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IJCRR - 6(9), May, 2014

Pages: 41-57

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PHOSPHORUS-31 MR SPECTROSCOPY OF THE HUMAN BRAIN : TECHNICAL ASPECTS AND BIOMEDICAL APPLICATIONS

Author: Celi S. Andrade, Maria C. G. Otaduy, Eun J. Park, Claudia C. Leite

Category: Healthcare

Abstract:Phosphorus-31 magnetic resonance spectroscopy (31P-MRS) is a non-invasive method that provides useful information about metabolism and phosphoenergetic status in both physiologic and pathologic conditions of the human brain. With the progressive advances in magnetic resonance imaging (MRI) technology, particularly with higher magnetic field strengths, 31P-MRS has been more easily implemented and more readily available in the past few years, which has increasingly extended its access and favored its use in different research fields. However, the current knowledge about this advanced neuroimaging modality is still scarce and fragmented in the literature. Hence, in order to contribute to future researches and to shorten the gap between neuroscientific studies and common clinical routines, we present a comprehensive review about the basic technical aspects and biomedical applications of 31P-MRS.v

Keywords: 31P-MRS, MRI, phosphorus spectroscopy, magnetic resonance imaging, neurometabolism, energetics, phospholipids, pH, magnesium, cell membrane

Full Text:

INTRODUCTION

Magnetic resonance spectroscopy (MRS) offers the unique ability to noninvasively measure, in vivo, the chemical composition of biological tissues. This method can be combined to the anatomic information provided by magnetic resonance imaging (MRI), giving functional data that can improve the understanding of the pathophysiological processes at a molecular level (1,2) . Most MRS studies have focused in the evaluation of proton (1H) signal, due to the intrinsic physical characteristics of this nucleus and because it is possible to perform the proton spectroscopic acquisition with the same coil used to obtain conventional magnetic resonance (MR) images. However, with the progressive technical improvements in recent years, such as the development of different MRS pulse sequences, improvement of data processing, as well as commercial availability of high and ultra-high magnetic field scanners, phosphorus-31 magnetic resonance spectroscopy (31P-MRS) has been more easily implemented (3-5) . Our purpose is to provide a comprehensive overview about the concepts, technical aspects and implementation of 31P-MRS. Thereafter, we summarize the metabolites identified and their roles in brain physiology and pathology. The aim of this review is not to be an exhaustive compendium, but rather to guide and familiarize researchers and students with the basic principles of 31P-MRS

