Radiochemical Synthesis and Evaluation of 13N-Labeled 5-Aminolevulinic Acid for PET Imaging of Gliomas

Article abstract of DOI:10.1021/acsmedchemlett.7b00311

The endogenous amino acid, 5-aminolevulinic acid (5-ALA), has received significant attention as an imaging agent, including ongoing clinical trials for image-guided tumor resection due to its selective uptake and subsequent accumulation of the fluorescent protoporphyrin IX in tumor cells. Based on the widely reported selectivity of 5-ALA, a new positron emission tomography imaging probe was developed by reacting methyl 5-bromolevulinate with [¹³N] ammonia. The radiotracer, [¹³N] 5-ALA, was produced in high radiochemical yield (65%) in 10 min and could be purified using only solid phase cartridges. In vivo testing in rats bearing intracranial 9L glioblastoma showed peak tumor uptake occurred within 10 min of radiotracer administration. Immunohistochemical staining and fluorescent imaging was used to confirm the tumor location and accumulation of the tracer seen from the PET images. The quick synthesis and rapid tumor specific uptake of [¹³N] 5-ALA makes it a potential novel clinical applicable radiotracer for detecting and monitoring tumors noninvasively.

Full text of DOI:10.1021/acsmedchemlett.7b00311

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Letter
Radiochemical Synthesis and Evaluation of 13N-Labeled
-Aminolevulinic Acid for PET Imaging of Gliomas
5
Adam B Pippin, Ronald J Voll, Yuancheng Li, Hui Mao, and Mark M. Goodman
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00311 • Publication Date (Web): 15 Nov 2017
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Radiochemical Synthesis and Evaluation of N-Labeled 5-
Aminolevulinic Acid for PET Imaging of Gliomas
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,1,†,‡
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Adam B. Pippin,*
Ronald J. Voll, Yuancheng Li, Hui Wu, Hui Mao, and Mark M. Goodman
1
Department of Radiology and Imaging Sciences, Emory University Center for Systems Imaging, Wesley Woods Health
Center, 1841 Clifton Road, NE, Atlanta, Georgia 30329, United States
KEYWORDS: 5-ALA, brain tumor, cancer, positron emission tomography, imaging
ABSTRACT: The endogenous amino acid, 5ꢀaminolevulinic acid (5ꢀALA), has received significant attention as an imaging agent,
including onꢀgoing clinical trials for imageꢀguided tumor resection due to its selective uptake and subsequent accumulation of the
fluorescent protoporphyrin IX in tumor cells. Based on the widely reported selectivity of 5ꢀALA, a new Positron Emission Tomogꢀ
raphy imaging probe was developed by reacting methyl 5ꢀbromolevuinate with [ N] ammonia. The radiotracer, [ N] 5ꢀALA, was
produced in high radiochemical yield (65%) in 10 minutes, and could be purified using only solid phase cartridges. In vivo testing
in rats bearing intracranial 9L glioblastoma showed peak tumor uptake occurred within 10 minutes of radiotracer administration.
Immunohistochemical staining and fluorescent imaging was used to confirm the tumor location and accumulation of the tracer seen
13
13
13
from the PET images. The quick synthesis and rapid tumor specific uptake of [ N] 5ꢀALA makes it a potential novel clinical appliꢀ
cable radiotracer for detecting and monitoring tumors nonꢀinvasively.
Brain tumors present additional challenges for diagnosis and
treatment due to the intracranial location, involvement of the
central nervous system and difficult to deliver imaging and
therapeutic agents through the blood brain barrier. Gliomas
make up 80% of malignant brain tumors and often result in
operative imaging probe for determining tumor margins durꢀ
ing the resection of malignant gliomas has been found to inꢀ
crease the rate of gross total resection to 65% compared to
35% without 5ꢀALA . The progressionꢀfree survival rate at 6
months is also increased to 41% when using 5ꢀALA versus
14
1
14,15
poor prognosis and decreased life expectancy . Diagnosing
21% without . In addition, 5ꢀALA shows promise for future
brain tumors heavily depends on invasive procedures of craniꢀ
al biopsy or after tumor resection. Current first pass diagnostic
therapeutic applications for cancer using photodynamic theraꢀ
py (PDT). In this therapy technique, the preferential accumulaꢀ
tion of PpIX in tumors provides a substrate which when exꢀ
posed to UV light, results in reactive oxygen species that can
2
imaging for brain tumors such as MRI and CT provide mostly
anatomical and morphological details, but lack of molecular
and functional information.
