Nuclear Medicine and Biology 34 (2007) 659 – 665
www.elsevier.com/locate/nucmedbio

Transport of D-[1- 14 C]-amino acids into Chinese hamster ovary(CHO-K1) cells: implications for use of labeled D-aminoacids as molecular imaging agents
Naoto Shikano a,⁎, Syuichi Nakajima a , Takashi Kotani a , Masato Ogura a , Jun-ichi Sagara b ,Yukio Iwamura c , Mitsuyoshi Yoshimotod , Nobuo Kubota a ,Nobuyoshi Ishikawa a , Keiichi Kawai daDepartment of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japanb
Center for Medical Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japanc
Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, JapandSchool of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Received 3 March 2007; received in revised form 28 April 2007; accepted 10 May 2007

Abstract
Introduction: The fact that D-amino acids have been found in various tissues and are involved in various functions is a clue to how todevelop new imaging agents. We examined D-amino acid transport mechanisms in Chinese hamster ovary (CHO-K1) cells because
CHO-K1 cells are widely used in biomedical studies and are thought to be useful for expression of genes involved in metabolism of Damino acids.
Methods: Uptake experiments were performed. CHO-K1 cells cultured in 60-mm plastic culture dishes under ordinary culture conditions
were incubated with 18.5 kBq of radiolabeled amino acid in 2 ml of phosphate-buffered-saline-based uptake solution at 37°C. The following
radiolabeled amino acid tracers were used: D-[1-14C]-alanine, L-[1-14C]-alanine, D-[1-14C]-serine, L-[1-14C]-serine, D-[1-14C]-methionine, L[1-14C]-methionine, D-[1-14C]-phenylalanine, L-[1-14C]-phenylalanine, D-[1-14C]-leucine, L-[1-14C]-leucine, D-[1-14C]-valine, L-[1-14C]valine, D-[1-14C]-tyrosine, L-[1-14C]-tyrosine, D-[1-14C]-glutamic acid, L-[1-14C]-glutamic acid, D-[1-14C]-lysine, L-[1-14C]-lysine, D[1-14C]-arginine and L-[1-14C]-arginine. We tested the inhibitory effects of the following compounds (1.0 mM) on transport: 2-(methylamino)
isobutyric acid (a specific inhibitor of system A, in Na+-containing uptake solution) and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (aspecific inhibitor of system L, in Na+-free uptake solution).
Results: D-[1-14C]-methionine, D-[1-14C]-phenylalanine and D-[1-14C]-tyrosine accumulated mainly via system L. D-[1-14C]-alanine and D[1-14C]-serine accumulated primarily via system ASC. High uptake of D-[1-14C]-alanine, D-[1-14C]-methionine, D-[1-14C]-phenylalanine and14
D-[1- C]-leucine was observed. The uptake of radiolabeled serine, valine, tyrosine, glutamic acid and arginine into CHO-K1 was highly
stereoselective for L-isomers.
Conclusions: We observed high uptake of D-[1-14C]-alanine via system ASC (most likely alanine–serine–cysteine-selective amino acidtransporter-1) and high uptake of D-[1-14C]-methionine and D-[1-14C]-phenylalanine via system L (most likely L-type amino acidtransporter-1).
© 2007 Elsevier Inc. All rights reserved.
Keywords: Chinese hamster ovary cells; D-Amino acids; Neutral amino acid transport systems; System L; System A; System ASC

1. Introduction
With the exception of glycine, all natural amino acids
have two optical isomers: L and D. After injection into
⁎ Corresponding author. Tel.: +81 29 840 2217; fax: +81 29 840 2317.
E-mail address: sikano@ipu.ac.jp (N. Shikano).
0969-8051/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2007.05.001

animals, 14C-labeled and 125 I-labeled D-amino acids localize
preferentially in tumors and the pancreas [1,2]. Previous
studies have used PET to image D-[1-11C]-methionine [3], D[1-11C]-tyrosine [4] and D-[1-11C]-phenylalanine [5] intumors and the pancreas. It is not yet known why D-aminoacids accumulate in tumors and the pancreas, but it is likelythat membrane transport plays a very important role.

