With the SL technique, CE contrast can be tuned to different labile protons by adjusting the irradiation parameters. Slow, intermediate, and fast exchange regimes are imaged optimally with low power and long duration, intermediate power and duration, and high power and short duration spin-locking pulses, respectively. From Eq. 
, the linewidth of Rex
as a function of the frequency offset Ω is equal to
. With slow exchange and low irradiation power, the Rex
peak is narrow so that the CE contrast can only be detected within a small offset range of the labile proton frequency (Ω = δ). Consequently, the slow-exchange sensitive CE method has high specificity, such as APT where amide proton resonance frequency is well-resolved from the water resonance. With intermediate- and fast-exchange sensitive CE contrast, high RF power and fast exchange rates reduce the specificity of CE contrast. Since the Rex
peak becomes broader, CE contrast can be detected at the frequency offset far away from the labile proton frequency (Jin et al., 2011
). As shown in our data (–), the APEX SLRasym
peak is much broader than the narrow peak from the APT effect, indicating a reduced specificity for APEX.
, there are a variety of amine-groups with different chemical shifts and exchange rates. For example, creatine has a much slower amine-water proton exchange rate than many amino-acids, such as glutamate (Jin et al., 2011
; Sun et al., 2008
). Roughly, the chemical shift between amine and water protons is 2–3 ppm (5026–7540 rad/s at 9.4 T) (Ward et al., 2000
), and the amine exchange rates span a wide range between 500 s−1
to 10000 s−1
(Liepinsh and Otting, 1996
; van Zijl and Yadav, 2011
). Thus, the in vivo
APEX signal represents a weighted summation of many different amine groups. The contribtuion to the APEX signal from a certain amine-group depends on several variables, including the chemical shift, concentration, and exchange rate which is dependent on pH and temperature, as well as experimental parameters including the power and duration of the irradiation pulse, and the frequency offset. Further study is necessary to systematically determine the amine-proton exchange rates of the protein/peptides and the different amino acids. In addition to APEX, a high power irradiation pulse may also be sensitive to other intermediate to fast CE, such as the hydroxyl-water proton exchange with exchange rates between 700 – 15000 s−1
(Liepinsh and Otting, 1996
; Ward et al., 2000
). Although some of the hydroxyl groups may be close to the amine Larmor frequencies, e. g., glucose has CEST peaks close to 2 and 3 ppm (van Zijl et al., 2007
), most hydroxyl groups have much smaller chemical shift (~ 1 ppm) than amines; therefore, the contamination from hydroxyl-water exchange to our APEX signal should be small, which we confirmed with phantom experiment II (see ). Finally, the CE effect is highly dependent on the ratio of exchange rate to the chemical shift (k
/δ), which will increase at lower magnetic fields. At lower fields, the frequency separation between amine and water protons is smaller so that many of the amine exchange rates may become too fast to be detected by the APEX experiment. Consequently, the APEX contrast may be smaller at 1.5 or 3 T.
At a high field of 9.4 T, the APT peak at 3.5 ppm can be well-resolved in the Z-spectra of brain due to the increased spectral resolution compared to 3 T and 4.7 T (Zhou et al., 2003
). However, a pronounced broad peak around −2 to −4 ppm was also observed, which greatly distorted the baseline of SLRasym
. A similar result was reported in homogenized rat brain at 9.4 T (Narvainen et al., 2010
). This upfield feature is not readily seen at 1.5 T and 3 T, but has been observed in humans at 7 T (Mougin et al., 2010
). The exact source of this broad peak is not clear, but it is likely due to an inter/intramolecular NOE which increases slowly with irradiation time (Chen et al., 2006
; van Zijl and Yadav, 2011
). The observed NOE, in addition to the recently reported problem of asymmetry with the conventional MT effect (Hua et al., 2007
) makes quantification of APT from the SLRasym
analysis challenging. The APEX contrast is obtained from the asymmetry analysis similar to APT. Since a much shorter irradiation pulse is used with APEX, the NOE contamination is smaller and the MT effect may also be less. As a result, no significant subtraction problem in SLRasym
was observed (as seen in ).
