After the demonstration of these basic localization methods, some advanced proton MRS techniques were developed to improve the quality of in vivo spectra. Some methods simplify the appearance of spectra for better observation and quantification of one or more metabolites of interest, which can be classified to the spectral editing techniques. Besides spectral editing techniques, the magnetic field stability is another factor that affects the quality of spectra, especially in high-field systems. Even small drift of B0 would lead to spectral distortion, including the broadening of spectral lines and the loss of phase coherence.
Most spectral editing techniques are dependent on the phenomenon of j-coupling.
For example, the doublet of lactate at around 1.3 ppm or the multiplet of GABA uses the concept to get higher spectral SNR. MEGA-PRESS is one of the spectral editing methods, which based on the fundamental spectral acquisition techniques PRESS and combined the ideas of water suppression and j-coupling spectral editing simultaneously. The solvent suppression sequence element MEGA can be placed within any pulse sequence with an element to refocus transverse magnetization, and it utilizes gradients surrounding the frequency selective pulses to dephase transverse
magnetization (Fig. 4(a)) [34]. There are some advantages of MEGA, including its relatively insensitive to the pulse flip angle errors of the frequency selective pulses, not introducing phase distortion to spectral peaks, and easy to implement because of these identical frequency selective pulses. Furthermore, MEGA can be used to suppress several resonances by applying multi-banded pulses [35]. Therefore, some metabolites, such as glutathione (GSH), GABA, and vitamin C are proposed to use this technique to observe their spectra more easily [23, 36]. Even simultaneously editing two metabolites, which is called double editing, is possible [37]. Although spectral editing is a solution to advance the spectral quality for target metabolites, it doubles scan time than traditional techniques and may not be so applicable for clinical uses.
Interleaved Navigator Scan (INS)-PRESS is one of the techniques to reduce the
spectral distortion resulting from field instabilities, which is a more critical problem for high field systems [38]. It uses the navigator signals to correct the spectrum distortions from field drifts, eddy current and susceptibility effects, and the acquisition of navigator signals for artifact and motion correction is well developed in several MR imaging techniques [39-41]. Typically, a non water suppression spectrum would be acquired after the spectral acquisition with water suppression to correct the effects
from eddy current. Due to the low SNR of the spectrum, it is necessary to increase the numbers of acquisition and takes several minutes to finish a spectrum. Therefore, it is impossible to get the non water suppression spectrum immediately to correct the effects from eddy current. In INS-PRESS, the navigator signals is prepared and acquired after each acquisition of the FID using small angle excitation to minimize saturation effects and leave the metabolite signal nearly undisturbed (Fig. 4(b)). It also brings more efficient eddy current correction, reduces the sensitivity to susceptibility changes and does not prolong the scan time for most clinical applications at general TR values (about 3 sec).
Figure 4. (a) The sequence diagram of MEGA-PRESS. Based on the structure of PRESS, G1 to G3 are used for MEGA implementation, and the RF 180°s represents the frequency selective pulse. A single-banded frequency selective pulse is used for only water suppression, and multiple-banded one is additionally used for editing of other metabolites. (b) INS-PRESS is also implemented based on PRESS. After the water suppression (not shown) and the typical PRESS spectral acquisition, the excitation and acquisition of navigator signal is executed right away. Only a low flip angle excitation (~20°) is used for navigator signals to minimize saturation effects.
Chapter 2
Vitamin C: Function and Detection
L-Ascorbic acid (vitamin C, Asc), which appears physiologically as the ascorbate anion in the body, is an important antioxidant, enzyme co-factor and neuromodulator in the brain. The recent studies of vitamin C included its role in clinical nutrition, pharmacokinetics, and regulation in plasma or brains [42-44]. However, the typical method for vitamin C detection and quantification is invasive, for example, taking blood samples, which are not applicable to acquire the in vivo data from human brains.
Recently, some of the non-invasive methods using MRS were developed to possibly acquire the spectrum from living brains [23, 45], but their time-consuming made this technique not practical for clinical applications.
