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Nuclear Medicine and Biology 37 (2010) 189 – 196
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Stimulation of 125 I-3-iodo-α-methyl-L-tyrosine uptake in Chinese hamster
ovary (CHO-K1) cells by tyrosine esters
Naoto Shikanoa,⁎,1 , Masato Oguraa , Jun-ichi Sagaraa , Syuichi Nakajimaa , Masato Kobayashib ,Takeshi Babaa , Naoto Yamaguchia , Yukio Iwamuraa , Nobuo Kubotaa , Keiichi KawaibaDepartment of Radiological Sciences, Center for Medical Sciences and Center for Humanities and Sciences,
Ibaraki Prefectural University of Health Sciences, Inashiki-gun, Ibaraki, Japanb
Division of Health Science, Graduate School of Health Sciences, Kanazawa University, Kanazawa, Ishikawa, Japan
Received 24 March 2009; received in revised form 17 September 2009; accepted 2 October 2009

Abstract
Introduction: Transport of the amino acid analog 123I-3-iodo-α-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly
via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, wemade a preliminary investigation of the effects of amino acid esters on enhancement of 125I-3-iodo-α-methyl-L-tyrosine (IMT) uptake via
LAT1 in Chinese hamster ovary (CHO-K1) cells.
Methods: Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq)
uptake mechanisms using specific inhibitors. L-Gly, L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys ethyl/methyl esters were
tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of L-Tyr ethyl andmethyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of L- and D-Tyr ethyl andmethyl esters in the cells (by enzymatic attack or other cause), the concentration of L- and D-Tyr was analyzed by high-performance liquid
chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37°C or under ice-cold conditions.
Results: Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of L-Tyr ethyl or
methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in
the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer.
Conclusions: The L-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by transstimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic
attack. We will use IMT and L-Tyr ethyl or methyl esters to examine LAT1 function in tumor cells or tissues in vivo.
© 2010 Elsevier Inc. All rights reserved.
Keywords: Amino acid transport; Amino acid ester; Chinese hamster ovary cells; 3-iodo-α-methyl-L-tyrosine; In vitro study

1. Introduction
The rapid accumulation of amino acids in tumor cells
enables active proliferation, and this fact has been exploitedfor the clinical imaging of tumors using 123 I-labeled 3-iodo-αmethyl-L-tyrosine by SPECT [1–5]. 125I-3-iodo-α-methyl-L⁎ Corresponding author. Department of Radiological Sciences, Ibaraki
Prefectural University of Health Sciences, Inashiki-gun, Ibaraki 300-0394,
Japan. Tel.: +81 298 40 2217; fax: +81 298 40 2317.
E-mail address: sikano@ipu.ac.jp (N. Shikano).
1
Current address: Division of Health Science, Graduate School of
Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa,
Ishikawa 9200-942, Japan.
0969-8051/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2009.10.003

tyrosine (IMT) has also been developed as a functional
imaging agent for analysis of the tyrosine (Tyr) transportmechanisms in the brain and pancreas [6–8]. Understandingtransport systems of amino acids forms the underlying basis
for developing enhanced imaging systems.
The transport of neutral amino acids involves several
distinct systems with overlapping substrate specificities [9].
The main Na+-dependent transport systems are systems A
and ASC (defined below), while the main Na+-independenttransport system is system L. System L is defined by its Na+
independency and inhibition by 2-amino-bicyclo[2,2,1]
heptane-2-carboxylic acid (BCH). System A is defined by
its Na+ dependency and inhibition by 2-(methylamino)