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N. Shikano et al. / Nuclear Medicine and Biology 34 (2007) 659–665

With the exception of certain mechanisms in earthworms,
silkworms [6,7] and a few bacteria [8], all known biologicalmechanisms involve only L-amino acids. However, due torecent advances in amino acid analysis, several D-aminoacids have been detected in high concentrations in the bodies
of mammals [9]. Free D-amino acids have been found invarious tissues, including the brain; their presence suggeststhat they perform physiological functions [10–12]. The
presence of D-amino acids in mammalian tissue is itself amajor finding that overturns previously accepted biochemical theories. D-Amino acids have been found in various
tissues, and studies indicate that they are involved in various
functions, but numerous issues about D-amino acids remain
unclear, including their membrane transport.
Membrane transport is a first step in metabolic
pathways of D-amino acids. Chinese hamster ovary(CHO-K1) cells are easy to culture and are widely usedin genetic and biochemical studies [13]. CHO-K1 cells
have been used as host cells for transient or stable
expression of genes [14]. This suggests that these cells
could be useful tools for the study of metabolism of Damino acids and the possibility of using D-amino acids as
molecular imaging agents, as well as studying the roles ofD-amino acids in biological functions.
In the present study, to examine transport of D-aminoacids, we performed uptake experiments using 14C-labeled
D-amino acids and CHO-K1, whose transport of L-aminoacids has been studied in detail by Shotwell et al. and Basset al. [15–17]. Those studies have revealed that CHO-K1 has
at least three cellular uptake mechanisms for neutral aminoacids: transport system L, transport system A and transportsystem ASC. Transport systems A and ASC are Na+dependent systems and can be differentiated from each otherbased on pH dependence and specific inhibition by artificial
amino acids. System A is specifically inhibited by 2(methylamino)isobutyric acid (MeAIB), whereas system
ASC is not inhibited by MeAIB. Transport system L is not
Na+ dependent and is specifically inhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) [16,17].
Recent molecular biology studies of amino acid transport
have revealed much about solute carrier (SLC) genes, which
are the genes encoding proteins that transport substances
found in liquids [18–20]. These genes are classified into
various SLC families according to the classification systemof the Human Gene Nomenclature Committee. Genes
encoding amino acid transporters belong to the SLC1,
SLC3, SLC6, SLC7, SLC16, SLC25, SLC38 and SLC43
families. Neutral amino acid transporters belong to the
SLC1, SLC6, SLC7, SLC16, SLC38 and SLC43 families.
SLC1, SLC7 and SLC38 are particularly important transporter families in CHO-K1 cells.
In the present study, we examined the transporter
isoforms involved in D-amino acid transport, based on thefindings of recent molecular biology studies involving CHOK1. The results for the D-amino acids were compared withthose of the corresponding L-amino acids. To the best of ourknowledge, this is the first report of the characterization oftransport of D-amino acids in CHO-K1, which is a useful cellline for biomedical studies of gene expression andmetabolism. The fundamental data acquired in such studies
should be useful for the development of molecular imaging
agents based on radiolabeled D-amino acids.
2. Materials and methods
2.1. Chemicals and labeled compounds
Reagent-grade chemicals (Aldrich Chemical Co., Milwaukee, WI, USA) were used in this experiment. We purchased
the following D-amino acids from American Radiolabeled
Chemicals Co. (St. Louis, MO, USA), along with their L[1-14C]-isomers: D-[1-14C]-alanine (D-14C-ALA), D-[1-14C]serine (D-14C-SER), D-[1-14C]-methionine (D-14C-MET), D[1- 14 C]-phenylalanine ( D - 14 C-PHE), D -[1- 14 C]-leucine(D-14C-LEU), D-[1-14C]-valine (D-14C-VAL), D-[1-14C]-tyrosine (D-14C-TYR), D-[1-14C]-glutamic acid (D-14C-GLU), D[1-14C]-lysine (D-14C-LYS) and D-[1-14C]-arginine (D-14CARG). Specific activity ranged from 1.85 to 2.22 GBq/mmol.
2.2. Cell culture
CHO-K1 cells were purchased from Riken Cell Bank
(Cat. No. RCB0285, Tsukuba, Japan). CHO-K1 cells (105cells/ml) were incubated in 6-cm dishes (Cat. No. 150288,
Nalge Nunc International, Roskilde, Denmark) with 5 ml ofDulbecco's modified Eagle's medium (Cat. No. D5796,
Sigma Chemical Co., St. Louis, MO, USA), which contained
10% fetal calf serum, at 37°C in a 10% CO2 atmosphere (pH7.4). A cell proliferation curve was prepared to determine theduration of the logarithmic growth phase.
2.3. Uptake experiments with CHO-K1 cells
To identify the transport systems involved in uptake ofvarious neutral D-amino acids and to assess the contributionsof each transport system, we investigated intracellular uptakeof 14C-labeled D-amino acids with or without Na+, using
artificial amino acids and a modification of the method of
Shotwell et al. [16].
When cells had been cultured to a density of about 80% ofconfluence, the medium was replaced with 5 ml of phosphatebuffered saline (PBS) containing Na+ (37°C, pH 7.4). The
cells were then incubated at 37°C for 10 min. Next, the PBS
was replaced with 2.0 ml of a solution containing a 14Clabeled D-amino acid and 1.0 mM of an amino acid transportsystem inhibitor: MeAIB (a specific inhibitor of system A) or
BCH (a specific inhibitor of system L). The cells were thenincubated for 10 min. Next, the medium was removed, and thecells were washed twice using 5.0 ml of cold PBS and werethen lysed using 2.0 ml of NaOH (0.1 N). A liquid scintillator
cocktail (Clear-sol II; Nacalai Tesque, Kyoto, Japan) was
mixed with 200 μl of the resulting lysate, and this mixture wasplaced in a scintillation vial. A liquid scintillation counter (LS6500; Beckman Instruments, Fullerton, CA, USA) was usedN. Shikano et al. / Nuclear Medicine and Biology 34 (2007) 659–665