TECHNICAL ASPECTS

The nucleus has an intrinsic magnetic spin that is resultant from the uneven number of protons or neutrons. When exposed to a strong magnetic field, there is an alignment of these spins in a parallel or antiparallel direction to the applied field. If a specific radiofrequency pulse is applied for few microseconds (with the Characteristic precession frequency for each nucleus studied), there is a misalignment of the total Magnetization vector. When the radiofrequency (RF) pulse ceases, there is a realignment of the magnetic field, which generates a small electric signal, known as free induction decay (FID). This signal is detected by a RF coil, and, by means of transformation from time domain to frequency domain through a mathematical equation (Fourier transform), the spectral graph is obtained (6,7) . The precession frequency of the nuclei can be calculated by the Larmor equation, and it is proportional to the intensity of the magnetic field and to the gyromagnetic constant, which is specific to each chemical element or isotope. The nuclei within the molecules, however, suffer from small shifts of the precession frequency due to the magnetic field generated by adjacent electrons, and this phenomenon is called chemical shift. Each molecule has then specific chemical shifts, measured in Hertz (Hz) or parts per million (ppm) (6) . The result of this process is not an anatomical image, but a spectral graph, in which each metabolite has its specific position corresponding to the variation of resonance frequency (chemical shift), expressed in ppm on the horizontal scale (X axis), while the amplitude of each metabolite is represented in the vertical axis (Y axis), which allows their relative quantification (2,7-11) . Albeit not fully explored, 31P-MRS provides unique and relevant information about the bioenergetics state, the composition of the cell membrane, intracellular pH and the concentration of magnesium (Mg 2+), which cannot be obtained with other conventional or spectroscopic techniques (12) . However, this method has not been implemented widespread because it is necessary that the MR equipment is prepared to work in the resonance frequency of the phosphorus-31 ( 31P) nucleus, and it is also required a dedicated brain coil (Fig. 1) to detect the specific signal (12,13) . Just like the 1H nucleus, the 31P nucleus also represents a nuclear spin number of ½, capable to produce an MRI signal (Table 1). However, because of the physical characteristics of 31P (for example, greater mass), its gyromagnetic ratio (that indicates the level of the interaction between the nucleus and the magnetic field of the MRI scanner) is approximately 2.5 times lower than for 1H. This results in a lower resonance frequency - 51.7 MHz as compared to 127.7 MHz in a field of 3.0 T - and in a much lower sensitivity, only 6.6% when compared to 1H signal (14,15) . These factors imply that to obtain a satisfactory 31P spectrum, comparable to 1H spectrum, it is necessary to repeat the same acquisition several times in order to increase the signal-to-noise ratio (SNR) of the spectrum, resulting in a much longer acquisition time (16) . It should also be noted that the concentration of 31P metabolites (1- 14 mM) is lower than that of the metabolites detected in the 1H-MRS, which makes it even more difficult to obtain a 31P spectrum with sufficient signal intensity (Table 2) (14, 17-21) . Due to the short transverse relaxation time (T2) of the 31P metabolites, the techniques most commonly used to obtain the 1H spectrum, like stimulated echo acquisition mode (STEAM) and point resolved spectroscopy (PRESS), which are based on the formation of an echo, are not recommended for the acquisition of the 31P spectrum. These techniques require a minimum TE around 8 to 20 ms, which would result in a large loss of signal due to the transverse relaxation times for most metabolites. For this reason, the most commonly used techniques are the image selected in vivo spectroscopy (ISIS) or the pulse acquire technique (direct acquisition of the FID signal immediately after the RF pulse), which allow to obtain the signal with a minimum TE around 300 ?s (22,23) . In the ISIS technique, the localization of a slice is made through the acquisition of two FIDs, one generated after the application of a 180º pulse selective for one dimension, and the other generated without the prior application of this pulse. The subtraction of these two signals corresponds to the signal of one single slice (onedimensional, 1D ISIS). For the localization of a volume (three-dimensional, 3D ISIS), it is necessary to acquire eight FIDs. The difference is only whether the selective180º pulse is applied or not in a determined direction, resulting in eight different combinations. In practice, instead of working with subsequent subtraction of the signals, they are acquired with alternating phases (phase cycling), and only the signal of interest is recorded (24,25) . On the other hand, the pulse acquire technique allows only the selection of one single slice, but not a volume, the reason why it is not suitable for the study of minute structures. However, when combined with twodimensional (2D) or three-dimensional (3D) acquisition of the spectrum (acquisition of multiple volumes of interest, or voxels), it can also be used for evaluation of smaller volumes. The metabolites present in the spectral curve as single, double, triple or multiple peaks. The factor that generates the division of a resonance signal in two or more peaks in the spectrum is the interaction known as J coupling between adjacent nuclei in the same molecule (4) . The J coupling effect can be reduced or completely canceled in the spectrum if, during the signal acquisition, the nucleus responsible for this effect is irradiated by a second RF channel. This technique, used to reduce the J coupling effect, is known as decoupling (26) . 31P-MRS in vivo presents two double peaks and one triple peak of the adenosine triphosphate molecule (ATP) (27) . However, in this case, the cause of the J coupling effect is the interaction of a 31P nucleus with another 31P nucleus, what precludes the reduction of this effect by decoupling in the frequency of 1H nucleus. However, it is observed that the interaction between 1H and 31P within the same molecules, or even with the adjacent water, is large enough to cause broadening of the peaks observed in the 31P spectrum. This effect is particularly important for the phosphodiester peak (Fig. 2), but may also have a lesser effect on the intensity of the other peaks (28,29-33) . Through previous irradiation of the 1H nucleus, it is possible to transfer some of the energy absorbed by 1H to 31P, and thus increase the basic signal from the 31P nuclei. This effect is known as nuclear Overhauser enhancement (nOe). The intensity of the basic signal can be increased depending on the relation between the gyromagnetic constants of the irradiated and the observed nucleus, on the relaxation times of the nuclei, and on the chosen irradiation method. The nOe effect can also be produced when it is used only a decoupling pulse (because the 1H spins absorb energy that can be transferred to the 31P spins), and thus it could become an extra factor of variability that may affect the reproducibility of the method (34-36) . Therefore, it is recommended that, whenever a decoupling technique is applied, the1H nuclei should be irradiated prior to the acquisition of 31P-MRS, in order to produce a larger and more controlled nOe effect (Fig. 2). The spectroscopic examinations benefit from the use of higher magnetic field strengths that increase the sensitivity of the study and the spectral resolution, with at least linear increases in SNR. On the other hand, there is also an increase in distortions of the field related to the effects of magnetic susceptibility, which can be minimized with a procedure known as "shimming", held in the preparation phase of the exam with the aim to increase the homogeneity of the magnetic field within the region of interest (37- 39) . In order to achieve a spatial resolution that allows assessment of multiple regions of the brain and still offers sufficient SNR, the ideal strategy is to acquire 3D volumes, with a non-selective and adiabatic radiofrequency pulse, where the spatial localization is done with application of phase encoding gradients(16,40-42) . Figure 3 shows the planning of a 31P-MRS acquired with a three-dimensional chemical shift imaging (3DCSI) sequence with a multivoxel matrix that had total exam duration of 36 minutes. However, despite the need for adjustments of multiple parameters and the technical challenges for the acquisition of 31P-MRS, there are also some advantages of this modality of spectroscopy. A convenience of the 31P-MRS, as compared to 1H-MRS, is that, because it does not present signals from the water molecules, it is not necessary to apply saturation methods (6) . Another convenience in favor of 31P-MRS is that it presents a large range of dispersion of the chemical shift, around 30 ppm (parts per million) or 2000 Hz (at 3.0 T) (43) . This contributes to a good spectral resolution, with a satisfactory differentiation between the different resonances in the spectrum and an easy identification of the various metabolites, explained in detail in the next section.

