional-enhancing region [20, 21].

Because histopathological examination requires biopsy/surgical resection which are invasive, it has morbidity of up to 3.6%, hemorrhage rate of up to 8%, and mortality of up to 1.7%, as assessed over a large number of studies [22].

Alternatively, many noninvasive or minimally invasive imaging technologies have been used to evaluate the malignancy of brain tumors [20, 21].

In our study, grades I and II were graded as low grade and grades III and IV were graded as high grade [19].

We have used the following criteria for grading gliomas by conventional MRI: margins of the lesion, perilesional edema, heterogeneity of lesion, mass effect, crossing the midline, hemorrhage, necrosis, and enhancement pattern. Other studies have also used the similar criteria [21].

In literature, Cho/Cr, Cho/NAA, and NAA/Cr were the commonly used metabolites [19, 21]. Lipid-lactate ratio was used for grading of gliomas in few studies [26]; we have used lactate/ Cr ration since lipid peak was not consistently seen in a majority of cases at intermediate TE. In our study, the values of mean and median for Cho/Cr, Cho/NAA, and NAA/Cr had statisti-

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Other studies have also shown significant difference of mean for Cho/Cr, Cho/NAA, and

NAA is a neuronal marker and decreases in all gliomas since the neurons are destroyed and/ or substituted by the malignant cells. The elevation of choline is due to the increased cellularity and cell membrane turnover. The decrease in creatine is due to the altered energy metabolism in the cells and can be attributed to the low energy status of the glycolyzing tumors. Therefore, the typical MRS characteristics of a glioma are elevated Cho with reduction in NAA and Cr signals. This results in an absolute increase in Cho/Cr and Cho/NAA and a decrease in NAA/

Lac/Cr ratio in our study had significant difference in mean between low- and high-grade

Studies conducted by Yoon et al. [26] and Kim et al. [27] showed significant difference in mean

Since lactate is a marker of anaerobic glycolysis, malignant transformation of glial tumors is accompanied by an increase in cell density with relative ischemia resulting in a higher level of lactate. There is a direct correlation between Lac levels and glioma grade with higher peaks seen in higher-grade tumors [3]. Therefore, Lac/Cr ratio increases with an increase in the

In a clinical setting, where decisions such as the extent of tumor resection, the dose of postoperative chemo radiation, and the interval of follow-up must be made, cutoff values can be

In our study, we have provided cutoff values for Cho/Cr, Cho/NAA, and lactate/Cr ratios to

A cutoff value of 1.76 for Cho/Cr and 2.22 for Cho/NAA ratio provided good sensitivity, specificity, PPV, and NPV with a total diagnostic accuracy of 78.05 and 82.93%, respectively,

Other studies in literature have given a cutoff value ranging from 1.56 to 2.04 for Cho/Cr and 1.6 to 2.49 for Cho/NAA. These values provided good sensitivity, specificity, PPV, and NPV

Even though the cutoff value for Cho/Cr and Cho/NAA provided by our study was comparable with other studies in literature [21], the small variation in cutoff value between studies is possibly because of differences in the MRS imaging methods, including magnetic resonance field strength, acquisition parameters, voxel size and location, heterogeneity of tumor, and

used as important supplementary information in noninvasive grading of gliomas [21].

cally significant difference between low- and high-grade gliomas.

values for lipid-lactate/Cr between low- and high-grade gliomas.

NAA/Cr between low- and high-grade gliomas [10].

Cr ratios [1].

malignant potential of tumor.

sample size and distribution.

differentiate low- from high-grade gliomas.

in differentiating low- from high-grade gliomas.

with a total diagnostic accuracy ranging from 81 to 88% [21].

gliomas.

Sensitivity, specificity, PPV, and NPV of conventional MRI in grading of gliomas were found to be 62.2, 78.8, 76.7, and 65%, respectively, with the total diagnostic accuracy of 70%.

In other studies conducted by Law et al. [21], Zou et al. [19], and Ellika et al. [23], the sensitivity ranged from 72 to 86% and the specificity ranged from 60 to 67%.

Other studies in literature have shown sensitivity ranging from 55 to 83% [19, 21].