Fig. 1. Growth curve of CHO-K1. The logarithmic growth phase is for 1 to 4
days after inoculation. It took 5 days to reach confluence.

to measure radioactivity. The experiment was repeated using
PBS without Na+. The result for each set of experimental
conditions was the mean of five dishes.
3. Results
Fig. 1 shows the growth curve of CHO-K1. For the uptake
experiments, we used CHO-K1 cells in the logarithmic
growth phase, 4 days after inoculation.
The highest uptake was observed for D-14C-ALA (about
30% of the administered dose), followed by D-14C-PHE(25%). Approximately 15% of the administered dose of
14
14
D- C-MET and D- C-LEU was taken up by CHO-K1 cells.
14
The uptake of D- C-TYR was about 5% of the administered

661

Fig. 3. Contributions of amino acid transport systems to total uptake of
14
14
D- C-LEU into CHO-K1 cells. The total uptake of D- C-LEU was
divided into the fractions mediated by different transport systems. System
A contributed the portion of the total uptake that was inhibited by MeAIB.
System ASC and other systems contributed the portion of the Na+dependent uptake that was not inhibited by MeAIB. System L contributed
the portion of the Na+-independent uptake that was inhibited by BCH.
Nonsaturable systems contributed the portion of the Na+-independent
uptake that was not inhibited by BCH. Each column represents mean±S.D.
of five CHO-K1 monolayers.