2.1 Background of Vitamin C
Vitamin C (formula: C6H8O6) is a water soluble, hexonic sugar acid with a molecular weight of 176.13, and the pKa values of its two dissociable protons are 4.2 and 11.8.
Therefore, the ascorbate anion exists at physiological pH (Fig. 5(a)) [46]. Via proton
MRS measurements, the resonance peaks of ascorbate occur at 3.73, 4.01, and 4.50 ppm resulting from the protons from C6, C5, and C4, respectively (Fig. 5(b)). In most animals, ascorbic acid can be synthesized from D-glucose, but can not in humans, nonhuman primates and guinea pigs because of lack of L-gulono-γ-lactone oxidase, which is required for the last step of ascorbate biosynthesis [47]. Although these species lose the ability of ascorbic acid synthesis, vitamin C is still very important to maintain the physiological functions and available from dietary sources.
Ascorbic acid plays an important role in many aspects. It is one of the essential antioxidants to provide protection from the toxic effects of reactive oxygen species (ROS), which are generated as by-products of normal aerobic respiration, during inflammation and after exposure. As an antioxidant, ascorbate protects the vascular endothelium and helps maintain normal cardiovascular function [48]. Besides, it decrease the damaging effects from nitrosamines in gut [49]. Some of important chronic diseases, chronic inflammatory diseases, and diabetes are related to oxidative stress [50, 51]. When the content of antioxidant is not sufficient to protect the body from ROS, oxidative stress would develop. Therefore, the reduced ascorbate levels would expose the vascular endothelium to oxidative stress, lead to endothelial dysfunction and raise the risk of cardiovascular disorders.
McGregor and Biesalski [42] considered that the restoration of ascorbate levels could be of significant clinical benefit. For example, the parenteral high-dose vitamin C reserved the effects of oxidative stress and allowed the recovery of cardiovascular function. There was also a study in heavy smokers during acute bronchial infection, giving a high oral dose (2g/day) treatment of vitamin C to improve impaired lung function [52]. Some research articles have reported that many cancer patients had extremely low vitamin C levels [53, 54]. Those patients of lower vitamin C status also showed shorter survival time. For the cancer patients suffering from acute oxidative stress during chemotherapy, the parenteral high-dose vitamin C has been shown to reduce the toxic side-effects of chemotherapy [55].
In recent two decades, the research about Alzheimer disease (AD) and multiple sclerosis (MS) was also very significant in image or metabolism analysis. Many evidences supported the role of oxidative stress in AD and MS. The oxidative stress, which is linked to abeta-lipid interactions, plays a pathogenetic role in AD. The research from Galbusera et al [56] treated the subgroup of mild and moderate AD patients with vitamin C and E for three months, and found that the plasma lipoperoxidation susceptibility decreased by 60%. A more direct evidence was found
by Charlton et al [57]: Plasma vitamin C levels were lower in the dementia and AD group compared to controls, which was not explained by their dietary vitamin C intakes. Such a phenomenon was also found in MS patients. The serum levels of ascorbate and other three antioxidant vitamins were significant lower in MS group compared to controls [58]. Besides, high-dose antioxidant supplementation (vitamin C 2g/day, vitamin E 480 mg/day) was also recommended for MS patients [59].
Ascorbate also shows significant functions in central nervous system (CNS). After ascorbate is acquired from the diet, it is distributed to all organs in the body via blood supply, with the concentration of plasma typically about 50 μM [60]. However, the concentration of ascorbate in brains is increased up to roughly 100-fold compared to plasma, which implies a large-amount demand of vitamin C there. Because human beings have lost the ability to synthesize vitamin C, the only source of vitamin C is from diet, and intestinal absorption capacity of it is limited by the amount of sodium-dependent transporter [61, 62]. Furthermore, the content of vitamin C in most tissues less than 100 μM are controlled with blood level corresponding to a steady state between intestinal uptake and renal clearance.