1
6
ultimately kill cancer cells . While the fluorescence of PpIX
can be useful for therapeutic applications, imaging PpIX for
diagnostic purposes is limited to superficial tumors or intraꢀ
operative procedures due to poor light penetration through the
The endogenous amino acid, 5ꢀaminolevulinic acid (5ꢀ
ALA), is the first compound in the biological synthesis of
3
heme in mammals . It is found that enhanced uptake of 5ꢀALA
17,18
skin
.
in cancerous cells provides an accumulation of a fluorescent
4
porphyrin known as Protoporphyrin IX (PpIX) . While the
Using a radiolabled version of 5ꢀALA for Positron Emission
Tomography (PET) not only can overcome the limitations of
optical detectability but also can potentially provide a highly
tumor specific PET tracer compared to existing [ F] fluorodeꢀ
oxyglucose (FDG), which often has strong background signals
preferential accumulation of 5ꢀALA in tumors still remains an
area of active investigation, the current proposed mechanisms
include: increased 5ꢀALA entry through a disrupted blood
18
5
6
brain barrier , upregulation of beta and oligopeptide transꢀ
7
19
porters (PEPT1 and PEPT2), increased expressions of enꢀ
due to the high uptake in brain parenchyma . Furthermore, 5ꢀ
zymes in the heme biosynthesis pathway and decreased
ALA PET scanning would be useful for preꢀoperative planꢀ
ning in conjunction to surgical resection procedures that use 5ꢀ
ALA to visualize tumors for fluorescenceꢀguided surgery.
Thus, an amino acidꢀbased PET tracer may be more useful for
8
amount of the enzyme ferrochetelase . In particular, 5ꢀALA is
useful for brain tumors because of its selective fluorescence in
9
gilomas . It is widely reported that PpIX is preferentially acꢀ
20ꢀ23
cumulated in glioblastoma cells with ratios of 20 to 50:1 comꢀ
imaging tumors in the brain . Additionally, a 5ꢀALA based
10,11
pared to normal brain cells . Primary transportation through
radioligand could also help determine the viability of a candiꢀ
date for PDT when using 5ꢀALA as a photosensitizer. There is
also evidence that the uptake of 5ꢀALA is variable in different
12
the blood brain barrier occurs through the choroid plexus ,
however, leaky vascular may also be a factor when tumors are
5
24,25
present in the brain . Therefore, there are increasing interests
tumor lines . A PET ligand may be used to quantitate the
2
6,27
and efforts in developing clinical applications of 5ꢀALA. For
rate of PpIX synthesis . This information could potentially
be used to nonꢀinvasively grade or differentiate brain tumors
based on the PpIX metabolism. These wide arrays of potential
applications lead us to seek a method to develop a radioactive
1
3
example, 5ꢀALA and its esters have found use in fluoresꢀ
cenceꢀguided surgery to help visualize tumorous tissue in neuꢀ
1
1
rosurgical procedures . The use of 5ꢀALA as an intraꢀ
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Scheme 1. Synthesis of the precursor, methyl 5ꢀbromolevulinate 2, and radiochemical synthesis of [ N] 5ꢀALA 1.
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version of the naturally occurring amino acid that would be
suitable for use with PET imaging.
(60 µA) produced around 250 mCi (9.25 GBq) of [ N] amꢀ
monia.
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The molecule 5ꢀALA lends itself to very few practical inꢀ
stallments of a PET radioisotope. Without altering the comꢀ
For the production of [ N] 5ꢀALA 1, the [ N] ammonia
was eluted off the cartridge with aqueous sodium acetate (1
mL, 3 M) into a vial containing the precursor 2 (8 mg) disꢀ
solved in acetonitrile (250 µL) (Scheme 1). Heating the vial
for 3 minutes at 50 ºC was sufficient for complete radioisotope
incorporation, but to ensure no [ N] ammonia was present, the
vial was purged with argon for 2 minutes to remove any volaꢀ
tile unreacted [ N] ammonia. Due to time constraints and the
1
1
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pound, the only usable PET isotopes are C, N, and O.