dose. Very little of the administered dose of D-14C-SER,
14
14
14
14
D- C-VAL, D- C-GLU, D- C-LYS and D- C-ARG was
taken up by CHO-K1 cells (Fig. 2).
Fig. 3 shows the contribution of each transport system tothe total uptake of each radiolabeled amino acid. The main
contributor to uptake of D-14C-ALA was system ASC. The
main contributor to uptake of D-14C-PHE and D-14C-MET
was system L (Tables 1 and 2).
For all of the tested amino acids, the uptake into CHO-K1
was stereoselective in favor of the L-enantiomer. The
following stereoselectivity ratios [(uptake of L-aminoacid)/(uptake of D-amino acid)] were observed: alanine,
1.3; serine, 31.0; methionine, 1.4; phenylalanine, 2.9;
Table 1
Contributions of systems A, ASC and L to transport of D-[1-14C]-aminoacids into CHO-K1
D-Isomer
14

D-

C-ALA
14
D- C-SER
14
D- C-MET
14
D- C-PHE
14
D- C-LEU
14
D- C-VAL
14
D- C-TYR
14
D- C-GLU
14
D- C-LYS
14
D- C-ARG
Fig. 2. Percent uptake of L-[1-14C]-amino acids (open column) and D[1-14C]-amino acids (filled column) into CHO-K1 cells. Each column
represents mean±S.D. of five CHO-K1 monolayers. All differences between
L-isomer and D-isomer were significant (Pb.005).

System A

System ASC

System L

Nonsaturable

15.39 ± 7.27
7.80 ± 7.47
4.48 ± 5.98
11.28 ± 11.86
5.69 ± 7.40
0.00 ± 4.98
10.30 ± 11.17
7.53 ± 15.62
14.36 ± 4.39
0.00 ± 10.18

71.88 ± 4.73
72.64 ± 6.05
0.00 ± 11.98
4.67 ± 9.61
38.09 ± 3.49
21.09 ± 14.07
0.00 ± 16.97
74.91 ± 9.66 a
21.04 ± 7.78 b
67.74 ± 9.31 b

10.78 ± 0.51
6.01 ± 4.63
95.36 ± 10.75
78.14 ± 3.79
46.33 ± 2.47
54.60 ± 11.51
79.68 ± 14.30
4.20 ± 4.99
12.41 ± 9.97
5.62 ± 5.36

1.9 ± 0.20
14 ± 1.75
9.3 ± 0.50
5.9 ± 0.36
9.9 ± 0.14
28 ± 5.49
17 ± 1.20
13 ± 2.27
52 ± 6.26
27 ± 3.67

Percentage of administered dose/monolayer of noncarrier added D-[1-14C]amino acid; mean±1 S.D. of five monolayers.
aAcidic amino acid transport systems.
b
Basic amino acid transport systems.

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N. Shikano et al. / Nuclear Medicine and Biology 34 (2007) 659–665

Table 2Contributions of systems A, ASC and L to transport of L-[1-14C]-aminoacids into CHO-K1
L-Isomer
14

L-

C-ALA
14
L- C-SER
14
L- C-MET14
L- C-PHE
14
L- C-LEU
14
L- C-VAL
14
L- C-TYR
14
L- C-GLU
14
L- C-LYS
14
L- C-ARG

System A

System ASC

System L

Nonsaturable

5.81 ± 13.17
0.00 ± 8.79
9.50 ± 6.40
11.56 ± 7.51
0.00 ± 3.73
16.82 ± 7.23
15.08 ± 4.71
9.74 ± 5.07
2.02 ± 4.46
0.00 ± 5.93

88.25 ± 1.28
93.43 ± 1.86
0.00 ± 4.02
22.92 ± 6.42
39.27 ± 4.41
0.00 ± 5.81
13.68 ± 5.17
76.84 ± 4.12a
33.15 ± 5.12b
75.14 ± 3.00b4.00 ± 0.53
4.70 ± 0.65
84.98 ± 3.22
57.66 ± 3.86
50.87 ± 2.82
78.85 ± 3.33
61.85 ± 4.32
2.96 ± 1.48
0.00 ± 4.79
5.40 ± 4.25

1.95 ± 0.36
2.02 ± 0.34
11.68 ± 0.60
7.85 ± 0.63
15.86 ± 0.19
9.79 ± 0.45
9.39 ± 0.48
10.46 ± 1.11
66.03 ± 1.69
26.16 ± 3.95

