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Noninvasive study of brain tumours metabolism using phosphorus-31 magnetic resonance spectroscopy

Noninvasive study of brain tumours metabolism using phosphorus-31 magnetic resonance spectroscopy DOI: 10.4149/BLL_2020_080 Bratisl Med J 2020; 121 (7) 488 – 492 A PILOT STUDY Noninvasive study of brain tumours metabolism using phosphorus-31 magnetic resonance spectroscopy 1 2 3 2 4 4 Hnilicova P , Richterova R , Zelenak K , Kolarovszki B , Majercikova Z , Hatok J Biomedical Centre Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia. [email protected] ABSTRACT Phosphorus-31 magnetic resonance spectroscopy ( P MRS) is currently not accepted as a diagnostic tool in the neuro-oncological practice, although it provides useful non-invasive information about biochemical processes ongoing in the intracranial tumours. This pilot study was aimed to present the diagnostic capability of the P MRS in brain tumour examination, even its application on clinical 1.5T MR scanner. Seven patients with brain tumorous lesions (four glioblastomas, one ependymoma, and two lung metastasis) underwent multivoxel in vivo P MRS performed on clinical 1.5 T MR scanner within measurement time of 20 minutes. Comparing two selected voxels, one in the tumour and the other one in the normal-appearing brain tissue, enabled to investigate their metabolic differences. Enhanced markers of membrane phospholipids synthesis (signifi cantly increased phosphomonoesters ratios) than markers of their degradation (signifi cantly decreased phosphodiesters ratios) manifested a higher cell proliferation ongoing in tumours. High energetic tumorous tissue demands leading to anaerobic metabolic turnover were present as a signifi cant decline in phosphocreatine ratios and adenosine triphosphates. Intracellular pH evaluation showed a tumorous tendency to alkalize. P MRS enables the non-invasive metabolic characterization of intracranial tumours and thus appears to be a clinically useful method for the determination of ongoing tumour pathomechanisms (Fig. 2, Ref. 26). Text in PDF www.elis.sk KEY WORDS: brain tumour, P MRS, 1.5 Tesla; energetic metabolism. Introduction and cell membrane phospholipids composition through phospho- monoesters (PME), and phosphhodiesters (PDE) (1, 3). Although Phosphorus-31 MR Spectroscopy ( P MRS) is a very use- it can be demonstrated that different concentrations of various ful clinical tool with great potential mostly in neuro-oncological phosphorus metabolites measured spectroscopically between the practice, because it provides non-invasive insight into the com- brain tumours and normal brain tissue (3, 4, 5), the clinical ap- position of the examined tissue in vivo, and allows to obtain in- plication of P MRS for the examination of brain tumours has formation about cellular energy or membrane metabolism (1, 2). limitations, until recently (1, 2, 6). One reason for this may be the 31 31 P MRS enables direct measurement of energy metabolites like relatively low sensitivity of P MRS at 1.5 T fi eld strength (1, 7). phosphocreatine (PCr), adenosine-triphosphates (ATP), inorganic Therefore, the longer scan time or larger single-voxels had to be phosphate (Pi), as well as and indirectly evaluate intracellular pH chosen (~ 40‒100 cc), making it impossible to study heteroge- neity of the intracranial tumours (4, 8, 9). In this pilot study, the P MRS signal was obtained using the multivoxel technique with relatively small voxel volume (~ 27 cm ) and in a tolerable scan Biomedical Centre Martin, Jessenius Faculty of Medicine in Martin, Co- menius University in Bratislava, Martin, Slovakia, Clinic of Neurosur- time of 20 minutes, including the scanning for localization. This gery, Jessenius Faculty of Medicine in Martin, Comenius University in 31 pilot study aimed to achieve clinically feasible P MRS brain tu- Bratislava, Martin, Slovakia, Clinic of Radiology, Jessenius Faculty of mours examination, despite application on basic clinical 1.5 T MR Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia, 4 scanner, showed the medical relevance of non-invasive metabolic and Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia characterization of intracranial tumours and investigated the most useful P MRS parameters, which may determinate the ongoing Address for correspondence: P. Hnilicova, Ing, PhD, Biomedical Centre Martin, Jessenius Faculty of Medicine