1
1
Ideally, C would be the bestꢀsuited isotope due to its extenꢀ
sive use in the field of PET imaging and its longer halfꢀlife (20
min). Unfortunately, at this time, there are no facile ways of
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synthesizing a C version of 5ꢀALA in high radiochemical
11
13
yield. To date, only a C derivative of 5ꢀALA, 5ꢀaminoꢀ4ꢀ
1
1
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oxoꢀ[6ꢀ C]hexanoic acid ([ C] MALA) has been syntheꢀ
lack of side products, the radiotracer was quickly purified usꢀ
ing only cartridges to remove any remaining precursor. The
crude reaction mixture was loaded by vacuum onto tC18 and
HLB cartridges (Waters) into a waste vial. A small amount of
[ N] 5ꢀALA 1 (<20 mCi or 0.74 GBq) was detected in the
waste vial but the majority of radioactivity was retained on the
two cartridges. Eluting 3 mL of an acidic solution through the
SepꢀPaks provided the radiotracer [ N] 5ꢀALA 1 in high radiꢀ
ochemical yield (65% decay corrected) (Scheme 1). Using
acidic phosphate (0.2 M, pH = 5), acetic acid/sodium acetate
(0.2 M, pH = 6), or HCl (10 mmol, pH = 2) for elution all proꢀ
vided similar yields (~100 mCi or 3.7 GBq). To ensure no
particles were present, the purified solution was pressure filꢀ
tered through a 0.2 µm filter into a sterile vial and subsequentꢀ
ly used immediately in studies. The total time from the end of
bombardment to dose was 10 minutes. The dose formulation
was intentionally made slightly acidic (pH = 6) to ensure diꢀ
merization did not occur. It is well known that when nonꢀ
radioactive 5ꢀALA is formulated at neutral or basic pH, it can
rapidly dimerize to 2,5ꢀdicarboxyethylꢀ3,6ꢀdihydropyrazine
17
11
sized . This novel radiotracer was created by installing a
C
methyl group on an oxime precursor using tetrabutyl ammoniꢀ
11
um fluoride and [ C] methyl iodide in DMSO. There were
several drawbacks to this radiosythesis: the precursor was
difficult to purify, the reaction results in a racemic mixture, the
synthesis is multiple steps, preparatory HPLC is required, raꢀ
diochemical yield was low (4.4% uncorrected decay), and the
resulting radiotracer is not a natural occurring amino acid.
Nonꢀradioactive MALA, was found to be an inhibitor of 5ꢀ
13
13
aminolevulinate dehydratase (K = 0.2 M), and thus can only
i
1
7,28
be used to estimate the synthesis rate of PpIX . Because of
these factors and our previous experience working with the
13
29
quick decaying N isotope , we sought to produce the natuꢀ
13
rally occurring [ N] 5ꢀALA radiotracer 1. Herein, we describe
13
the radiochemical synthesis of [ N] 5ꢀALA 1 and demonstrate
its use in tumor imaging in a rat model of glioma using the 9L
glioma cell line.
13
The precursor for [ N] 5ꢀALA 1, methyl 5ꢀbromolevulinate
2
, was synthesized by reacting levulinic acid with bromine in
methanol (Scheme 1). This reaction has been studied at great
(DHPY)
and
further
oxidize
to
produce
2,5ꢀ
33
dicarboxyethylpyrazine (PY) . In an acidic formulation, the
radiotracer shelfꢀlife was longer than its decay.
length in a variety of different solvents including ionic liqꢀ
3
0,31
uids . The reaction of bromine with levulinic acid typically
produces a mixture of 3ꢀbromo, 5ꢀbromo, and 3,5ꢀdibromo
isomers depending on the solvent used. Using methanol as a
solvent, the nonꢀoptimized reaction resulted in a 30% yield of
the desired isomer after separation using silica column chroꢀ
matography. The amount of methyl 5ꢀbromolevulinate proꢀ
duced was sufficient for labeling studies.
13
Due to the short halfꢀlife of N, the quality control on the
amino acid was conveniently performed using radio thin layer
chromatography. This is the type of analysis routinely used for
the quality control of polar radiotracers. For a more inꢀdepth
analysis, the polar nature of this amino acid requires the use of
hydrophilic interaction chromatography to assay the quality of
the dose produced . The 5ꢀALA HCl salt standard lacks a
significant chromophore and requires that low wavelengths
210 nm) be used. This also makes determining specific activiꢀ
17
1
3
[ N] Ammonia is produced by bombarding water containꢀ
3
2
ing ethanol (0.25%) as a reductant . After eluting the cycloꢀ
tron product through a QMA cartridge to remove residual [ F]
18
(
13
ty difficult. The only source of ammonia should be from the
cyclotron target, we estimate the specific activity of [ N] 5ꢀ
ALA 1 is similar to [ N] ammonia (> 2 Ci/µmol or 74
fluoride, [ N] ammonia can be trapped on a CM cartridge in
13
1
3
near quantitative recovery. The [ N] ammonia can be eluted
off the CM cartridge in aqueous form by an increase in ionic
strength. In a typical experiment, a 30 minute bombardment
13
GBq/µmol) for the end of synthesis.