To maintain such high levels of vitamin C in brains, active transport is essential
for its regulation. Ascorbate uptakes from blood into cerebrospinal fluid (CSF) at the choroids plexus via the active, stereo-specific, sodium-dependent transport [63]. The ascorbate concentration of CSF and extracellular fluid (ECF), about tenfold higher than that of plasma, is in equilibrium because of diffusion of CSF ascorbate to ECF.
Then ascorbate is transported from ECF into neurons or glia. The average estimated concentration of ascorbate is about 10 mM in neurons and 1mM in glia. Therefore, the ascorbate concentration of overall brain tissue is several millimolar [44], and the cortical ascorbate content increases with the increase of neuron density across species (Fig. 6(a)). Besides, Fig. 6(b) shows the relationship between the change in neuron density and the ascorbate concentration in the developing rat brain. In cerebral cortex, tissue ascorbate content is highest after birth with nearly a pure neuronal population and only few immature glia cells. With the cortical gliogenesis, ascorbate levels decrease in the next few weeks.
Normally the turnover of ascorbate in brains is about 2% per hour [60]. Although the concentration of ascorbate is much higher in brain than in other tissues, it is retained tenaciously even under the condition of ascorbate deficiency, with decrease of less than 2% per day [64]. As a result of that ascorbate prevents several diseases originating from oxidative stress, it is suspect the ascorbate levels of brains relate to
AD or MS, which have been reported the lower plasma ascorbate levels in these patients compared to normal volunteers.
(a)
(b) C6
C5
C4
Figure 5. (a) Molecular structure of L-ascorbic acid as the ascorbate anion (left). After the loss of two electrons and one proton, the oxidation form, dehydroascorbate, is produced. (b) The proton spectrum of vitamin C. Three resonance peaks reflect the proton resonance from three different chemical environments, C4 (at 4.5 ppm), C5 (at 4.01 ppm), and C6 (at 3.73 ppm).
Figure 6. (a) The relation between ascorbate content in adult cerebral cortex and neuron density in different species. The intercept of y-axis was used to estimate the concentration of ascorbate in glia, which would be the theoretical cell population when neuron density was zero. Data are n = 9-61 samples per mean and R2 = 0.997. (b) The development changes in ascorbate content, including actual (square points) and calculated (circles) data, during gliogenesis in the rat cortex. In order to calculate an ascorbate concentration in neurons of 10 mM, cortical ascorbate content at postnatal day 3 (P3) and cerebellar content at P15 were used with appropriate intracellular and extracellular volume fraction data [44].
2.2 Detection of Vitamin C
The detection and quantification of vitamin C is essential and helpful for research, especially the non-invasive methods. However, the invasive methods are commonly used due to their higher sensibility to possibly determine the metabolites of low concentrations even though it is not applicable to all aspects to collect the data from the living subjects. For example, plasma ascorbate concentration is often determined by directly collecting blood samples and then using high-performance liquid chromatography. Biopsy is used to measure its concentration of some specific organs, but not available to collect the data from living brains.
The development of MRS provides one of the non-invasive methods to measure chemicals within the body. Because of the limitation of MRS, the chemicals would be detectable at least the concentration larger than hundreds of micro-molar (Table 1).
However, the concentration of vitamin C in human brain is within the range of several millimolar [44], it is hence high enough to be detectable via proton MRS. Dr. Terpstra and her colleagues [23, 37, 45] have proposed to use a spectral editing method to detect and quantify the concentration of ascorbate and other antioxidants, and their results are comparable to those estimated by using other methods.