in Martin, Comenius University in tumour pathomechanisms. Bratislava, Mala Hora 4D, SK-036 01 Martin, Slovakia. Phone: +421.43.2633 660 Materials and methods Acknowledgments: This work was supported by the Slovak Research and Development Agency under Contract No. APVV-18-0088 and by the Study participants project implementation: “Centre of Excellence for Research in Personal- All examinations of this P MRS pilot study were carried out ized Therapy (Cevypet),” Itms: 26220120053 supported by the Operational Programme Research and Innovation funded by the ERDF. in compliance with the local institutional medical ethics com- Indexed and abstracted in Science Citation Index Expanded and in Journal Citation Reports /Science Edition Hnilicova P et al. Noninvasive study of brain tumours metabolism using phosphorus-31 MR spectroscopy xx Fig. 1. P MRS of the tumorous and normal-appearing brain tissue. For all participants, one voxel in the tumour (red voxel) and another one in normal-appearing brain tissue (green voxel) were chosen, and P MR spectra were evaluated in jMRUI. In fi tted spectra measured on 1.5T are detectable peaks of phosphomonoesters (PME), phosphodiesters (PDE), adenosine triphosphates (α-,β-,γ-ATP), phosphocreatine (PCr, and inorganic phosphate (Pi). mittee and after obtaining a written informed consent from study (4096 points) and 5 Hz exponential line broadening, the P MR participants. In the study, seven patients were enrolled (the mean spectra were obtain (Fig. 1). The integrals of measured P me- age: 65 ± 12 years; 3 males and 4 females), who had suspected tabolites were evaluated, and the following ratios were calculated: brain tumour lesions detected during the standard clinical MRI PCr/Pi, PCr/ATP, Pi/ATP, PME/PDE, PME/PCr, PME/Pi, PME/ examination. All the patients underwent P MRS before any of ATP, PDE/PCr, PDE/Pi, and PDE/ATP. The intracellular pH of the the surgical nor therapeutic treatment procedures. After a sub- examined tissue was calculated by using the chemical shift of Pi sequent histological analysis, four grade IV glioblastomas, one relative to the PCr peak (10, 11). grade III ependymomas, and two metastasis of lung carcinoma were confi rmed. Statistics The mean values of the intracellular pH and metabolite ratios Data measurement and analysis between the normal-appearing brain tissue group (normal) and All measurements were performed on a clinical 1.5 T MR the brain tumours group (tumour) were compared by using the scanner Siemens Magnetom Symphony (Siemens, Erlangen, Ger- two-tailed paired t-test. p value of less than 0.05 was considered many) using the P quadrature volume head coil (Stark contrast, signifi cant. The statistical analysis was performed by using the Erlangen, Germany) operating at 25.7 MHz for P-signal detec- SPSS v.15.0 software package (Chicago, IL, USA). tion. The whole examination protocol was in total duration of 20 min and consisted of multi-slice T -weighted MRI (2D spin multi- Results echo, 24 slices with a 3.5 mm slice thickness and 5 mm interslice 2 31 gap, 245x280 mm fi eld of view, repetition time TR/echo time Comparing P MRS metabolite ratios and pH in the tumour TE = 3000/51 ms) and P MRS. For multivoxel spectroscopy, and in the normal-appearing brain tissue (Fig. 2), proved metabolic the 3D free induction decay (FID) chemical shift imaging (CSI) differences of these groups. In tumorous tissue, signifi cantly in- sequence was carried out in an axial plane with the following pa- creased phosphomonoesters ratios (PME/PDE, PME/PCr, PME/Pi, rameters: TR/TE = 290/2.3 ms, CSI grid 8x8x8 interpolated to and PME/ATP) and signifi cantly decreased phosphodiesters ratios 16x16x16, nominal voxel size 32.5x32.5x32.5 mm . All spec- (PDE/PCr, PDE/Pi, and PDE/ATP) was observed. Furthermore, a troscopic data were evaluated in jMRUI 5.0 software (10). The signifi cant decline in PCr/Pi and PCr/ATP ratios and a signifi cant software allows for overlapping the T -weighted MRI with the increase in Pi/ATP were shown, compared to the normal-appear- spectroscopic grid, enabled to select voxels in the desired tissue ing brain tissue. The intracellular pH in the brain tumours tended area. For each of study participants, one voxel inside the tumour towards alkalization. The highest differences between tumorous and one voxel in normal-appearing brain tissue (contralaterally to and normal brain tissue refl ected PME/PDE (p < 0.001), followed