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Figure 2. Summed coronal PET images (0ꢀ15 min) of a rat bearꢀ
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ing intracranial 9L glioma: (A) [ N] 5ꢀALA in the brain, (B)
coregistered PETꢀCT, (C) CT template. The arrow indicates the
location of the tumor.
Figure 1. HPLC profiles of 5ꢀALA. (A) UV absorbance (210 nm)
of unlabeled 5ꢀALA. (B) Radioactivity (mV) of [ N] 5ꢀALA.
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Studies reported by Hebeda et al showed 5ꢀALA was readiꢀ
ly taken up in 9L glioma cells implanted in rats by using fluoꢀ
3
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rescence to detect PpIX . Therefore, we chose to use the 9L
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cells to prepare the rat intracranial tumor model to test [ N] 5ꢀ
ALA in vivo using microPET imaging. The studies were conꢀ
ducted in male rats with 9L cells surgically implanted into the
right hemisphere of the brain. After 12 to 14 days, in which
the 9L tumors typically grow to 3ꢀ5 mm in size, dynamic PET
imaging was performed for 60 minutes followed by a CT scan
to obtain the anatomic images.
13
Figure 3. Ratio of [ N] 5ꢀALA uptake in the 9L tumor (right
hemisphere) vs normal brain tissue (left hemisphere) (n = 3).
PET images (Figure 2) show that the 9L tumor takes up
N] 5ꢀALA, resulting in observation of high signal intensity
13
[
at the tumor region with a signal intensity ratio of 1.5 comꢀ
pared to the symmetrical contralateral normal brain tissue in
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the left hemisphere (Figure 3). Peak uptake of [ N] 5ꢀALA in
the tumor cells occurred within 10 minutes. To confirm and
validate the imaging results, the animals were sacrificed imꢀ
mediately after the imaging experiments to collect the whole
brains and snap frozen before storing in ꢀ80 ˚C. Each brain
was then sectioned to 1 mm thick slices for fluorescent imagꢀ
ing using the wavelength of PpIX to determine the distribution
in the brain, followed by the H&E staining for tumor region
identification. Figure 4 shows PET, optical images and H&E
staining of the brain slice from one of the animals. H&E
staining indicate the location of the 9L tumor and correspondꢀ
ing PET image which correlates well with the high signal reꢀ
gion in the PpIX fluorescent image.
13
In conclusion, we have successfully synthesized [ N] 5ꢀ
ALA in high radiochemical yield and purity within 10
minutes. The stability of the radiotracer was high for imaging
experiments and no dimerization was observed. This rapid
synthesis time and lack of HPLC purification makes the radioꢀ
Figure 4. (A) Transverse PET summed image (0ꢀ15 min) of the
brain. (B) Post mortem brain slice. (C) Optical image of the brain
slice shown in B. (D) Immunohistochemical (H&E) staining of
the brain slice B. The circle indicates the location of the tumor.
13
tracer [ N] 5ꢀALA suitable for additional imaging experiꢀ
ments of other tumor cell lines.
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ASSOCIATED CONTENT
Supporting Information
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Supporting information provides full experimental details and
characterization data for organic compounds, and radiochemical
procedures. This material is available free of charge via the interꢀ
net at http://pubs.acs.org
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Ocheltree, S. M.; Shen, H.; Hu, Y.; Xiang, J.; Keep, R. F.;
Smith, D. E. Role of PEPT2 in the Choroid Plexus Uptake of Glycylsar-
cosine and 5-Aminolevulinic Acid: Studies in Wild-Type and Null
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AUTHOR INFORMATION
Corresponding Author
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Ohgari, Y.; Nakayasu, Y.; Kitajima, S.; Sawamoto, M.; Mori,
*
Email: Pippin.17@osu.edu
H.; Shimokawa, O.; Matsui, H.; Taketani, S. Mechanisms Involved in
Aminolevulinic Acid (ALA)-Induced Photosensitivity of Tumor Cells:
Relation of Ferrochelatase and Uptake of ALA to the Accumulation of
Protoporphyrin. Biochem. Pharmacol. 2005, 71 (1-2), 42–49.
Present Addresses
Laboratory for Translational Research in Imaging Pharmaceutiꢀ
cals. The Ohio State University. 1216 Kinnear Rd. Columbus, OH
3212. Email: Pippin.17@osu.edu
†
(
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Ma, R.; Watts, C. Selective 5-Aminolevulinic Acid-Induced
Protoporphyrin IX Fluorescence in Gliomas. Acta Neurochirurgica
2016, 158, 1935–1941.