When blood ascorbate levels are below 70 μM, the kidney’s sodium-dependent vitamin C transporters would reabsorb ascorbate to prevent the loss by renal clearance [65]. However, when the ascorbate concentration is higher than 70 μM, the body would excrete ascorbate more rapidly [66]. The plasma half-life of ascorbate is dependent on the intake. During the period of deficient intake, the plasma half-life of ascorbate is between 8 to 40 days [67], and ascorbate has a half-life time of about 30 minutes when the higher intake levels lead to more rapid excretion [68]. Some early clinical studies of vitamin C reported the benefits to use high-dose treatment on some diseases suffering from oxidative stress, such as cancer. However, studies about its pharmacokinetics of high-dose treatment are still rare in the recent decade. How much vitamin C needs to be administrated via the intravenous or oral route to achieve the desired concentration and how long it takes are questions that remain to be answered.
Dr. Padayatty and his colleagues [43] discussed the plasma vitamin C concentration varying with the route of administration, oral and intravenous use, in healthy volunteers. Vitamin C concentrations are tightly controlled when using oral dose and only intravenous administration can produce high plasma vitamin C concentrations (Fig. 7). Besides, the plasma concentrations reach the maximal values
immediately after the intravenous administration, while they arrive at the peaks with about 3 hours delay after oral supplements. The pharmacokinetic data at high intravenous dose of vitamin C in cancer patients are still sparse, and such research about brains is even rare. Fig. 8 shows the preliminary results from two healthy volunteers [69]. The concentrations of vitamin C in human brains reach the maximum in about 24 hours after intravenous bolus, with only 30% to 40% of increment, which implies that the concentration of vitamin C is also strictly controlled in human brains.
It is essential to collect more in vivo spectra data from healthy volunteers and patients for further investigations.
Figure 7. Plasma vitamin C concentrations in healthy subjects after intravenous or oral administration of vitamin C. The larger subfigure shows the change of plasma vitamin C concentration after the 1.25-g oral or intravenous dose administrated at steady state (N = 12). The inset subfigure shows peak plasma vitamin C concentrations as a function of dose after oral or intravenous administration of vitamin C [43].
Figure 8. It shows the normalized concentration change of ascorbic acid and the sum of glutamine, glutamate and glucose after an intravenous bolus of 3-g ascorbic acid.
The spectral data were acquired from occipital lobe. The ascorbate concentration reaches a maximal value in about 24 hours after the intravenous bolus [69].
Table 1. Ranges of some steadily MR-detectable metabolite concentrations reported for normal adult human brains and biopsy tissues [70].
Metabolites Concentration range (mmol/kgww)
NAA 7.9-16.6 (average 10.3)
NAAG 0.6-2.7 Choline (total) 0.9-2.5
Creatine 5.1-10.6 Glutamate 6.0-12.5 Glutamine 3.0-5.8 Myo-inositol 3.8-8.1 Phosphocreatine 3.2-5.5 Aspartate 1.0-1.4 GABA 1.3-1.9
Chapter 3
Detectability and Reliability of Vitamin C Using MRS
3.1 Motives
The importance of vitamin C has shown in many previous studies. It relates to the storage of iron and stimulation of the immune system. It also has the connection with some brain diseases, such as AD and MS as well as its correlation to age. Furthermore, it is the most concentrated non-enzymatic antioxidant in the central nervous system.
Since vitamin C plays a significant role in brain functioning [44, 71], a non-invasive and quantitative detection method of vitamin C in the human brain would be highly desirable.
The concentration of ascorbate in brains is about proportional to neuron density, and its concentration in human brain is roughly 1.0 mM [44]. This level is sufficiently high to be detectable with MRS. However, in traditional clinical routines of MRS, the concentration of ascorbate was not discussed and even its component was not
included during MR spectral analysis. Table 2 shows the chemical shifts of ascorbate and other metabolites, which possess the chemical shifts close to it [70, 72, 73]. Due to the multiple overlaps with the spectra of other metabolites, such as Gln (C2H group at 3.75 ppm), Glu (C2H group at 3.74 ppm) and myo-inositol (mI), therefore, it is not trivial to quantify the concentration of ascorbate (Fig. 9). To evaluate the detectability of ascorbate and the possible impacts on other evaluated metabolite concentrations, we focused on Gln, Glu, and mI as they all own resonance peaks close to the most prominent ascorbate signal at 3.73 ppm.