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isobutyric acid (MeAIB), while system ASC is a Na+dependent transport system, which is intolerant to Nmethylation of substrates. While systems L and A havespecific inhibitors, system ASC does not [9].
At the molecular level, the genes of the proteins thattransport substances are classified into various solute carrier(SLC) families according to the standards established by the
Human Gene Nomenclature Committee [10–12]. System L
is divided into two classes [13]: system L1 belonging to the
SLC7 family and system L2 belonging to the SLC43
family. There are two known isoforms of system L1,
namely, L-type amino acid transporter (LAT) types 1 and 2(LAT1 and LAT2). The SLC7 family comprises twoclasses: the LAT family and the cationic amino acidtransporter family [10–12]. To express transport function,
LAT1 and LAT2 of system L1 require an additional
protein, the heavy chain of 4F2 antigen (CD98; 4F2hc/
SLC3A2), to form a heterodimeric functional complex. By
obligatory exchange mechanisms, LAT1 and LAT2 selectively transport neutral amino acids. On the other hand,
members of system L2, LAT3 and LAT4, do not require
4F2hc. These transporters possess broad substrate selectivity for various neutral amino acids. The expression
patterns and the substrates of these transporters partly
overlap among the systems but do differ [10–12].
It is noteworthy that LAT1 is conspicuously up-regulated
in many tumors and transformed cell lines, which isconsistent with the observed increase in uptake of aminoacids needed for rapid cell growth and proliferation [14,15].
In the future, we aim to develop a technique for IMT andamino acid ester enhancement of amino acid uptake via theamino acid exchanger LAT1 in tumor tissue or cell samples
from patients as an alternative to the current immunohistochemical quantification of transporter proteins. As apreliminary investigation was needed to develop thisexperimental system, we examined the mechanisms of
IMT uptake in Chinese hamster ovary (CHO-K1) cells.
Although transport of IMT has been better studied in othercell lines [16–20] than in CHO-K1, we chose this cell line as
it has been well characterized [21] and would thus enablebiological comparisons of the transport properties ofartificial amino acid tracers. We modified the methods of
Shotwell et al. [21] to examine the CHO-K1 cells. Because it
should be possible to detect the isoform of the system L
transporter that mediates IMT transport by assessing theinhibition profiles of the 20 D- or L-amino acids, we
conducted an inhibition study using 20 naturally occurring
amino acids including the mother amino acid, Tyr. To
confirm the expression of LAT1 in human cells, RT-PCR
should be used to detect the heavy chain of LAT1 mRNA,but homologues of CHO cells have not yet been sequenced.
We investigated the effects of amino acid methyl/ethyl esters
in combination with IMT on IMT accumulation in CHO-K1
cells. To confirm the extent of de-esterification, we analyzed
the concentration of amino acids produced through hydrolysis of the esters in the cell homogenates.

2. Materials and methods
2.1. Materials and preparation of labeled compounds
CHO-K1 cells were purchased from the Riken Bioresource center (Tsukuba, Japan). Reagent-grade α-methylL-tyrosine was acquired from Sigma-Aldrich Japan K.K.
(Tokyo, Japan). Instead of 123I-labeled IMT, which is forclinical SPECT studies, 125I-labeled IMT (IMT) was used.
125
I-NaI (8.1×107 GBq/mmol) was purchased from Muromachi Chemical Co. (Tokyo, Japan). Chloramine-T andother chemicals of reagent grade were purchased from
Kanto Chemical Co. (Tokyo, Japan). No-carrier-added IMT
was prepared by the conventional chloramine-T method,using α-methyl-L-tyrosine as a precursor, as previously
reported [12–14].
2.2. Cell culture
Cell line studies were performed using a modification of
the methods described by Shotwell et al. [21], as follows.
CHO-K1 cells were maintained by serial passage in 25-cm2
cell culture flasks. Cells were fed with Dulbecco's modified
Eagle's medium (Sigma-Aldrich Japan K.K.) supplemented
with L-glutamine (2 mM) and 10% fetal bovine serum
without antibiotics in an atmosphere of 5% CO2 and 95% air
at 37°C (pH 7.4). Subculturing was performed every 5 days
using 0.02% EDTA and 0.05% trypsin.
2.3. Measurement of IMT transport in CHO-K1 cells
CHO-K1 cells were seeded on 60-mm-diameter plastic
tissue culture dishes at a cell density of 5×105 cells/dish in
5 ml of complete medium and were used on the fourth day
after inoculation (semi-confluent phase cells). Uptake into
cells of 11C-methionine and IMT has been reported to differ
considerably depending on whether the cells are in thegrowth phase or the confluent phase [11].
After removal of culture medium, each dish was washedonce with 5 ml of incubation medium, which was
Dulbecco's phosphate-buffered saline for 10 min at 37°C
(pH 7.4). Uptake was measured in 2 ml of Na+-containing
incubation medium or in Na+-free incubation medium as thenormal control with 18.5 kBq of non-carrier-added IMT for
10 min at 37°C. The incubation medium was phosphatebuffered saline (PBS; pH 7.4) containing 137 mM NaCl, 3
mM KCl, 8 mM Na2HPO2, 1 mM CaCl2 and 0.5 mM MgCl,
with pH adjusted by the addition of HCl or NaOH solution.
In the Na+-free medium, NaCl and Na2HPO4 normallypresent in PBS were replaced with choline chloride andK2HPO4, respectively.
For the inhibition study, the following inhibitors (at aconcentration of 1.0 mM) were tested: MeAIB (a system Aspecific inhibitor), BCH (a system L-specific inhibitor),p-aminohippurate (PAH, an organic anion transporter
specific inhibitor), tetraethylammonium chloride (TEA, anorganic cation transporter specific inhibitor), 2,4-dinitrophenol (DNP, an inhibitor of anaerobic mitochondrial energy