A study conducted by Dean et al. [24] and Atkinson et al. [25] showed that a high-grade glioma may be mistaken for a low-grade when it demonstrates minimal edema, no contrast enhancement, no necrosis, and no mass effect. Conversely, low-grade gliomas sometimes can be mistaken for a high-grade when it demonstrates peritumoral edema, contrast material enhancement, central necrosis, and mass effect.

Low diagnostic accuracy in our study was because of the overlapping imaging features in low- and high-grade gliomas, predominantly between grades II and grade III. Fourteen cases of grade III tumors were classified as low grade because they had imaging features favoring low grade such as minimal perilesional edema, no significant mass effect, no contrast enhancement, no necrosis, and no hemorrhage.

Seven cases of low-grade gliomas were classified as high grade since they had imaging features favoring high grade such as significant perilesional edema, mass effect, presence of necrosis or hemorrhage, and heterogeneous contrast enhancement.

In our study, the most consistent characteristics on MRI to predict the grade were necrosis, mass effect, and crossing the midline.

A study conducted by Dean et al. [24] has shown that mass effect and necrosis are the most important characteristic for predicting tumor grade by conventional MRI.

Because of the limitations of conventional MRI in differentiating high- from low-grade gliomas, advanced multi-parametric magnetic resonance techniques have been used in grading of gliomas and they are diffusion-weighted imaging (DWI), proton MR spectroscopy, and perfusion imaging [19].

Magnetic resonance spectroscopy is one of the advanced adjuvant MR techniques and because of its safe and noninvasive nature it is of great advantage in characterizing gliomas.

Because glial tumors have some specific metabolic characteristics which further differ according to the grade, there is growing interest in MR spectroscopy that could further increase the sensitivity of routinely used diagnostic imaging [3].

In our study, we have used Cho/Cr, Cho/NAA, NAA/Cr, and lactate/Cr ratios for grading since the study was done at intermediate TE, prominent peaks seen were Cho, Cr, NAA, and lactate.

In literature, Cho/Cr, Cho/NAA, and NAA/Cr were the commonly used metabolites [19, 21]. Lipid-lactate ratio was used for grading of gliomas in few studies [26]; we have used lactate/ Cr ration since lipid peak was not consistently seen in a majority of cases at intermediate TE.

We have used the following criteria for grading gliomas by conventional MRI: margins of the lesion, perilesional edema, heterogeneity of lesion, mass effect, crossing the midline, hemorrhage, necrosis, and enhancement pattern. Other studies have also used the similar

Sensitivity, specificity, PPV, and NPV of conventional MRI in grading of gliomas were found

In other studies conducted by Law et al. [21], Zou et al. [19], and Ellika et al. [23], the sensitiv-

A study conducted by Dean et al. [24] and Atkinson et al. [25] showed that a high-grade glioma may be mistaken for a low-grade when it demonstrates minimal edema, no contrast enhancement, no necrosis, and no mass effect. Conversely, low-grade gliomas sometimes can be mistaken for a high-grade when it demonstrates peritumoral edema, contrast material

Low diagnostic accuracy in our study was because of the overlapping imaging features in low- and high-grade gliomas, predominantly between grades II and grade III. Fourteen cases of grade III tumors were classified as low grade because they had imaging features favoring low grade such as minimal perilesional edema, no significant mass effect, no contrast

Seven cases of low-grade gliomas were classified as high grade since they had imaging features favoring high grade such as significant perilesional edema, mass effect, presence of

In our study, the most consistent characteristics on MRI to predict the grade were necrosis,

A study conducted by Dean et al. [24] has shown that mass effect and necrosis are the most

Because of the limitations of conventional MRI in differentiating high- from low-grade gliomas, advanced multi-parametric magnetic resonance techniques have been used in grading of gliomas and they are diffusion-weighted imaging (DWI), proton MR spectroscopy, and

Magnetic resonance spectroscopy is one of the advanced adjuvant MR techniques and because

Because glial tumors have some specific metabolic characteristics which further differ according to the grade, there is growing interest in MR spectroscopy that could further increase the

In our study, we have used Cho/Cr, Cho/NAA, NAA/Cr, and lactate/Cr ratios for grading since the study was done at intermediate TE, prominent peaks seen were Cho, Cr, NAA, and

of its safe and noninvasive nature it is of great advantage in characterizing gliomas.

to be 62.2, 78.8, 76.7, and 65%, respectively, with the total diagnostic accuracy of 70%.