the tumour voxel) were selected (Fig.1). For data quantifi cation, by PME/PCr with PME/ATP (p = 0.001; p = 0.007), and PCr/Pi apriori information fi les were entered, which were implemented (p = 0.010) ratios (Fig. 2). into the AMARES evaluation algorithm (10). After zero-fi lling 489 Bratisl Med J 2020; 121 (7) 488 – 492 Fig. 2. Differences between tumorous and normal brain tissue based on P MRS. Visualization of differences between tumours (tumour) and normal-appearing brain tissue (normal) among all P MRS metabolite ratios and pH. The statistical signifi cance of these differences was de- picted as follows: * signifi cant (0.01 ≤ p < 0.05), ** very signifi cant (0.001 ≤ p < 0.01), and *** extremely signifi cant (p < 0.001). Abbreviations: PME = phosphomonoesters, PDE = phosphodiesters, PCr = phosphocreatine, ATP = adenosine triphosphates, Pi = inorganic phosphate. Discussion 16). Furthermore, the basic clinical 1.5 T fi eld strength limits the P MRS detection and thus enables to evaluate only the joined The multivoxel P MRS examination implemented under PME-signal consisting mainly of phosphocholine with phospho- the basic clinical condition on 1.5 T magnetic fi eld strength com- ethanolamine and joined PDE-signal composed of glycerophos- bined with semi-automatic data quantifi cation in jMRUI provides phocholine and glycerophosphoethanolamine (1, 5, 16). Never- a straightforward method for spatial-mapping of P-containing theless, both PME, as well as PDE, are considered as markers for metabolite ratios across the brain. Similar H MRS metabolite tumour detection (1, 4, 5). In our study, a signifi cant elevation mapping of intracranial tumours has been previously studied in in all evaluated PME ratios (PME/PCr, PME/Pi and PME/ATP) our group (12), showing a clinical utility to extend the palette of as well as in PME/PDE ratio in tumours compared to that in the detected metabolites for comprehensive non-invasive tumorous normal-appearing brain tissue was observed. On contrary, all PDE tissue characterization. Using P MRS enables to provide infor- ratios (PDE/PCr, PDE/Pi, and PDE/ATP) were signifi cantly de- mation about the energetic and phospholipid metabolism or in- creased in tumours. Considering elevated PME ratios as a marker tracellular pH, as was previously declared (4,5,9). Although the of membrane synthesis (4, 9, 17) combined with the decline in most useful P-metabolite marker for clinical oncology was not PDE ratios as an indicator of membrane catabolism (1, 5, 18), the agreed yet (1, 4, 6). tumorous tissue examined in our study manifested a shift toward membrane synthesis and tumour growth. According to another Membrane phospholipids metabolism experimental tumour studies, an increase of the PME is related to Cell membranes, especially membrane phospholipids, which malignant progression, relapses, and increasing grade of tumour are involved in membrane structure, signal transduction mecha- malignancy, and the signifi cant increase in the PDE may indicate nisms, regulation of cell proliferation, and lipoprotein metabo- therapy-induced membrane degradation in dying cells (4, 17, 18). lism are important for tumorigenesis (13, 14). Although it is not However, a typical feature of phosphorus spectra of proliferating feasible to directly measure membrane phospholipids due to their intracranial tumours are predominant PME peaks, but contrary to fi xed membrane integration, using P MRS is possible to detect healthy tissue, also increased values of PDE peaks as the result of phospholipids precursors as well as their degradation products (15, the overall metabolism of higher cellular density (1, 5, 9). There- 490 Hnilicova P et al. Noninvasive study of brain tumours metabolism using phosphorus-31 MR spectroscopy xx fore, the ratio of PME/PDE can serve as an index of the metabo- 11). The Pi position is determined by its form of conjugated pair of - 2- lism of membrane phospholipid and refl ect changes in the rate of anions H PO and HPO , which change rapidly depending on the 2 4 4 membrane synthesis or metabolic turnover (1, 9, 16). dissociation reactions (4, 24). Despite the assumption that tumour cells have acidic metabolism due to an increased lactate production, Cellular energy metabolism measurements of intracellular tumours pH by P MRS reported Cellular energy metabolism is a crucial determinant of cell pro- more alkaline values (11, 22, 25). In this pilot