N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 189–196

production), and sodium azide (NaN3, an inhibitor of aerobic
energy production). We also investigated the effect of low
temperature (4°C) [20,22–25].
To identify the system L transporter isoform that mediates
IMT transport, test inhibition was conducted with each of the
20 D- or L-amino acids (final concentration, 1.0 mM) [21],and 125 I radioactivity was determined.
2.4. Screening effective amino acid esters and optimizing
concentration and incubation duration for enhanced
IMT transport
To identify effective amino acid esters and their optimal
concentration and incubation duration for enhanced IMTtransport, cells were incubated in Na+-containing medium at
37°C for 1 h (except in the time-course analysis) with
18.5 kBq of non-carrier-added IMT and one of the following:(1) 1.0 mM of the ethyl/methyl ester chloride salts L-Gly,
L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys;(2) 1 mM of the ethyl and methyl esters of L-Met and L-Tyrfor 5, 15, 30, 45, 60, 120 or 180 min for time-course analysis;(3) 0.001 to 10 mM L-Tyr ethyl and methyl esters forconcentration dependence analysis; and (4) preloading the
L-Tyr esters so that esters are present 10 or 30 min prior to
the start of incubation and not during the incubation for timecourse analysis for 5, 10, 15, 30, 45 or 60 min. For all
experiments, 125 I radioactivity was determined as below.
2.5. Determination of radioactivity
Following the conclusion of incubations, the medium was
aspirated and the cells were rinsed twice in rapid succession
with 5 ml of ice-cold incubation medium. Cells weresolubilized in 1.5 ml of 1 N NaOH, and the radioactivity ofeach aliquot was counted. The 125I radioactivity of thesolubilized cells was determined using an ARC-1000M welltype scintillation counter (Alokam, Tokyo, Japan). Cell
numbers were counted simultaneously on a subcultured dishusing a Coulter counter (Coulter Counter Electronics, Inc.,
London, England).

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studied using cell homogenates at 37°C in a time-course
study for 0, 30 or 60 min. Concentrations of L- and D-Tyr inthe homogenates were analyzed by HPLC with the following
conditions: Nova-Pak C18; UV detection at 280 nm; eluent,water:ethanol:acetic acid (7:3:1); flow rate, 0.5 ml/min. The
retention times were as follows: L- and D-Tyr, 4.0–4.3 min;L- and D-Tyr ethyl esters, 5–6 min; L- and D-Tyr methyl
esters, 4.5–4.75 min.
2.7. Statistical analysis
Data were collated as mean±standard deviation of fivemeasurements, and each experiment was performed in
duplicate. Results were analyzed using Student's t test, andPb.01 was considered to be statistically significant.
3. Results
The contribution of individual transport systems to total
uptake of IMT is shown by the differences in IMT uptake(Fig. 1). Low temperature inhibited IMT uptake, suggestingthat uptake is carrier mediated. The Na+ -independenttransport system was the main contributor to IMT uptake.
These carriers could be energy independent because neither
DNP nor NaN3 inhibited IMT uptake. Moreover, neither
PAH nor TEA inhibited IMT uptake, suggesting that organic
anion and organic cation transport systems were not
involved. However, BCH significantly inhibited uptake,suggesting that IMT is a substrate of system L. IMT was nota substrate of system A because inhibition of IMT uptake
with MeAIB was not significant. IMT appears to be a
substrate of system ASC as well as system L, but not to asignificant degree.
As shown in Fig. 2, IMT uptake was inhibited by both Land D-neutral amino acids (Ala, Ser, Thr, Cys, Leu, Ile, Phe,
Met, Tyr, His, Trp Val and Asn) in CHO-K1 cells. In