Other studies in literature have shown sensitivity ranging from 55 to 83% [19, 21].

ity ranged from 72 to 86% and the specificity ranged from 60 to 67%.

118 GABA And Glutamate - New Developments In Neurotransmission Research

necrosis or hemorrhage, and heterogeneous contrast enhancement.

important characteristic for predicting tumor grade by conventional MRI.

enhancement, central necrosis, and mass effect.

enhancement, no necrosis, and no hemorrhage.

sensitivity of routinely used diagnostic imaging [3].

mass effect, and crossing the midline.

perfusion imaging [19].

lactate.

criteria [21].

In our study, the values of mean and median for Cho/Cr, Cho/NAA, and NAA/Cr had statistically significant difference between low- and high-grade gliomas.

Other studies have also shown significant difference of mean for Cho/Cr, Cho/NAA, and NAA/Cr between low- and high-grade gliomas [10].

NAA is a neuronal marker and decreases in all gliomas since the neurons are destroyed and/ or substituted by the malignant cells. The elevation of choline is due to the increased cellularity and cell membrane turnover. The decrease in creatine is due to the altered energy metabolism in the cells and can be attributed to the low energy status of the glycolyzing tumors. Therefore, the typical MRS characteristics of a glioma are elevated Cho with reduction in NAA and Cr signals. This results in an absolute increase in Cho/Cr and Cho/NAA and a decrease in NAA/ Cr ratios [1].

Lac/Cr ratio in our study had significant difference in mean between low- and high-grade gliomas.

Studies conducted by Yoon et al. [26] and Kim et al. [27] showed significant difference in mean values for lipid-lactate/Cr between low- and high-grade gliomas.

Since lactate is a marker of anaerobic glycolysis, malignant transformation of glial tumors is accompanied by an increase in cell density with relative ischemia resulting in a higher level of lactate. There is a direct correlation between Lac levels and glioma grade with higher peaks seen in higher-grade tumors [3]. Therefore, Lac/Cr ratio increases with an increase in the malignant potential of tumor.

In a clinical setting, where decisions such as the extent of tumor resection, the dose of postoperative chemo radiation, and the interval of follow-up must be made, cutoff values can be used as important supplementary information in noninvasive grading of gliomas [21].

In our study, we have provided cutoff values for Cho/Cr, Cho/NAA, and lactate/Cr ratios to differentiate low- from high-grade gliomas.

A cutoff value of 1.76 for Cho/Cr and 2.22 for Cho/NAA ratio provided good sensitivity, specificity, PPV, and NPV with a total diagnostic accuracy of 78.05 and 82.93%, respectively, in differentiating low- from high-grade gliomas.

Other studies in literature have given a cutoff value ranging from 1.56 to 2.04 for Cho/Cr and 1.6 to 2.49 for Cho/NAA. These values provided good sensitivity, specificity, PPV, and NPV with a total diagnostic accuracy ranging from 81 to 88% [21].

Even though the cutoff value for Cho/Cr and Cho/NAA provided by our study was comparable with other studies in literature [21], the small variation in cutoff value between studies is possibly because of differences in the MRS imaging methods, including magnetic resonance field strength, acquisition parameters, voxel size and location, heterogeneity of tumor, and sample size and distribution.

A cutoff value of 0.68 for Lac/Cr ratio provided an excellent diagnostic accuracy of 95.12% which was the most useful metabolite ratio in differentiating low- from high-grade gliomas in our study.

Advanced multi-parametric magnetic resonance techniques, including DWI, DTI, and MR perfusion and multimodal imaging including PET/SPECT, also compliment MRS to distinguish HGGs from LGGs. Though it is controversial which imaging technique is superior, most authors concluded that combined techniques would improve the diagnostic accuracy [19].