The detection of ascorbate was recently reported in human subjects at 4T and in rat brain at 9.4T by MEGA-PRESS edited spectroscopy [23, 45]. Although the technique is based on the typical 3D localization spectral acquisition method, PRESS, it is more time-consuming for several reasons. Such a spectral editing method needs to be acquired twice, one time for with and the other for without frequency selective RF pulse. Thus, the scan time at least becomes double (Fig. 4(a)). Besides, the longer TE acquisition of spectra sacrifices the SNR, so it is essential to increase the number of average to compensate the loss of the signal.
In order to avoid these disadvantages and apply MR spectroscopy techniques to
vitamin C quantification, we use traditional PRESS sequence on a clinical 3T MR scanner to acquire in vivo spectra. The goal of this thesis is to evaluate the possibility and to verify the reliability of detecting vitamin C in human brains by using clinical standard 3T MR spectroscopy methods.
Figure 9. Basis spectra of NAA, mI, Glu, Gln, and Asc. These spectra were used in LCModel analysis and the resonance peaks of Glu, Gln, and Asc are close to each other at about 3.75 ppm.
Table 2. 1H chemical shifts of ascorbic acid and other metabolites. The metabolites that possess resonance peaks close to ascorbic acid are listed. DD, double-doublet; M, multiplet; Q, quartet; T, triplet [70, 72, 73]
Metabolites Group Chemical shift Multiplicity
Asc C6H2 3.73 M
3.2 Material and Methods
3.2.1 In Vivo Spectra Collection and Analyses
From our data archives we collected 76 in vivo single voxel spectra (SVS) of 49 subjects from different brain regions. These included cerebellum (25 cases, average age = 27.50 ± 6.35 years), frontal lobe (29 cases, average age = 28.56 ± 6.77 years), occipital lobe (7 cases, average age = 29.43 ± 16.28 years), parietal lobe (10 cases, average age = 18.60 ± 16.09 years), and others (5 cases, average age = 26.40 ± 15.61 years) (Fig. 10). These spectra were acquired on a 3T Siemens Magnetom Trio system (Erlangen, Germany) with a common PRESS sequence, voxel size = 8 cm3, TE = 30 ms, TR = 3000 ms, NEX = 128 (with water-suppression), 1024 complex data points, a receiver bandwidth = 1200 Hz (centered on the water resonant frequency) with a standard 1H quadrature single-channel coil.
In order to observe the impact on other metabolite concentrations if Asc was included in the LCModel (v. 6.1-4A) estimation [74], these data were fitted twice within the spectral range of 0.2-4.2 ppm in the frequency domain. We included 14 metabolites in the standard basis-set: aspartate (Asp), Cre, GABA, glucose (Glc), Gln,
Glu, glycerophosphocholine (GPC), guanidoacetate (Gua), phosphocholine (PCh), mI, NAA, N-acetylaspartylglutamate (NAAG), scyllo-inositol (Scyllo), and taurine (Tau).
The analyses were operated once using the basis-set with an Asc basis spectrum (15 metabolites included in total) and once without the Asc basis spectrum (14 metabolites). To simplify the discussion of this approach we will use the description
“with” and “without” Asc basis throughout the following.
Metabolite concentrations were evaluated as creatine ratio (1/Cre). The differences of the estimated concentrations using a “with” Asc and a “without” Asc basis set analyses were calculated for each metabolite. This was done by deducting the “with”
Asc results from the “without” Asc results. The inter-individual standard deviations (SDs) of these estimated metabolite concentrations were calculated to depict variations of these metabolites in various regions. Furthermore, we evaluated the consistency of the estimated concentrations of different metabolites through
Asc results from the “without” Asc results. The inter-individual standard deviations (SDs) of these estimated metabolite concentrations were calculated to depict variations of these metabolites in various regions. Furthermore, we evaluated the consistency of the estimated concentrations of different metabolites through