study, a tendency liferation or cell death (5, 14). Therefore, P MRS is regarded as of tumour alkalization (pH = 7.16 ± 0.1) compared to normal-ap- a useful clinical application enabling the non-invasive disclosure pearing brain tissue (pH = 7.04 ± 0.03) was also observed. Several of cells energetic processes through ATP, PCr, and Pi, compounds studies reported intracellular pH in the healthy human brain in the that are metabolically interconnected (14, 19, 20). ATP hydrolysis range of 7.01‒7.07 (4, 9, 25). Furthermore, in intracranial tumours is the reaction, by which chemical energy from the high-energy it was previously described that solid tumorous part was alkaline phosphoanhydridic bonds in ATP is released, and by which the Pi (pH = 7.15‒7.48), contrary to the acidic (pH = 6.45‒6.87) extra- and ADP are formed (19, 21). Whereas this energy is utilized for cellular tumour environment (9, 25, 26). It may be explained by most cellular processes and functions (14, 21, 22), ATP is consid- the function of tumour cells to export the excessive lactate along ered as a marker of the momentary energetic state of the cell (1, with protons via cell-specifi c monocarboxylate transporters in- 22). The metabolic pathways of ATP and PCr are tightly coupled stead of utilizing it as a nutrient (13, 14, 26). Besides, in tumour via the enzyme creatine kinase that generates ATP by the transfer cells, an enhanced activity of H extruding pathways was found, + + of a phosphate group from PCr to ADP under conditions of higher like the Na /H exchanger or by buffering intracellular protons energy demands and/or insuffi cient ATP production through oxi- via the transmembrane carbonic anhydrases, both counteracting dative phosphorylation in the cellular mitochondria (20, 21, 23). the intracellular proton accumulation (15, 22). Consequently, the Although the PCr is in the brain tissue under physiological condi- extracellular environment gets more acidic, enhancing the inva- tion stable, it is rapidly utilized for fast ATP recovery in the case of siveness of tumour cells, and promoting angiogenesis (21, 23). energetic needs (1, 19, 22). Therefore, PCr is considered as a sensi- tive marker for mitochondrial effi ciency. Its decline usually suggests Conclusion a mitochondrial dysfunction and/or tissue excessive requirements (5, 14, 23). Under physiological brain cell conditions, the majority This pilot study declared the usefulness of multivoxel P MRS of glucose is metabolized oxidatively, leading to a stable PCr-ATP- examination applied under the basic 1.5 T fi eld strength as a clini- Pi chemical exchange system (13, 14, 21). But tumour cells have cally feasible method for P-containing metabolite mapping across a higher demand for metabolic energy than normal cells (4, 5, 9). the brain and thus a useful diagnostic tool enabling tumorous tis- Nevertheless, they rely mostly on anaerobic glycolysis (Warburg sue characterization. From all investigated parameters, the PME effect) even in the presence of adequate oxygen supply (21, 22). (mostly PME/PDE), followed by PCr/Pi ratios seems to be the Tumour cells meet their increased demand for ATP by increasing most useful markers for the detection of the brain tumorous tissue. glucose transport into the cells and by the acceleration of glycoly- References sis (14, 21, 23). Recent studies of metabolic pathways showed that in the presence of abundant nutrients, anaerobic glycolysis could 1. Wijnen JP. Multi-nuclear magnetic resonance spectroscopy of human potentially produce ATP in greater amounts and faster than mito- brain tumours: Radboud University Nijmegen 2010, 193. chondrial oxidative phosphorylation (21, 23). In this pilot study 2. Faghihi R, Zeinali-Rafsanjani B, Mosleh-Shirazi MA et al. Magnetic of brain tumours, the PCr/Pi and PCr/ATP ratios were signifi cant- Resonance Spectroscopy and its Clinical Applications: A Review. 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Java-based graphical user interface for MRUI, a software package for Molecular Basis of Tumor Metabolism: a Continuing Challenge Since quantitation of in vivo/medical magnetic resonance spectroscopy signals. Warburg’s Discovery. Cell Physiol Biochem 2011; 28 (5): 771‒792. Comput Biol Med 2001; 31 (4): 269‒286. 22. Beloueche-Babari M, Chung YL, Al-Saffar NMS, Falck-Miniotis 11. Zhang X, Lin Y, Gillies RJ. Tumor pH and Its Measurement. J Nucl M, Leach MO. Metabolic assessment of the action of targeted cancer Med 2010; 51 (8): 1167–1170. therapeutics using magnetic resonance spectroscopy. Brit J Cancer 2010; 102 (1): 1‒7. 