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**1.** Our study was done at a magnetic field strength of 1.5 T. A higher-field strength of 3 T or

**2.** Small sample size may reduce or bias the power of the results. Further studies with a larger

**3.** Our sample volume predominantly consists of astrocytomas, and other glial tumors like

**4.** Our study was carried at intermediate TE, so we could not evaluate smaller metabolite peaks like myoinositol and glutamate. MRS at short TE would have given spectra with

**5.** Comparison of MRS with other advanced MR techniques like perfusion and DTI would

MRS is an important diagnostic and research tool in clinical neuroscience. MRS is a very useful tool in combination with conventional MRI for grading of gliomas. Lactate/creatine ratio has highest diagnostic accuracy followed by choline/NAA and choline/creatine ratios. NAA/creatine has least diagnostic significance in grading of gliomas. Lipid peak on MRS is more frequently found in high-grade gliomas. MRS could differentiate grade II and grade III gliomas more effectively in comparison to only conventional MRI. Apart from tumor grading, MRS can support the diagnosis in many brain infections by showing the specific metabolite peaks. It is also helpful in diagnosis of post-radiotherapy patients when differentiation between recurrent brain tumor and radiation change/injury is the concern. It also has a significant role in narrowing the differential diagnosis of metabolic brain disorders. MRS is also

more would have yielded a better spatial resolution and high SNR.

sample size are necessary to extend these results.

have given additional value in grading of gliomas.

important in the diagnosis of stroke and demyelination in brain.

Case 1. A 46-year-old female presented with headache since 1 year.

multiple metabolite peaks.

oligodendrogliomas and ependymomas were less frequent.

**8. Limitations**

**9. Conclusion**

**A. Appendix A**

**A.1. Representative cases**

Limited studies in literature have given cutoff for lipid-lactate/Cr ratio in grading of gliomas. A study performed by Kim et al. gave a cutoff value of 0.59 for lipid-lactate/Cr [28]. A study performed by Yoon et al. gave a cutoff of 1.39 with a sensitivity of 64.6% and a specificity of 91.7% [26].

There was no cutoff value given for NAA/Cr ratio in our study even though the mean values were statistically significant in differentiating high- and low-grade gliomas because of overlapping of NAA/Cr values between the grades; out of 37 people in high-grade gliomas, 29 (78% of high-grade glioma cases) had values higher than the lowest value in low grade.

Zeng et al. [28] and Liu et al. [29] have given cutoff values of 0.72 and 0.97 for NAA/Cr ratio to differentiate low- and high-grade gliomas with a diagnostic accuracy of 73–75%.

In our study, lipid peak was found in 46% of high-grade and 12% of low-grade gliomas. A prominent lipid peak in high-grade gliomas was due to necrotic component.

Other studies have also shown that gliomas are heterogeneous tumors and they include areas of viable tumor, necrosis, and hemorrhage. Elevated lipid peaks are often found in high-grade gliomas due to the necrotic portion [30, 31].

Since our study is carried out at intermediate TE (135 ms), we could not evaluate other peaks like myoinositol and glutamate. Few cases had myoinositol peak but the peak was not consistently seen to comment upon.

Differentiation between grade II and III gliomas is clinically important since the treatment plan and prognosis are different but it is difficult to differentiate them on conventional MRI because of the overlapping imaging characteristics [32].

Literature review showed limited studies to differentiate grade II and grade III gliomas. In our study, we analyzed and tried to establish cutoff values to differentiate grade II and grade III gliomas.

Lactate/Cr had excellent diagnostic value followed by Cho/NAA and Cho/Cr ratios. NAA/Cr had poor diagnostic value in differentiating the grade II and grade III because of the overlapping of values.

Cutoff values for Cho/Cr, Cho/NAA, and lactate/Cr ratios were 1.76, 2.22, and 0.685 with good sensitivity, specificity, PPV, and NPV. Diagnostic accuracy was 78.05, 82.93, and 95.12% for Cho/Cr, Cho/NAA, and lactate/Cr, respectively.

Stadlbauer et al. [23] found that there was significant difference in Cho, Cr, and NAA levels between grade II and grade III gliomas, and all patients with a grade II glioma had a Cho/ NAA ratio of less than 0.8, whereas all patients with a grade III glioma had a Cho/NAA ratio of greater than 0.8.

Zonari et al. [33] found the sensitivity, specificity, PPV, and NPV for Cho/Cr ratio to be 75.6 45, 60.7, and 62.1 and NAA/Cr ratio to be 64.4, 65, 67.4, and 61.9 in differentiating grade II from grade III gliomas by a logistic regression analysis.