2.6. Confirmation of de-esterification of L- and D-Tyr ethyl
and methyl esters in the cell homogenates
All subsequent steps were conducted at 4°C. To prepare
the cell homogenates, cells in the 60-mm dishes were
scraped out with a rubber policeman, and the cells were
homogenized with five volumes of ice-cold 3-mM Tris
buffer (pH 7.4) supplemented with 0.25 M sucrose and
0.1 mM EDTA. The homogenates were centrifuged for
10 min at 9,000×g, and the pellets were discarded. The
supernatants were centrifuged at 105,000×g for 60 min, andmicrosomal pellets were discarded. The resulting cytosolic
fractions were stored at −20°C and used in the deesterification study [26].
We studied de-esterification of L- and D-Tyr ethyl andmethyl esters of the cells in the cell homogenates at 37°C andon ice in the Tris buffer (see above). De-esterification wasFig. 1. Effects of various inhibitors and conditions on non-carrier-added
IMT transport into CHO-K1 cells. Contributions of individual transportsystems to total uptake of non-carrier-added IMT. Open and filled columns
represent uptake of non-carrier-added IMT into CHO-K1 cells in Na+containing and Na+-free uptake medium, respectively. *Pb.005, **Pb.0001
versus control.

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N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 189–196

Fig. 2. Effects of natural L- and D-amino acids (1 mM) on non-carrier-added
IMT transport into CHO-K1 cells. Open and filled columns represent uptakeof non-carrier-added IMT into CHO-K1 cells in Na+-containing and Na+free uptake medium, respectively.

contrast, the D-amino acids Ala, Ser and Thr had weaker or
statistically insignificant inhibitory effects on IMT uptake.
The L-isomers demonstrated higher inhibitory effects thanthe D-isomers.
Of the tested ethyl/methyl ester chloride salts coadministered with IMT, IMT uptake was significantly
increased (Pb.001) by the ethyl esters of L-Tyr, L-Phe and
L-Ser (Fig. 3A) and the methyl esters of L-Tyr, L-Ser and
L-Lys (Fig. 3B). For the L- and D-Tyr esters, the L-isomers
enhanced uptake to a greater degree than the D-isomers(Fig. 3C).
In the time-course study with the ethyl and methyl estersof L-Tyr, both L-Tyr esters demonstrated significantly greater
uptake after 15 to 180 min (Fig. 4A). On the other hand, theethyl and methyl esters of L-Met did not increase IMT
uptake. Co-administration of 1 mM of the L-Tyr ethyl or
methyl ester with IMT induced the greatest enhancement(Fig. 4B). Preloading of the L-Tyr esters caused earlier
uptake enhancement (Fig. 4C) as compared with simultaneous loading (Fig. 4A). Longer preloading interval
produced a longer and enhanced steady state.
De-esterification kinetics of D-Tyr ethyl and methyl esters
were very slow under the same conditions compared to L-Tyr

Fig. 3. Influence of amino acid esters co-administered with IMT. One millimolar of amino acid ethyl esters (A), methyl esters (B) and Tyr esters (C) on uptake of
non-carrier-added IMT into CHO-K1 cells. *Pb.01 versus uptake when not incubated in amino-acid-free medium, **not significant versus uptake when notincubated in amino-acid-free medium.

N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 189–196

193

Fig. 4. Time course of uptake into CHO-K1 cells of non-carrier-added IMT co-administered with 1 mM L-Tyr ethyl ester or L-Tyr methyl ester (A), concentration
dependence (B) and preloading of the esters: that is, esters present in preloading and not in incubation conditions (C).

ethyl and methyl esters (Fig. 5A and B). De-esterification
kinetics of L-Tyr ethyl and methyl esters were very fast in thehomogenates at 37°C (pH 7.4) and slow in ice-cold
homogenates and the phosphate buffer (pH 7.4; Fig. 5C
and D). Thus, the reaction was stereo selective andtemperature dependent.

4. Discussion
There is no published data describing an in vitro method
to observe amino acid exchanger function by enhancementof amino acid tracer uptake in tissue or cell samples—for
example, by using IMT in combination with an amino acid
ester. We believe that the advantage of such a method would
be “function enhancement reflecting the malignancy of thetumor” [27,28] compared to methods such as immunohistochemical quantification of transporter protein using LAT1
antibody. The development of such a method is our next goal
and, to that end, the focus of this preliminary study was theinterventional control of IMT uptake in vitro in a cell line inorder to enhance IMT uptake by stimulation of the aminoacid exchanger function.
The magnitude of system L and A amino acid transporter
expression reportedly indicates tumor malignancy [27,28].