One feature of the SL technique is its ability to tune to a specific pH or exchange rate (see ). Note that Rex
in Eq. 
peaks at ω1
. Hence, Rex
can be tuned to a particular k
by adjusting SL power to be ω1
(Jin et al., 2011
). Our data suggests that when the pH decreases, the exchange rate shifts from fast to intermediate for APEX, and from slow to slower for APT. Thus, the APEX contrast increases as pH decreases, while the APT contrast decreases with pH (McMahon et al., 2006
). This inverse relationship between CE contrast and pH has also been observed for hydroxyl-water proton exchange with poly-L-threonine (McMahon et al., 2008
). In practice, the optimization of ω1
to achieve maximum sensitivity depends on the type of application. To detect a change of labile proton concentration with minimal variation in the exchange rate, the ω1
that gives the maximum SLRasym
should be selected. In the example of , that would be 250 Hz for pH = 5.8, and 500 Hz for pH = 6.3 to 7.4. Conversely, to detect a change in exchange rate without a change in the labile proton concentration, ω1
should be selected that has the largest slope in SLRasym
for the pH range of interest, i. e.
250 Hz (or similarly, 500 Hz) in the example of . Note that in contrast to Rex
, which is proportional to pB
does not linearly increase with the concentrations of protein or metabolites when concentrations are high (, and ), similar to a previous CEST study on glycogen (van Zijl et al., 2007
). With high labile proton concentration, linearity only holds for very small irradiation power and/or very short irradiation durations ().
Although both are chemical exchange-based techniques, APEX shows positive contrast during ischemia, which is preferable to the negative contrast observed with APT, and better sensitivity than APT in many brain regions. More importantly, different spatial-temporal patterns were observed for these two approaches in our preliminary studies. In many animals, the lesion areas indicated by APEX maps were smaller (specifically, in the subcortical region) than those observed with APT (and ADC) maps. The APEX maps also showed greater temporal variation during the evolution of ischemia. Since the lesion volume and tissue outcome of the focal ischemia model are strongly dependent on the surgical procedure, the larger variation found with APEX maps suggests that the APEX signal may be more sensitive to physiological variations. Further studies will be performed to systematically compare the APEX maps with APT and ADC, as well as histology. The discrepancies between APEX and APT image contrast during focal ischemia suggest different underlying physiological sources. With APT, the CE agent is presumably derived from the amide protons of proteins and peptides, and the decrease of APT in the lesion area mainly reflects tissue acidosis since the concentration of proteins and peptides does not change significantly during initial hours of ischemia (Zhou et al., 2003
). With APEX, the source of contrast is more complex because in addition to the proteins and peptides, free amino-acids also contribute to the signal, and these concentrations can vary significantly during the evolution of ischemia and may also be highly region-dependent. For example, it has been reported that for MCAO of 30 min to 4 hr, there were strong regional and temporal dependent changes in the concentrations of glutamate, glutamine, and GABA. Specifically, in the lateral caudate-putamen and lower parietal cortex regions, the decrease of glutamate concentration was close to 50%, whereas the GABA concentration increased more than 100% at 4 hours post MCAO (Haberg et al., 2006). Hence, the spatial-temporal mismatch between APT and APEX maps may be indicative of amino acid concentration change during ischemia and helps to understand disease evolution. Further study would be necessary to examine the physiological source of the APEX signal and its potential application in ischemia research.
Besides potential applications of APEX to pH-related research, such as ischemia and cortical spreading depression, the sensitivity of APEX to protein and amino acid concentration suggests that it may also be applicable to the study of many other diseases or physiological conditions. For example, the concentration of amino acids, especially neurotransmitters like glutamate and GABA, may change significantly in pathological conditions such as neurodegenerative diseases, epilepsy or psychiatric disorders. As illustrated by proton spectroscopy studies, the concentration of glutamate, compared with total creatine level, decreases in the hippocampus of transgenic Alzheimer’s disease (AD) mice (Marjanska et al., 2005
), and can be used to distinguish the mild cognitive impairment with AD in human patients (Rupsingh et al., 2011
). In addition, Zhou et al. (2008)
showed that APT imaging can distinguish low- and high-grade glioma due to an increase of protein concentration with tumor grade. Due to its sensitivity in amino acid and protein concentrations, the APEX contrast may also be a potential biomarker in these applications.