Advanced multi-parametric magnetic resonance techniques, including DWI, DTI, and MR perfusion and multimodal imaging including PET/SPECT, also compliment MRS to distinguish HGGs from LGGs. Though it is controversial which imaging technique is superior, most authors concluded that combined techniques would improve the diagnostic accuracy [19].

#### **8. Limitations**

A cutoff value of 0.68 for Lac/Cr ratio provided an excellent diagnostic accuracy of 95.12% which was the most useful metabolite ratio in differentiating low- from high-grade gliomas

Limited studies in literature have given cutoff for lipid-lactate/Cr ratio in grading of gliomas. A study performed by Kim et al. gave a cutoff value of 0.59 for lipid-lactate/Cr [28]. A study performed by Yoon et al. gave a cutoff of 1.39 with a sensitivity of 64.6% and a specificity of 91.7% [26]. There was no cutoff value given for NAA/Cr ratio in our study even though the mean values were statistically significant in differentiating high- and low-grade gliomas because of overlapping of NAA/Cr values between the grades; out of 37 people in high-grade gliomas, 29 (78% of high-grade glioma cases) had values higher than the lowest value in low grade.

Zeng et al. [28] and Liu et al. [29] have given cutoff values of 0.72 and 0.97 for NAA/Cr ratio to

In our study, lipid peak was found in 46% of high-grade and 12% of low-grade gliomas. A

Other studies have also shown that gliomas are heterogeneous tumors and they include areas of viable tumor, necrosis, and hemorrhage. Elevated lipid peaks are often found in high-grade

Since our study is carried out at intermediate TE (135 ms), we could not evaluate other peaks like myoinositol and glutamate. Few cases had myoinositol peak but the peak was not consis-

Differentiation between grade II and III gliomas is clinically important since the treatment plan and prognosis are different but it is difficult to differentiate them on conventional MRI

Literature review showed limited studies to differentiate grade II and grade III gliomas. In our study, we analyzed and tried to establish cutoff values to differentiate grade II and grade

Lactate/Cr had excellent diagnostic value followed by Cho/NAA and Cho/Cr ratios. NAA/Cr had poor diagnostic value in differentiating the grade II and grade III because of the overlap-

Cutoff values for Cho/Cr, Cho/NAA, and lactate/Cr ratios were 1.76, 2.22, and 0.685 with good sensitivity, specificity, PPV, and NPV. Diagnostic accuracy was 78.05, 82.93, and 95.12% for

Stadlbauer et al. [23] found that there was significant difference in Cho, Cr, and NAA levels between grade II and grade III gliomas, and all patients with a grade II glioma had a Cho/ NAA ratio of less than 0.8, whereas all patients with a grade III glioma had a Cho/NAA ratio

Zonari et al. [33] found the sensitivity, specificity, PPV, and NPV for Cho/Cr ratio to be 75.6 45, 60.7, and 62.1 and NAA/Cr ratio to be 64.4, 65, 67.4, and 61.9 in differentiating grade II from

differentiate low- and high-grade gliomas with a diagnostic accuracy of 73–75%.

prominent lipid peak in high-grade gliomas was due to necrotic component.

gliomas due to the necrotic portion [30, 31].

120 GABA And Glutamate - New Developments In Neurotransmission Research

because of the overlapping imaging characteristics [32].

Cho/Cr, Cho/NAA, and lactate/Cr, respectively.

grade III gliomas by a logistic regression analysis.

tently seen to comment upon.

III gliomas.

ping of values.

of greater than 0.8.

in our study.


#### **9. Conclusion**

MRS is an important diagnostic and research tool in clinical neuroscience. MRS is a very useful tool in combination with conventional MRI for grading of gliomas. Lactate/creatine ratio has highest diagnostic accuracy followed by choline/NAA and choline/creatine ratios. NAA/creatine has least diagnostic significance in grading of gliomas. Lipid peak on MRS is more frequently found in high-grade gliomas. MRS could differentiate grade II and grade III gliomas more effectively in comparison to only conventional MRI. Apart from tumor grading, MRS can support the diagnosis in many brain infections by showing the specific metabolite peaks. It is also helpful in diagnosis of post-radiotherapy patients when differentiation between recurrent brain tumor and radiation change/injury is the concern. It also has a significant role in narrowing the differential diagnosis of metabolic brain disorders. MRS is also important in the diagnosis of stroke and demyelination in brain.

