Ryan T Oglesby1,2, Wilfred W Lam2, and Greg J Stanisz1,2
1Medical Biophysics, University of Toronto, Toronto, ON, Canada, 2Physical Sciences, Sunnybrook Research Institute, Toronto, ON, Canada
Synopsis
This study
demonstrates the in vitro characterization of tryptophan, 5-HTP,
serotonin (5-HT), 5-HIAA (all members of the serotonin biosynthesis pathway),
and melatonin at precise pH, temperature, and concentration. At pH 5.5, CEST
contrast between 0.6 and 1.9 ppm, originating from the NH3+
side chain, is exhibited by tryptophan, 5-HTP, and 5-HT. All five molecules at
pH 7.4 exhibit CEST contrast between 5.11
and 5.47 ppm, originating from the NH proton on the
indole ring. If sensitive enough in vivo, these measurements could improve
the objectivity of clinically diagnosed psychiatric disorders and could provide
a new biological understanding of serotonergic dysfunction.
Introduction
Serotonin (5-HT) receptor
dysfunction has been implicated in numerous psychiatric disorders such as
anxiety1, depression2, schizophrenia3, Alzheimer’s disease4, and autism5. Currently, the diagnosis of such psychiatric
disorders is based on the phenomenological evaluation of symptoms and behavior
by clinicians6. In this study, we evaluate the feasibility of using chemical
exchange saturation transfer (CEST) MRI, a non-invasive tool, to quantify the
metabolites involved in the serotonin biosynthesis and breakdown pathway (Fig.
1) with the goal of aiding the diagnostic assessment of psychiatric disorders. CEST
is a contrast mechanism in which magnetization in compounds containing
exchangeable protons is selectively saturated and detected indirectly through
the bulk water signal with enhanced sensitivity7. If sufficiently sensitive in vivo, these
measurements would be invaluable in psychiatric research and could lead to a new
biological understanding of serotonergic dysfunction.Methods
Phantom Preparation: Phantoms of
the following five metabolites (30 mM each) were prepared: tryptophan, 5-HTP, 5-HT,
5-HIAA, and melatonin. Separate phantoms were made at the following three pHs: 5.5
(intravesicular8), 6.7, and 7.4
(extracellular) for a total of 15 unique phantoms. Additionally, tryptophan was
prepared and evaluated in eight pH environments ranging from 5.5 to 8.0. Each
phantom was prepared in triplicate.
MRI Acquisition and Data Analysis: The phantoms were
scanned at 7 T (Ascend 300WB, Bruker BioSpin, Billerica, MA) using a
temperature-controlled phantom holder9 stabilized at 37.0 ±
0.2 °C. All Z-spectra were acquired using a single slice, saturation transfer-prepared
(CW block saturation pulse, tsat = 490 ms per line of k-space) FLASH
sequence (TR = 500 ms; TE = 3 ms; FA = 30°; matrix = 64 × 64). CEST- and MT-sensitive
Z-spectra were obtained using B1 = 0.30, 0.50, 0.75, 1.00, 1.50 µT
and Δω spanning a ±7 ppm range in steps of 0.05 ppm. MT-sensitive spectra were
obtained using B1 = 3 and 6 µT with Δω logarithmically spaced
between ±3 ppm and ±300 ppm in 21 steps. A WAter Saturation Shift Referencing
(WASSR)10 spectrum was obtained
to correct for voxelwise B0 inhomogeneity and to measure the direct
effect without CEST and MT contamination, using B1 = 0.1 µT with Δω
between ± 0.5 ppm in 1/60 ppm steps. A T1 map was calculated from eleven
inversion recovery-prepared RARE sequences (TR = 20,000 ms; TE = 6 ms; TI = 30
- 10,000 ms; RARE factor = 2) and a T2 map was calculated from a
CPMG sequence (TR = 5000 ms; TE = 20 - 3600 ms, 180 echoes; FA = 90°). A B1
map was calculated from seven 3D FLASH (TR = 200 ms; TE = 4 ms; FA = 120° - 240°)
sequences for inhomogeneity correction. Non-linear least squares fitting to three-
and four-pool Bloch-McConnell11 models was done in
MATLAB.Results
The Z-spectra and MTRasym
for each metabolite in their various pH environments are illustrated in Fig. 2.
At pH 5.5 (Fig. 2a), tryptophan, 5-HTP, and 5-HT exhibit similar CEST effects
with a measured resonance frequency offset between 2.63 and 2.71 ppm, albeit
the MTRasym (Fig. 2 and 3) is the largest (20%) for 5-HT at
approximately 2.50 ppm. At pH 6.7 (Fig. 2b), tryptophan, 5-HTP, 5-HT, and
5-HIAA Z-spectra are characterized by two peaks: one between 5.30 and 5.46 ppm
with CEST asymmetry amplitude of 1.0–2.3% and another between 0.6 and 1.9 ppm
with CEST asymmetry amplitude of 0.9–1.3%. At
pH 7.4 (Fig. 2c), all five metabolites demonstrate similar CEST contrast with a
measured resonance frequency offset between 5.11 and 5.47 ppm. Again, the 5-HT
CEST asymmetry is the largest (9.5%) at 5.29 ppm. The estimated parameters from fitting the three-pool CEST/MT
model is provided in Fig. 4. For tryptophan the exchange rates, RC1
and RC2, of the two peaks observed in each pH environment were
evaluated individually to illustrate a linearly increasing relationship between
RC and pH (Fig. 5).Discussion
At pH 5.5,
tryptophan, 5-HTP, and 5-HT share similar CEST characteristics, while 5-HIAA
and melatonin differ. Therefore, the origin of their CEST contrast between 2.63
and 2.71 ppm is likely the NH3+ group, which is common to
tryptophan, 5-HTP, and 5-HT. The source of the CEST effect measured from 5-HIAA
at pH 5.5 remains unexplained by our NH3+ hypothesis. At
pH 7.4, each of the five metabolites share a common CEST peak between 5.11 and 5.47
ppm, implying their shared exchangeable proton (in the
NH group) is the source. Translation of these in vitro results to in
vivo studies will not be straightforward because in vivo serotonin concentration
is orders of magnitude less than 30 mM. Furthermore, there will be competing metabolites
(other breakdown products of serotonin) found in vivo which may
contaminate the desired CEST signal with unwanted CEST and MT effects.Conclusions
This study has
demonstrated the feasibility of in vitro detection and characterization
of five molecules associated with the serotonin biosynthesis and breakdown
pathway at precise pH, temperature, and metabolite concentration. At the
intravesicular pH of 5.5, CEST contrast originating from the NH3+
side chain is exhibited by tryptophan, 5-HTP, and 5-HT (serotonin). Under
extracellular pH conditions of 7.4, CEST contrast originating from the NH
proton on the indole ring is exhibited by all five molecules.Acknowledgements
We thank the Canadian
Institutes for Health Research (PJT156252) for financial support.References
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