Percentage of administered dose/monolayer of noncarrier added L-[1-14C]amino acid; mean±1 S.D. of five monolayers.
aAcidic amino acid transport systems.
b
Basic amino acid transport systems.

leucine, 2.0; valine, 28.9; tyrosine, 6.9; glutamic acid,
17.9; lysine, 11.2; arginine, 17.2. The highest stereoselectivity was observed for uptake of serine and valine. The
lowest stereoselectivity was observed for uptake of alanine
and methionine.
4. Discussion
Understanding membrane transport of D-amino acids andtheir roles in biological functions is a gateway to exploring
the possibility of using them as molecular imaging agents.
Na+-dependent transporters belong to the SLC1 family
and consist of the alanine–serine–cysteine (asc)-selective
transporter family that transports neutral amino acids andother groups of transporters for acidic amino acids. CHO-K1
cells contain cloned ASCT1/SLC1A4 complementary DNA(cDNA) [21]. System ASC is a transport system resembling
the asc-selective sodium-independent transporter family(asc, asc-type amino acid transporter family). However, asc
is Na+ independent and belongs to the SLC7 family. The
SLC7 family is further divided into two classes: the L-typeamino acid transporter (LAT) family and the cationic aminoacid transporter family [20]. The LAT family consists ofeight proteins that form a heterodimeric functional complex
together with an additional protein, the heavy chain of 4F2
antigen (CD98; 4F2hc/SLC3A2). Of the eight known aminoacid transporters, the most important transporter in CHO-K1
is thought to be L-type amino acid transporter-1 (LAT1;SLC7A), which is a major transporter of system L. Four LAT
isoforms have been cloned: LAT1 and LAT2 (belonging to
the SLC7 family) as well as LAT3 and LAT4 (belonging to
the SLC43 family). System L activity in CHO-K1 has been
examined at the cell biology level in experiments using theinhibitor BCH. Recent studies, using RT-PCR, haveexamined the expression of LAT1 and 4F2hc at themolecular level [22,23]. SLC38 is a Na + -dependenttransporter family. SLC38 contains the alanine-selective

transporter family (system A) and other transporters(glutamate transporters).
In the present study, D - 14 C-ALA and D- 14 C-SER
exhibited system ASC-selective transport. D- 14 C-ALA
exhibited high uptake, but D-14C-SER exhibited low uptake.
The uptake of serine via system ASC was highly
stereoselective for L-14C-SER. It is unclear which isoform
of system ASC transported D-14C-ALA. Two isoforms ofsystem ASC have been identified: ASCT1 and ASCT2. DAlanine appears to have a strong inhibitory effect on Lalanine transport via ASCT1 [24]. D-Alanine has a weak
inhibitory effect on L-alanine transport via ASCT2 [25].
Thus, it seems likely that the present transport of D-14C-ALA
occurred via ASCT1.
In the present study, D-14C-MET, D-14C-PHE and D-14CTYR exhibited system L-selective transport. D-14C-MET,
14
14
D- C-PHE and D- C-LEU exhibited high uptake, but
14
D- C-TYR exhibited low uptake. The uptake of tyrosine
via system L was highly stereoselective for L-14C-TYR.
Previous reports indicate that D-14C-MET, D-14C-PHE and
14
D- C-LEU are transported via LAT1, which is an isoform ofsystem L [13]. Studies using expression cloning of LAT1 in
Xenopus oocytes indicate that transport of serine, valine,
tyrosine and arginine is highly stereoselective for the Lisomer and that transport of methionine, phenylalanine andleucine is not stereoselective [13]. Makrides et al. [26]recently reported preferred transport of O-(2-[18F]fluoroethyl)-D-tyrosine into the porcine brain in comparison to Lisomer, in addition to considerable differences in stereoselective amino acid transport at the blood–brain barrier
among various species.
In the present study, system A exhibited lower transport
activity than systems ASC and L. The greatest contributionof system A to transport of an amino acid was for D-14CALA, 15% of whose total uptake was due to system A; 14%of the uptake of D-14C-LYS was due to system A. Recent
studies have examined functional expression of system A in
Xenopus oocytes injected with mRNA from CHO-K1 cells[27]. To the best of our knowledge, homologues of cDNA of
SLC38A1, A2, A4 and A6 have not yet been cloned in CHOK1 cells.
In the present study, D-14C-ALA, D-14C-MET, D-14CPHE and D-14C-LEU exhibited high uptake. Free L-alanine is
one of the largest pools of natural amino acids in the humanbody; free L-methionine, L-phenylalanine and L-leucine arepresent in smaller pools [28]. It appears that there is no direct
relationship between the rate of uptake of a free amino acidand the size of its pool. Moreover, it appears that the affinityof a transporter for a free amino acid is a more importantfactor in uptake of that free amino acid than the size of its
pool. We anticipate that these issues will be clarified by
future studies using D-amino acids.
In general, stereoselectivity of amino acid transport iswell documented, especially at epithelial barriers such as
those of the blood–brain barrier, renal proximal tubule andplacenta epithelial cells. CHO-K1 is an epithelial-like cell