12. Hnilicová P, Richterová R, Kantorová E, Bittšanský M, Baranovičová E, Dobrota D. Proton MR spectroscopic imaging of hu- 23. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The man glioblastomas at 1.5Tesla. Gen Physiol Biophys 2017; 36: 531–537. Biology of Cancer: Metabolic Reprogramming Fuels Cell Growth and Proliferation. Cell Metab 2008; 7 (1): 11‒20. 13. 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China: InTech, Chapter 15, MR-Based Methods 2012; 205‒216. for pH Measurement in Brain Tumors: Current Status and Clinical Poten- 17. van der Kemp WJM, Klomp DWJ, Wijnen JP. 31P T2s of Phos- tial 2011; 288‒302. phomonoesters, Phosphodiesters, and Inorganic Phosphate in the Human Brain at 7T. Magnetic Resonance in Medicine. 2018; 80: 29–35. Received February 17, 2020. Accepted March 26, 2020. 18. Chawla S, Kim S, Loevner LA. Proton and Phosphorous MR Spec- troscopy in Squamous Cell Carcinomas of the Head and Neck. Acad Ra- diol 2009; 16 (11): 1366–1372. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bratislava Medical Journal Unpaywall

Noninvasive study of brain tumours metabolism using phosphorus-31 magnetic resonance spectroscopy

Hnilicova, P.; Richterova, R.; Zelenak, K.; Kolarovszki, B.; Majercikova, Z.; Hatok, J.
Bratislava Medical Journal – Jan 1, 2020
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DOI: 10.4149/BLL_2020_080 Bratisl Med J 2020; 121 (7) 488 – 492 A PILOT STUDY Noninvasive study of brain tumours metabolism using phosphorus-31 magnetic resonance spectroscopy 1 2 3 2 4 4 Hnilicova P , Richterova R , Zelenak K , Kolarovszki B , Majercikova Z , Hatok J Biomedical Centre Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia. [email protected] ABSTRACT Phosphorus-31 magnetic resonance spectroscopy ( P MRS) is currently not accepted as a diagnostic tool in the neuro-oncological practice, although it provides useful non-invasive information about biochemical processes ongoing in the intracranial tumours. This pilot study was aimed to present the diagnostic capability of the P MRS in brain tumour examination, even its application on clinical 1.5T MR scanner. Seven patients with brain tumorous lesions (four glioblastomas, one ependymoma, and two lung metastasis) underwent multivoxel in vivo P MRS performed on clinical 1.5 T MR scanner within measurement time of 20 minutes. Comparing two selected voxels, one in the tumour and the other one in the normal-appearing brain tissue, enabled to investigate their metabolic differences. Enhanced markers of membrane phospholipids synthesis (signifi cantly increased phosphomonoesters ratios) than markers of their degradation (signifi cantly decreased phosphodiesters ratios) manifested a higher cell proliferation ongoing in tumours. High energetic tumorous tissue demands leading to anaerobic metabolic turnover were present as a signifi cant decline in phosphocreatine ratios and adenosine triphosphates. Intracellular pH evaluation showed a tumorous tendency to alkalize. P MRS enables the non-invasive metabolic characterization of intracranial tumours and thus appears to be a clinically useful method for the determination of ongoing tumour pathomechanisms (Fig. 2, Ref. 26). Text in PDF www.elis.sk KEY WORDS: brain tumour, P MRS, 1.5 Tesla; energetic metabolism. Introduction and cell membrane phospholipids composition through phospho- monoesters (PME), and phosphhodiesters (PDE) (1, 3). Although Phosphorus-31 MR Spectroscopy ( P MRS) is a very use- it can be demonstrated that different concentrations of various ful clinical tool with great potential mostly in neuro-oncological phosphorus metabolites measured spectroscopically between the practice, because it provides non-invasive insight into the com- brain tumours and normal brain tissue (3, 4, 5), the clinical ap- position of the examined tissue in vivo, and allows to obtain in- plication of P MRS for the examination of brain tumours has formation about cellular energy or membrane metabolism (1, 2). limitations, until recently (1, 2, 6). One reason for this may be the 31 31 P MRS enables direct measurement of energy metabolites like relatively low sensitivity of P MRS at 1.5 T fi eld strength (1, 7). phosphocreatine (PCr), adenosine-triphosphates (ATP), inorganic Therefore, the longer scan time or larger