We selected a system L amino acid exchanger as the target
transporter and attempted to enhance its function with transstimulation of neutral amino acid exchange by an amino acidmethyl or ethyl ester. As shown in Fig. 6, we proposed to use
one of the methyl or ethyl esters of Tyr with IMT, on thebasis that a methyl or ethyl ester of Tyr might act as aprodrug for the trans-stimulation of neutral amino acidexchange by the system L amino acid exchanger. Accordingly, we hoped that these lipophilic esters would diffuse
into cells, allowing the ester to act as a prodrug, which could
be metabolized into an amino acid by intracellular hydrolysis
enzyme(s). We expected that, compared to amino acidpreloading, amino acid ester loading would cause very little
competitive inhibition of IMT influx via the amino acidtransporter. Esterification of amino acids could mask thecompetitive inhibition of IMT uptake (influx transport).
LAT1, a system L transporter, uses an amino acid gradient toactivate exchange of intracellular amino acids generated
from the prodrug for extracellular IMT. We also anticipatedthat the prodrug would have a durable effect within the timespan of hydrolysis. The findings shown in Fig. 5 suggest thatde-esterification in the cells is caused by stereo-selective,
temperature-dependent enzymatic attack.
Membrane transport of IMT has already been relatively
well studied with other cell lines — cultured human 86HG-

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N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 189–196

Fig. 5. De-esterification of L- and D-Tyr ethyl and methyl esters in the cell homogenates. Time-course study on de-esterification of 1 mM D/L-Tyr ethyl ester (A)or L-Tyr methyl ester (B) in the cell homogenates at 37°C. The de-esterification of L- and D-Tyr ethyl (C) and methyl (D) esters in the cells in cell homogenates at
37°C or at ice-cold conditions and in a 3-mM Tris buffer (pH 7.4).

39 glioma cells [16], rat C6 glioma cells [17], human GOS3
glioma cells [18], human Ewing's sarcoma cells [19] andhuman U266 myeloma cells [20] — but has not beencharacterized with CHO-K1. Although IMT is reportedly
primarily mediated by system L in most tissues [4], it isimportant to determine what kind of membrane transport
mediates IMT transport; therefore, we sought to verify themechanisms of IMT uptake into CHO-K1 cells. The present
results with CHO-K1 demonstrate that IMT uptake ispredominantly (N70%) mediated by a temperature-dependent and energy-independent carrier, system L. Na+ dependent systems are responsible for a relatively minor
contribution to transport (b20%); IMT appears to be a
substrate of system ASC but not to a significant extent.
Neither system A nor organic anion and cation transportsystems contribute to IMT uptake into CHO-K1 cells. As
indicated by the inhibition profile shown by neutral L- and Damino acids under Na+ -free conditions, the system L
transport of IMT in CHO-K1 cells most likely involves
LAT1 [29,30].
IMT uptake was significantly increased by the ethyl estersof L-Tyr, L-Phe and L-Ser and by the methyl esters of L-Tyr,
L-Ser and L-Lys in the tested candidate amino acid esters.
The most effective amino acid esters were the methyl andethyl esters of L-Tyr. Generally, the esters of the aromatic
amino acids were more effective than those of other aminoacids such as L-Ser or L-Lys, successfully demonstrating asignificant increase in IMT uptake after 15 to 180 min. The
accumulation of IMT was effectively enhanced by the L-Tyr
esters but not by the corresponding D-isomers, indicating thatenzymes may be involved in the hydrolysis of L-Tyr ethyl/
methyl esters. It is unclear why the L-Met ethyl and methyl
esters did not induce activated IMT uptake; however, one
possible reason is the velocity of hydrolysis of the L-Met
ethyl and methyl esters in the cells and the affinity of thegenerated L-Met with the counter transport system. As
shown in Fig. 4B, the effective interventional ester levels on
IMT uptake in vitro were estimated to range from 0.1 to 1
mM for the L-tyrosine methyl/ethyl esters. However, IMT
uptake decreased at excess ester concentration (over 1 mM).
In Fig. 4A, the control shows a curve typical for depletion of
the cells during the experiment, as LAT1 influx and efflux isreversible, showing a steady state. This can be due to the preincubation followed by incubation in absence of necessary
amino acids.
As shown in Fig. 5, de-esterification kinetics of L-Tyr
methyl ester were 1.8-fold faster than that of ethyl ester. Deesterification in cells may be approximately six times fasterN. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 189–196