N. Shikano et al. / Nuclear Medicine and Biology 34 (2007) 659–665

line derived from ovary tissue. In the present study, uptake ofserine, valine, tyrosine, glutamic acid and arginine into
CHO-K1 was highly stereoselective for the L-isomers. It is
unclear whether endogenous D-14C-SER and isomerase arepresent in CHO-K1 cells. Blood-to-brain transfer of D-14CSER and D-14C-TYR at the blood–brain barrier occurs atextremely low levels [29], and it is assumed that D-serine in
the brain is generated by isomerization of L-serine [30,31]. In
an investigation of the differential cerebral uptake of the Dand L-isomers of a PET tracer, Langen et al. [32] concluded
that in rat and human brains, D-cis-4-[18F]fluoroproline and3
D-[ H]proline are preferably transported at the blood–brain
barrier in comparison with their L-isomers and are isomerized to the L-form. Therefore, D-proline in plasma might be asource of intracerebral L-proline.
The present results suggest that in CHO-K1 cells, acidic
amino acid transporters contribute to the uptake of D-14CGLU and that basic amino acid transporters contribute to theuptake of D-14C-LYS and D-14C-ARG. These amino acids
exhibited Na+-dependent uptake that was not inhibitedby MeAIB.
The biodistribution of a functional molecular imaging
agent should be such that it reflects tissue-specific metabolicor physiological function. Fig. 4 shows the general metabolic
pathway of amino acids. The main amino acid metabolic
pathway consists of membrane transport, the free amino acidpool and protein synthesis. Membrane transport and protein
incorporation or other functions are important targets offunctional imaging with labeled L-amino acids. The available
evidence about D-amino acids suggests that future studies
will reveal relationships between diseases and the biological
functions and activities of D-amino acids, including theirmembrane transport, racemization and catabolism.
D-Amino acids are much less common than L-amino acids
but are widely distributed in most organisms [33]. Recent
studies suggest that in vivo distribution of D-amino acids is
related to their catabolism and possible neurochemicalfunctions. For example, D-serine and serine racemase arepresent in the vertebrate retina and contribute to thephysiological activation of N-methyl-D-aspartate receptors[34]. D-Alanine is catabolized by D-amino acid oxidase in663