single-voxels had to be phosphate (Pi), as well as and indirectly evaluate intracellular pH chosen (~ 40‒100 cc), making it impossible to study heteroge- neity of the intracranial tumours (4, 8, 9). In this pilot study, the P MRS signal was obtained using the multivoxel technique with relatively small voxel volume (~ 27 cm ) and in a tolerable scan Biomedical Centre Martin, Jessenius Faculty of Medicine in Martin, Co- menius University in Bratislava, Martin, Slovakia, Clinic of Neurosur- time of 20 minutes, including the scanning for localization. This gery, Jessenius Faculty of Medicine in Martin, Comenius University in 31 pilot study aimed to achieve clinically feasible P MRS brain tu- Bratislava, Martin, Slovakia, Clinic of Radiology, Jessenius Faculty of mours examination, despite application on basic clinical 1.5 T MR Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia, 4 scanner, showed the medical relevance of non-invasive metabolic and Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Martin, Slovakia characterization of intracranial tumours and investigated the most useful P MRS parameters, which may determinate the ongoing Address for correspondence: P. Hnilicova, Ing, PhD, Biomedical Centre Martin, Jessenius Faculty of Medicine in Martin, Comenius University in tumour pathomechanisms. Bratislava, Mala Hora 4D, SK-036 01 Martin, Slovakia. Phone: +421.43.2633 660 Materials and methods Acknowledgments: This work was supported by the Slovak Research and Development Agency under Contract No. APVV-18-0088 and by the Study participants project implementation: “Centre of Excellence for Research in Personal- All examinations of this P MRS pilot study were carried out ized Therapy (Cevypet),” Itms: 26220120053 supported by the Operational Programme Research and Innovation funded by the ERDF. in compliance with the local institutional medical ethics com- Indexed and abstracted in Science Citation Index Expanded and in Journal Citation Reports /Science Edition Hnilicova P et al. Noninvasive study of brain tumours metabolism using phosphorus-31 MR spectroscopy xx Fig. 1. P MRS of the tumorous and normal-appearing brain tissue. For all participants, one voxel in the tumour (red voxel) and another one in normal-appearing brain tissue (green voxel) were chosen, and P MR spectra were evaluated in jMRUI. In fi tted spectra measured on 1.5T are detectable peaks of phosphomonoesters (PME), phosphodiesters (PDE), adenosine triphosphates (α-,β-,γ-ATP), phosphocreatine (PCr, and inorganic phosphate (Pi). mittee and after obtaining a written informed consent from study (4096 points) and 5 Hz exponential line broadening, the P MR participants. In the study, seven patients were enrolled (the mean spectra were obtain (Fig. 1). The integrals of measured P me- age: 65 ± 12 years; 3 males and 4 females), who had suspected tabolites were evaluated, and the following ratios were calculated: brain tumour lesions detected during the standard clinical MRI PCr/Pi, PCr/ATP, Pi/ATP, PME/PDE, PME/PCr, PME/Pi, PME/ examination. All the patients underwent P MRS before any of ATP, PDE/PCr, PDE/Pi, and PDE/ATP. The intracellular pH of the the surgical nor therapeutic treatment procedures. After a sub- examined tissue was calculated by using the chemical shift of Pi sequent histological analysis, four grade IV glioblastomas, one relative to the PCr peak (10, 11). grade III ependymomas, and two metastasis of lung carcinoma were confi rmed. Statistics The mean values of the intracellular pH and metabolite ratios Data measurement and analysis between the normal-appearing brain tissue group (normal) and All measurements were performed on a clinical 1.5 T MR the brain tumours group (tumour) were compared by using the scanner Siemens Magnetom Symphony (Siemens, Erlangen, Ger- two-tailed paired t-test. p value of less than 0.05 was considered many) using the P quadrature volume head coil (Stark contrast, signifi cant. The statistical analysis was performed by using the Erlangen, Germany) operating at 25.7 MHz for P-signal detec- SPSS v.15.0 software package (Chicago, IL, USA). tion. The whole examination protocol was in total duration of 20 min and consisted of multi-slice T -weighted MRI (2D spin multi- Results echo, 24 slices with a 3.5 mm slice thickness and 5 mm interslice 2 31 gap, 245x280 mm fi eld of view, repetition time TR/echo time Comparing P MRS metabolite ratios and pH in the tumour TE = 