#### **A. Appendix A**

#### **A.1. Representative cases**

Case 1. A 46-year-old female presented with headache since 1 year.

Case 2: A 70-year-old male presenting with seizures since 2 months.

enhancement is seen.

Diagnosed as high-grade glioma on conventional MRI.

Diagnosed as high-grade glioma on conventional MRI.

MRI shows relatively well-defined heterogeneous lesion noted in the left parietal lobe with moderate perilesional edema. There is mass effect on the adjacent neuroparenchyma without midline shift. Areas of necrosis and hemorrhage noted within the lesion. Peripheral nodular

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Relatively well-defined heterogeneous lesion noted in the left parietal lobe with moderate perilesional edema. There is mass effect on the adjacent neuroparenchyma without midline shift. Areas of necrosis and hemorrhage noted within the lesion. Peripheral nodular enhancement is seen.

MRI shows well-defined cystic lesion with enhancing peripheral mural nodule in the right cerebellar hemisphere with mild perilesional edema. Local mass effect on the adjacent neuroparenchyma. No areas of hemorrhage on SWI. No areas of necrosis.

Diagnosed as low-grade glioma on conventional MRI.

MRS: Elevated choline, reduced NAA, and Cr. Cho/Cr: 1.63; Cho/NAA: 1; NAA/Cr: 1.09; lactate/Cr: 0.18. No lipid peak.

Histopathological diagnosis: Pilocytic astrocytoma (grade I glioma).

Case 2: A 70-year-old male presenting with seizures since 2 months.

MRI shows relatively well-defined heterogeneous lesion noted in the left parietal lobe with moderate perilesional edema. There is mass effect on the adjacent neuroparenchyma without midline shift. Areas of necrosis and hemorrhage noted within the lesion. Peripheral nodular enhancement is seen.

Diagnosed as high-grade glioma on conventional MRI.

MRI shows well-defined cystic lesion with enhancing peripheral mural nodule in the right cerebellar hemisphere with mild perilesional edema. Local mass effect on the adjacent neuro-

MRS: Elevated choline, reduced NAA, and Cr. Cho/Cr: 1.63; Cho/NAA: 1; NAA/Cr: 1.09; lac-

Histopathological diagnosis: Pilocytic astrocytoma (grade I glioma).

parenchyma. No areas of hemorrhage on SWI. No areas of necrosis.

Diagnosed as low-grade glioma on conventional MRI.

122 GABA And Glutamate - New Developments In Neurotransmission Research

tate/Cr: 0.18. No lipid peak.

Relatively well-defined heterogeneous lesion noted in the left parietal lobe with moderate perilesional edema. There is mass effect on the adjacent neuroparenchyma without midline shift. Areas of necrosis and hemorrhage noted within the lesion. Peripheral nodular enhancement is seen.

Diagnosed as high-grade glioma on conventional MRI.

MRS: Elevated choline, reduced NAA, and Cr.

Cho/Cr: 2.47; Cho/NAA: 3.5; NAA/Cr: 0.7; lactate/Cr: 1.

Lipid peak present.

Histopathological diagnosis: GBM (grade IV glioma).

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Paraffin section with hematoxylin and eosin stain showing marked nuclear pleomorphism and mitotic figures.

#### **Author details**

B C Hamsini1 , Bhavana Nagabhushana Reddy2 , Sankar Neelakantan2 and Sunitha Palasamudram Kumaran1,2\*

\*Address all correspondence to: drsunitha27@gmail.com

1 Neuroimaging, Toronto Western hospital, Toronto, Ontario, Canada

2 Department of Radiology, Sri Sathya Sai Institute of higher medical sciences, Bangalore, India

#### **References**

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**Author details**

Sunitha Palasamudram Kumaran1,2\*

B C Hamsini1

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India

Cho/Cr: 2.47; Cho/NAA: 3.5; NAA/Cr: 0.7; lactate/Cr: 1.

124 GABA And Glutamate - New Developments In Neurotransmission Research

Histopathological diagnosis: GBM (grade IV glioma).

, Bhavana Nagabhushana Reddy2

1 Neuroimaging, Toronto Western hospital, Toronto, Ontario, Canada

\*Address all correspondence to: drsunitha27@gmail.com

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