mouse tissues including the kidneys, liver, brain, leucocytes
and heart [35,36]. Nagata et al. [37] detected substantial
amounts of D-serine, D-alanine and D-proline in plasma frompatients with renal disease and in tissues from mutant micelacking D-amino acid oxidase [35, 38]. Studies indicate thatin humans, free D-amino acids exist in substantial amounts invarious tissues, including the brain, during the growth anddevelopment stages, suggesting that they perform physiological functions. However, much remains unknown about thebiological functions of D-amino acids, including their roles inbrain function and diseases.
In addition, there have been interesting findings in studiesof D-amino acids in proteins. D-Amino acids are present inproteins in tooth enamel [39], dentin [40], cerebral whitematter [41] and the ocular lens of old animals and humans[42]. It has been reported that in the brains of deceased
Alzheimer's patients, the ratio of D-amino acids in βamyloid protein or tau protein was higher than that of healthy
individuals [43], suggesting that D-amino acids are related toage-related diseases such as cataracts and dementia [39,44].
It appears that D-amino acids are not incorporated directly
into protein during translation. Jilek et al. [45] recently
reported cloning the cDNA of an isomerase that catalyzes
posttranslational L–D-isomerization.
For more than 25 years, 11C-labeled amino acids havebeen used for PET imaging, especially for tumor imaging
using L-isomers (e.g., [S-methyl-11C]-L-methionine) [3].
There has been much less use of labeled D-amino acids forimaging because D-amino acids are much less common in
biological systems than are L-amino acids. Given how little is
known about them, 11C-labeled natural D-amino acids andtheir derivatives are interesting compounds for study. Studiesof parent D-amino acids are generally essential, comparing
transport characteristics between the natural parent aminoacids and their corresponding derived amino acids, to clarify
whether amino acids labeled with the relatively long-lived
123
I or 18F exhibit different transport characteristics thantheir natural parent amino acids.
We believe that clarification of the roles of D-amino acids
may lead to the revision of common biological theories andthe development of new diagnostic molecular imaging
agents for use in studying biosystems involving D-aminoacids. We plan to examine the relationship between thepotential of D-amino acids as molecular imaging agents andtheir biological functions in disease, by performing experiments in which genes are introduced into CHO-K1.

5. Conclusions

Fig. 4. General metabolic relationship of free amino acids. (1) Membranetransport, (2) racemization, (3) free D-/L-amino acid pool, (4) protein
synthesis, (5) biogenic amine synthesis, (6) energy-producing metabolismand (7) catabolism. TCA cycle, tricarbonic acid cycle.

We studied the transport of D-[1-14C]-amino acids, using
CHO-K1 cells. CHO-K1 cells accumulated neutral D[1-14C]-amino acids mainly via transport system L (D-14CMET, D-14C-PHE and D-14C-TYR) and system ASC (D-14CALA and D-14C-SER). Although it was previously thoughtthat D-amino acids have low affinity for biological systems,

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N. Shikano et al. / Nuclear Medicine and Biology 34 (2007) 659–665
14

we found that uptake of D-[1- C]-amino acids (especially
14
14
14
14
D- C-ALA, D- C-MET, D- C-PHE and D- C-LEU) by
CHO-K1 cells was comparable to uptake of the corresponding L-amino acids. Uptake of serine, valine, tyrosine,
glutamic acid and arginine into CHO-K1 was highly
stereoselective for the L-isomers. To develop D-amino acids
as diagnostic molecular imaging agents, there is a need forstudies of specific D-amino acids in which genes are
introduced into CHO-K1.

Acknowledgments
We wish to thank Sae Fujimoto, Misako Tanaka, Jun-ichi
Ohyama, Yusuke Kishi, Asami Teraoka, Natsumi Mibuka,
Yuki Kitamura and Yuzou Taguchi of Ibaraki Prefectural
University for their excellent technical assistance. This work
was supported by Grants-in-Aid for Scientific Research(#10770451, #14770498, #13557075, #15659283,#16659322 and #17390336) from the Ministry of Education,
Science, Sports and Culture of Japan and the Japan Society
for the Promotion of Science. Financial support was also
provided by the Ibaraki Prefectural University Research
Project (9808, 0118, 0220 and 0634) and Grants-in-Aid for
the Encouragement of Young Scientists from Ibaraki
Prefectural University of Health Sciences in 2001, 2002,
2004, 2005 and 2006.

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Monoiodo-D-tyrosine, an artificial amino acid radiopharmaceutical for
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