3000/51 ms) and P MRS. For multivoxel spectroscopy, and in the normal-appearing brain tissue (Fig. 2), proved metabolic the 3D free induction decay (FID) chemical shift imaging (CSI) differences of these groups. In tumorous tissue, signifi cantly in- sequence was carried out in an axial plane with the following pa- creased phosphomonoesters ratios (PME/PDE, PME/PCr, PME/Pi, rameters: TR/TE = 290/2.3 ms, CSI grid 8x8x8 interpolated to and PME/ATP) and signifi cantly decreased phosphodiesters ratios 16x16x16, nominal voxel size 32.5x32.5x32.5 mm . All spec- (PDE/PCr, PDE/Pi, and PDE/ATP) was observed. Furthermore, a troscopic data were evaluated in jMRUI 5.0 software (10). The signifi cant decline in PCr/Pi and PCr/ATP ratios and a signifi cant software allows for overlapping the T -weighted MRI with the increase in Pi/ATP were shown, compared to the normal-appear- spectroscopic grid, enabled to select voxels in the desired tissue ing brain tissue. The intracellular pH in the brain tumours tended area. For each of study participants, one voxel inside the tumour towards alkalization. The highest differences between tumorous and one voxel in normal-appearing brain tissue (contralaterally to and normal brain tissue refl ected PME/PDE (p < 0.001), followed the tumour voxel) were selected (Fig.1). For data quantifi cation, by PME/PCr with PME/ATP (p = 0.001; p = 0.007), and PCr/Pi apriori information fi les were entered, which were implemented (p = 0.010) ratios (Fig. 2). into the AMARES evaluation algorithm (10). After zero-fi lling 489 Bratisl Med J 2020; 121 (7) 488 – 492 Fig. 2. Differences between tumorous and normal brain tissue based on P MRS. Visualization of differences between tumours (tumour) and normal-appearing brain tissue (normal) among all P MRS metabolite ratios and pH. The statistical signifi cance of these differences was de- picted as follows: * signifi cant (0.01 ≤ p < 0.05), ** very signifi cant (0.001 ≤ p < 0.01), and *** extremely signifi cant (p < 0.001). Abbreviations: PME = phosphomonoesters, PDE = phosphodiesters, PCr = phosphocreatine, ATP = adenosine triphosphates, Pi = inorganic phosphate. Discussion 16). Furthermore, the basic clinical 1.5 T fi eld strength limits the P MRS detection and thus enables to evaluate only the joined The multivoxel P MRS examination implemented under PME-signal consisting mainly of phosphocholine with phospho- the basic clinical condition on 1.5 T magnetic fi eld strength com- ethanolamine and joined PDE-signal composed of glycerophos- bined with semi-automatic data quantifi cation in jMRUI provides phocholine and glycerophosphoethanolamine (1, 5, 16). Never- a straightforward method for spatial-mapping of P-containing theless, both PME, as well as PDE, are considered as markers for metabolite ratios across the brain. Similar H MRS metabolite tumour detection (1, 4, 5). In our study, a signifi cant elevation mapping of intracranial tumours has been previously studied in in all evaluated PME ratios (PME/PCr, PME/Pi and PME/ATP) our group (12), showing a clinical utility to extend the palette of as well as in PME/PDE ratio in tumours compared to that in the detected metabolites for comprehensive non-invasive tumorous normal-appearing brain tissue was observed. On contrary, all PDE tissue characterization. Using P MRS enables to provide infor- ratios (PDE/PCr, PDE/Pi, and PDE/ATP) were signifi cantly de- mation about the energetic and phospholipid metabolism or in- creased in tumours. Considering elevated PME ratios as a marker tracellular pH, as was previously declared (4,5,9). Although the of membrane synthesis (4, 9, 17) combined with the decline in most useful P-metabolite marker for clinical oncology was not PDE ratios as an indicator of membrane catabolism (1, 5, 18), the agreed yet (1, 4, 6). tumorous tissue examined in our study manifested a shift toward membrane synthesis and tumour growth. According to another Membrane phospholipids metabolism experimental tumour studies, an increase of the PME is related to Cell membranes, especially membrane phospholipids, which malignant progression, relapses, and increasing grade of tumour are involved in membrane structure, signal transduction mecha- malignancy, and the signifi cant increase in the PDE may indicate nisms, regulation of cell proliferation, and lipoprotein metabo- therapy-induced membrane degradation in dying cells (4, 17, 18). lism are important for tumorigenesis (13, 14). Although it is not However, a typical feature of phosphorus spectra of proliferating feasible to directly measure membrane phospholipids due to their intracranial tumours are predominant PME peaks, but contrary to fi xed membrane integration, using P MRS is possible to detect healthy tissue, also increased values of PDE peaks as the result of phospholipids precursors as well as their degradation products (15, the overall metabolism of higher cellular density (1, 5, 9). There- 490 Hnilicova P et al. Noninvasive study of brain tumours metabolism using phosphorus-31 MR spectroscopy xx fore, the ratio of PME/PDE can serve as an index of the metabo- 11). The Pi position is determined by its form of conjugated pair of - 2- lism of membrane phospholipid and refl ect changes in the rate of anions H PO and HPO , which change rapidly depending on the 2 4 4 membrane synthesis or metabolic turnover (1, 9, 16). dissociation reactions (4, 24). Despite the assumption that tumour cells have acidic metabolism due to an increased lactate production, Cellular energy metabolism measurements of intracellular tumours pH by P MRS reported Cellular energy metabolism is a crucial determinant of cell pro- more alkaline values (11, 22, 25). In this pilot study, a tendency liferation or cell death (5, 14). Therefore, P MRS is regarded as of tumour alkalization (pH = 7.16 ± 0.1) compared to normal-ap- a useful clinical application enabling the non-invasive disclosure pearing brain tissue (pH = 7.04 ± 0.03) was also observed. Several of cells energetic processes through ATP, PCr, and Pi, compounds studies reported intracellular pH in the healthy human brain in the that are metabolically interconnected (14, 19, 20). ATP hydrolysis range of 7.01‒7.07 (4, 9, 25). Furthermore, in intracranial tumours is the reaction, by which chemical energy from the high-energy it was previously described that solid tumorous part was alkaline phosphoanhydridic bonds in ATP is released, and by which the Pi (pH = 7.15‒7.48), contrary to the acidic (pH = 6.45‒6.87) extra- and ADP are formed (19, 21). Whereas this energy is utilized for cellular tumour environment (9, 25, 26). It may be explained by most cellular processes and functions (14, 21, 22), ATP is consid- the function of tumour cells to export the excessive lactate along ered as a marker of the momentary energetic state of the cell (1, with protons via cell-specifi c monocarboxylate transporters in- 22). The metabolic pathways of ATP and PCr are tightly coupled stead of utilizing it as a nutrient (13, 14, 26). Besides, in tumour via the enzyme creatine kinase that generates ATP by the transfer cells, an enhanced activity of H extruding pathways was found, + + of a phosphate group from PCr to ADP under conditions of higher like the Na /H exchanger or by buffering intracellular protons energy demands and/or insuffi cient ATP production through oxi- via the transmembrane carbonic anhydrases, both counteracting dative phosphorylation in the cellular mitochondria (20, 21, 23). the intracellular proton accumulation (15, 22). Consequently, the Although the PCr is in the brain tissue under physiological condi- extracellular environment gets more acidic, enhancing the inva- tion stable, it is rapidly utilized for fast ATP recovery in the case of siveness of tumour cells, and promoting angiogenesis (21, 23). energetic needs (1, 19, 22). Therefore, PCr is considered as a sensi- tive marker for mitochondrial effi ciency. Its decline usually suggests Conclusion a mitochondrial dysfunction and/or tissue excessive requirements (5, 14, 23). Under physiological brain cell conditions, the majority This pilot study declared the usefulness of multivoxel P MRS of glucose is metabolized oxidatively, leading to a stable PCr-ATP- examination applied under the basic 1.5 T fi eld strength as a clini- Pi chemical exchange system (13, 14, 21). But tumour cells have cally feasible method for P-containing metabolite mapping across a higher demand for metabolic energy than normal cells (4, 5, 9). the brain and thus a useful diagnostic tool enabling tumorous tis- Nevertheless, they rely mostly on anaerobic glycolysis (Warburg sue characterization. From all investigated parameters, the PME effect) even in the presence of adequate oxygen supply (21, 22). 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Published: Jan 1, 2020

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