METABOLITES

The great interest in 31P-MRS relies on the role that the phosphorylated molecules play in brain biochemistry, energy metabolism, and composition of cell membranes. Three main types of information can be obtained with this examination. The first one is related to the energy pool itself, with the resonances of phosphocreatine (PCr), inorganic phosphate (Pi) and the three isotopomers of adenosine triphosphate (α-, β-, and γ- ATP). Second, the phospholipids, represented by phosphomonoesters (PME) and phosphodiesters (PDE), inform about the synthesis and degradation of the cell membrane, respectively. Finally, it is possible to obtain the value of intracellular pH and the concentration of magnesium (Mg2+ ) (11). Figure 4 shows a typical 31P spectrum of the brain with identification of the main metabolites. Adenosine Triphosphate and Phosphocreatine 31P-MRS is able to distinguish ATP isotopomers in the form of three distinct peaks, from left to right in the curve: a doublet γ-ATP, a doublet α-ATP and a triplet β-ATP(43) .The ATP is mainly synthesized in the mitochondria (Fig. 5) from the oxidative phosphorylation of ADP (adenosine diphosphate) catalyzed by the enzyme ATP-synthase, and to a lesser extent by mechanisms of glycolysis, besides the synthesis from the creatinekinase reaction (44-46) . The PCr peak is the most prominent of the 31P spectrum of the brain, resonates at zero ppm, and, therefore, it is the reference to the localization of the other metabolites. PCr is a high-energy molecule, very abundant in the neural tissues, serving as a buffer to maintain a constant level of ATP and to support the demand of energy through the reaction catalyzed by creatinekinase (47) , as illustrated in Figure 5. Membrane Phospholipids The phosphomonoesters (PME) represent the anabolic activity of cell membranes and their main constituents are the phosphoethanolamine (PE) and phosphocholine (PC), precursors of membrane synthesis. The phosphodiesters (PDE) indicate, in turn, the catabolism of cell membranes, and are constituted by their degradation products, the glycerophosphoethanolamine (GPE) and glycerophosphocholine (GPC). The PDE are products of  the phospholipase enzyme activity and are converted into PME by the activity of the enzyme phosphodiesterase. The ratio PME / PDE is an indicator of the turnover of cell membranes, and it is representative of changes in the phospholipids double-layer (48,49) . The functioning and the plasticity of the brain are dramatically influenced by the physical and chemical properties of the neuronal membrane. This membrane is formed by a double layer of phospholipids, with immersed receptors, ion channels and other proteins involved in signal transduction and maintenance of cellular homeostasis (50) . The structure of the cell membrane determines its fluidity, as well as the number, density and affinity of receptors that modulate the signaling mechanisms. In addition, phospholipids serve as a substrate for the synthesis of intra and intercellular mediators, which indicates their relevance in the mechanisms of neurotransmission (51-54) .