Fig. 6. Proposed mechanism of IMT uptake activation into cells by system L
amino acid exchanger: ◘, amino acid esters penetrate the cell membrane as aresult of the membrane's lipophilicity; ○, amino acids are metabolized byesterase within the cells; ●, IMT (system L substrates). System L amino acidexchanger function is stimulated by amino acids due to amino acid methyl or
ethyl ester. The lipophilic amino acid esters could diffuse into cells, allowing
the esters to act as prodrugs, which would be metabolized into amino acids
by intracellular hydrolysis enzymes. A system L transporter uses an aminoacid gradient for obligatory exchange of intracellular amino acids generated
from the prodrugs for extracellular IMT.

than that in homogenates diluted to 1/6 cell cytosolic
concentration by 3-mM Tris buffer. Because ethanol is lesstoxic than methanol, L-Tyr ethyl ester is a better candidate.
As shown in Fig. 4A and C, the timing of reaching thesteady state of IMT accumulation may depend on when andhow long the ester is loaded. Magnitude of the enhancementof preloading was similar to that of simultaneous loading.
Longer preloading interval produced a longer and enhanced
steady state. Preloading of L-Tyr esters caused earlier uptake
enhancement as compared with simultaneous loading.
We think that the results of this study give us very
important suggestions, not only for diagnosis methods but
also for potential therapies with 131 I-labeled amino acids.
However, in the in vivo situation, amino acid esters may bedifficult to use because the amino acid esters would be
distributed and hydrolyzed in the cells of most tissues as wellas tumors, and then some kind of transport system on the cellsurface would most likely carry the de-esterified amino acids
out of the cell. As a result, a portion of the amino acids fromthe interventional amino acid esters might enter thecirculation. There is also the possibility of an inhibitory
effect of de-esterified amino acids on IMT uptake by in vivo
tumors. The inhibitory effect of intrinsic amino acids on theintervention in the vitro study may be less than that in an
vivo study.
In the clinical setting, patient preparation and interventions may improve amino acid imaging as flowing. Increased
tumor uptake, by threefold, of 11C-5-hydroxytryptophan byneuroendocrine tumors after administration of carbidopa was

195

observed by a group from Uppsala [31]. This waspresumably caused by inhibition of peripheral metabolism(decarboxylation), which leads to prolonged and higher
exposure of the tracer to the tumor. Prior amino acidadministration increases IMT tumor accumulation and image
contrast. This effect can be explained by the increased
antiporter activity of the amino acid transport system L in
preloaded conditions [32]. Recently, we examined thefundamental changes in the pharmacokinetics of IMT afterprobenecid loading in mice implanted with colon cancer
DLD-1 cells using IMT. We found that preloading withprobenecid yields better tumor images with good tumor/
normal tissue radioactivity ratios by blocking IMT excretionfrom the kidney [33].
In conclusion, to propose a method to quantify amino aciduptake function via amino acid exchanger LAT1, whichindicates tumor malignancy, we conducted a preliminary
study on the effect of amino acid esters in combination with
IMT on accumulation in CHO-K1 cells. LAT1 was selected
as the target transporter to enhance the amino acid exchangerfunction with trans-stimulation by amino acid esters because
it is the main contributor to IMT uptake. IMT uptake wassignificantly increased by the ethyl esters of L-Tyr, L-Phe and
L-Ser and by the methyl esters of L-Tyr, L-Ser and L-Lys (infact, uptake was increased by two- to threefold with the
L-Tyr methyl/ethyl esters). Thus, the L-Tyr methyl/ethyl
esters were the most effective compounds for enhancing
IMT uptake. We are planning to use L-Tyr ethyl ester andother related compounds (e.g., dipeptides) co-administeredwith IMT to enhance amino acid uptake for in vitro cell
examination of tumor tissue.
Acknowledgments
We wish to thank Akihiro Imura and Shinya Nakazawa(Ibaraki Prefectural University) for their excellent technicalassistance. This work was supported by Grants-in-Aid for
Scientific Research (#10770451, #14770498, #13557075,#15659283, #16659322 and #17390336) from the Ministryof Education, Science, Sports and Culture of Japan and theJapan Society for the Promotion of Science. Financial
support was also provided by the Ibaraki Prefectural
University Research Project (9808, 0118 and 0220); Ibaraki
Prefectural University Grants-in-Aid for the Encouragementfor Young Scientists 2001, 2002, 2004, 2005, 2006 and2008; and Japan Atherosclerosis Research Foundation
Grant 2008.
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