4
Author Contributions
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Pichlmeier, U.; Bink, A.; Schackert, G.; Stummer, W.; the
ALA Glioma Study Group. Resection and Survival in Glioblastoma
Multiforme: an RTOG Recursive Partitioning Analysis of ALA Study
Patients. Neuro-Oncology 2008, 10 (6), 1025–1034.
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manuꢀ
script. / ‡These authors contributed equally.
(
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Li, Y.; Rey-Dios, R.; Roberts, D. W.; Valdés, P. A.; Cohen-
Funding Sources
Gadol, A. A. Intraoperative Fluorescence-Guided Resection of High-
Grade Gliomas: a Comparison of the Present Techniques and Evolu-
tion of Future Strategies. World Neurosurgery 2014, 82 (1-2), 175–
185.
This research was financially supported with funds from the
Emory School of Medicine “Endowed Chair in Imaging Science”
for Mark M. Goodman and National Cancer Institute
(12)
Ennis, S. R.; Novotny, A.; Xiang, J.; Shakui, P.; Masada, T.;
(R01CA169937ꢀ04) to Hui Mao.
Stummer, W.; Smith, D. E.; Keep, R. F. Transport of 5-Aminolevulinic
Acid Between Blood and Brain. Brain Res. 2003, 959 (2), 226–234.
Notes
(
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Hexyl Aminolevulinate: 5-ALA Hexylester, 5-ALA Hexyles-
The authors declare no competing financial interest.
ther, Aminolevulinic Acid Hexyl Ester, Hexaminolevulinate, Hexyl 5-
Aminolevulinate, P 1206. Drugs R D 2006, 6 (4), 235–238.
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Colditz, M. J.; van Leyen, K.; Jeffree, R. L. Aminolevulinic
ACKNOWLEDGMENT
Acid (ALA)–Protoporphyrin IX Fluorescence Guided Tumour Resec-
tion. Part 2: Theoretical, Biochemical and Practical Aspects. Journal
of Clinical Neuroscience 2012, 19 (12), 1611–1616.
We thank Ronald J. Crowe, Karen B. Dolph, and Michael S.
Waldrep for assistance with radioisotope production and Margie
Jones, Jonathon A. Nye, and Jaekeun Park for assistance with
PETꢀCT imaging experiments.
(15)
Pirotte, B. J. M.; Levivier, M.; Goldman, S.; Massager, N.;
Wikler, D.; Dewitte, O.; Bruneau, M.; Rorive, S.; David, P.; Brotchi, J.
Positron Emission Tomography-Guided Volumetric Resection of Su-
pratentorial High-Grade Gliomas: a Survival Anaylsis in 66 Consecu-
tive Patients. Neurosurgery 2009, 64 (3), 471–481.
ABBREVIATIONS
5
ꢀALA, 5ꢀaminolevulinic acid; PPIX, Protoporphyrin IX; MRI,
(
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Wachowska, M. G.; Muchowicz, A.; Firczuk, M. G.; Gab-
Magnetic Resonance Imaging; PET, Positron Emission Tomograꢀ
phy; CT, Computed Tomography; PEPT1, Peptide Transporter 1;
PEPT2, Peptide Transporter 2; PDT, Photodynmaic Therapy;
FDG, Fluorodeoxyglucose; MALA, methyl aminolevulinic acid;
QMA, Quaternary Methyl Amine; CM, Carboxy Methylate.
rysiak, M.; Winiarska, M.; Wa czyk, M. G.; Bojarczuk, K.; Golab, J.
Aminolevulinic Acid (ALA) as a Prodrug in Photodynamic Therapy of
Cancer. Molecules 2011, 16 (12), 4140–4164.
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Labeled 5-Aminolevulinic Acid Derivative to Estimate the Induced
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Scheme 1. Synthesis of the precursor, methyl 5-bromolevulinate 2, and radiochemical synthesis of [13N] 5-
ALA 1.
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Figure 1. HPLC profiles of 5-ALA. (A) UV absorbance (210 nm) of unlabeled 5-ALA. (B) Radioactivity (mV) of
[13N] 5-ALA.
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Figure 2. Summed coronal PET images (0-15 min) of a rat bearing intracranial 9L glioma (arrow): (A) [13N]
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Figure 3. Ratio of [13N] 5-ALA uptake in the 9L tumor (right hemisphere) vs normal brain tissue (left
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Figure 4. (A) Transverse PET summed image (0-15 min) of the brain. (B) Post mortem brain slice. (C)
Optical image of the brain slice shown in B. (D) Immunohistochemical (H&E) staining of the brain slice B.
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