Intracellular pH and Magnesium

The peak of inorganic phosphate (Pi) is localized between the PME and PDE peaks. It is directly involved in the synthesis of ATP (Fig. 5), and its chemical shift relative to PCr peak (δ1) is used to calculate the intracellular pH, according to the formula (55-58):

Modulation of pH in the human brain is a puzzling combination of countless osmotic and metabolic mechanisms that are primarily related to the transport and diffusion of ions, buffer systems, activity of carbonic anhydrase and energy consumption (59-63) . Free cytosolic Mg2+ (pMg) can be estimated by in vivo 31P-MRS from the β-ATP chemical shift (δβ), which in turn depends on the fraction of total ATP linked to Mg2+, according to the equation below (55,64):

Table 3 summarizes the main metabolites obtained with 31P-MRS, indicating their position in the spectral curve and their roles in brain metabolism.

BIOMEDICAL APPLICATIONS

31P-MRS has been used in metabolic evaluation of the heart (65,66), liver (67-69), skeletal muscle (70-72) , and brain (73-75) in humans and animal models. In the investigation of the human brain, in particular, this method has shown some peculiarities in the pattern of physiological distribution of the phosphate metabolites. It was identified higher levels of PCr and PCr/ATP in gray matter compared to the white matter (76,77) . Another study found significant differences in the values of PME and PDE, which were higher in white matter compared to gray matter (78) . On the other hand, it does not seem to exist significant differences in the levels of Pi, intracellular pH or the concentration of Mg2+ between the white and gray matters (18,76). Most authors assume that the tissue specificity (gray matter versus white matter) is more important than the topography of the tissue (for example, occipital lobe versus frontal lobe). There is also no evidence of variations between the cortical or deep gray matters (77-79) . Evidence from studies in animals and humans suggest that the mitochondria undergo progressive morphological and functional changes with aging (80-82). The most consistent findings of studies that evaluated the effects of aging on the quantification of metabolites with 31P-MRS were increased levels of PCr and decreased intracellular pH. These investigations have also reported a reduction in PME and an increase in PDE, probably reflecting reduced synthesis and increased degradation of cell membranes (83-87) . In a study of 34 healthy volunteers, there were no significant differences between males and females for any of the brain metabolites quantified with 31P-MRS (85) . Because human brain is highly dependent on energy production in comparison to other organs, it is not surprising that energetic abnormalities are related to various brain disorders. 31P-MRS has been used in the investigation of a variety of neurological disorders: multiple sclerosis (88,89) , cerebral ischemia (90-92), migraine (93-95), and various neurodegenerative disorders (96-100) . 31P-MRS was also used in various studies to determine the metabolic profile of brain tumors. The results demonstrated trends to alkalinization in different histologic types, such as meningiomas, pituitary adenomas and glial tumors (101-105) . However, it is in the field of neuropsychiatric research that 31P-MRS has played a greater role. Indeed, it is believed that the phospholipid membrane plays a major role in some deterministic hypotheses of these diseases (52, 106, 107) . Some studies have shown a variety of abnormalities, mainly related to the membrane phospholipids and to measurements of intracellular pH in various diseases, such as schizophrenia (48, 108, 109), attention-deficit/ hyperactivity disorder (110, 111) , depression (107, 112) , and bipolar disorder (113) . Most studies of 31P-MRS in epilepsy were directed to the evaluation of mesial temporal sclerosis (MTS) (114- 118) . Despite some controversial findings and methodological differences in previous studies, it is believed that 31P-MRS will become a potential tool to aid in the lateralization of the epileptogenic focus, in the monitoring of clinical treatments, in defining the extent of surgical resection, and to predict the postoperative result (118-121). Our group has recently demonstrated several abnormalities in patients with epilepsy secondary to cortical malformations detected with 31P-MRS at 3.0 T (122) .

CONCLUSIONS

In conclusion, phosphorus metabolites play an important role in brain metabolism. However, the exact mechanisms in which they are involved in different neurological disorders remain to be determined. In the future, 31P-MRS may be a useful diagnostic tool, and may also help in the follow-up of patients and on the decision-making process. New studies are needed to better evaluate this method, and to ultimately shorten the distances between neuroscience and routine clinical practices. We believe that a better understanding of the 31P-MRS methodology and its applications is critical in the development of upcoming researches.

ACKNOWLEDGEMENTS

We are very grateful to FAPESP (São Paulo Research Foundation, ClnAPCe project 05/56464- 9) and CNPq (National Council for Scientific and Technological Development) for funding and support. Dr. Celi Santos Andrade is a recipient of a post-doctoral grant from FAPESP (2012/00398- 1 and 2013/1552-9). Dr. Claudia Costa Leite is supported by CNPq (308267/008-7).

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