An Artificial Amino Acid, 4-Iodo-L-meta-Tyrosine:
Biodistribution and Excretion via Kidney
Naoto Shikano, MS1; Keiichi Kawai, PhD2; Leo Garcia Flores II, MD, PhD3; Ryuichi Nishii, MD, PhD3;
Nobuo Kubota, PhD1; Nobuyoshi Ishikawa, MD, PhD1; and Akiko Kubodera, PhD4
1Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan; 2School of Health

Sciences, Faculty of Medicine, Kanazawa University, Ishikawa, Japan; 3Department of Radiology, Miyazaki Medical College,
Miyazaki, Japan; and 4Faculty of Pharmaceutical Sciences, Science University of Tokyo, Tokyo, JapanWe evaluated the use of radiolabeled 4-iodo-L-meta-tyrosine as
an amino acid transport marker. The pharmacologic features ofthis compound, particularly the biodistribution and excretion,
were examined by conducting in vivo and in vitro studies using
4-125I-iodo-L-meta-tyrosine (4-125I-mTyr). Results obtained for
L-14C-Tyr and 3-125I-iodo-␣-methyl-L-tyrosine (125I-IMT) wereused for comparison. Methods: In vivo biodistribution studiesof 4-125I-mTyr were performed in male ddY mice. Urinary excretion of 4-125I-mTyr and 125I-IMT with administration of probenecid was studied. Local distribution of 4-125I-mTyr and 125I-IMT in
kidney was visualized by autoradiography. We performed metabolite analysis of 4-125I-mTyr in mice. For in vitro studies,reabsorption mechanisms of 4-125I-mTyr were compared withthose of 125I-IMT and the parent L-14C-Tyr using superconfluent
monolayers of the porcine kidney epithelial cell line LLC-PK1 inmedium containing inhibitor (L-Tyr, D-Tyr, and 2,4-dinitrophenol), in Na⫹-free medium, and at 4°C. Results: 4-125I-mTyrdemonstrated high accumulation in the pancreas and kidneyand comparable brain uptake to that of 125I-IMT. Blood clearance of 4-125I-mTyr was faster than that of 125I-IMT. Three hours
after administration, ⬎70% of 4-125I-mTyr was excreted via theurine, whereas ⬍5% was found in the feces. Renal autoradiography revealed moderate accumulation of 4-125I-mTyr and highaccumulation of 125I-IMT in the renal cortex. Probenecid further
reduced accumulation of 4-125I-mTyr and 125I-IMT in the kidney
as well as urinary excretion. At 30 min after tracer injection,
intact free 4-125I-mTyr accounted for ⬎98.1% of the total
present in kidney and ⬎96.3% in urine. Protein incorporation
was not observed. Uptake of 4-125I-mTyr into LLC-PK1 cellmonolayers was remarkably reduced by 5 mmol/L L-Tyr (4.6%)
and incubation at 4°C (15.6%) but was reduced by 5 mmol/L
D-Tyr (50.0%). L-14C-Tyr and 125I-IMT showed similar results;
however, uptake of 125I-IMT was enhanced by 0.1 mmol/L 2,4dinitrophenol (165.1%), an inhibitor of generation of energy-rich
phosphates. Conclusion: The artificial amino acid 4-125I-mTyrdemonstrated high metabolic stability, rapid blood clearance,
rapid urinary excretion, and similar biodistribution to other radiolabeled L-Tyr analogs. 4-125I-mTyr can be a competitive substrate of L-Tyr reabsorption. However, 4-125I-mTyr demonstrates
different pharmacologic features than those of 125I-IMT, partic-

Received May 14, 2002; revision accepted Nov. 14, 2002.
For correspondence or reprints contact: Naoto Shikano, MS, Department ofRadiological Sciences, Ibaraki Prefectural University of Health Sciences,4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japan.
E-mail: sikano@ipu.ac.jp

ularly in renal handling. 4-125I-mTyr may potentially be applied
as a new amino acid transport marker.
Key Words: amino acid transport; artificial amino acid;
LLC-PK1; 4-iodo-L-meta-tyrosine; SPECTJ Nucl Med 2003; 44:625– 631

W

e recently studied the feasibility of iodinated L-metatyrosine (I-L-mTyr) for measuring amino acid transport in
the brain and pancreas (1). The results indicated that I-LmTyr is resistant to deiodination and accumulates in thebrain. This was thought to be because the L-amino acids
penetrate the brain and the hydroxy group at position 3
increases the metabolic stability (1). According to themethod of Adams et al. (2), radioiodination of L-mTyrresults in 2 major geometric isomers, 6-iodo- and 4-iodo-LmTyr (6-I-mTyr, 4-I-mTyr). We separated these isomers
using high-performance liquid chromatography (HPLC) (3).
The new radiopharmaceutical 6-I-mTyr was reported to be
useful in assessing cerebral amino acid transport mechanisms and quantifying metabolically active dihydroxyphenylalanine (DOPA) decarboxylase (3).
In this article, we present the pharmacologic features of
the artificial amino acid 4-125I-iodo-L-meta-tyrosine (4-125ImTyr) (Fig. 1) (2,3). We investigated the biodistribution andexcretion and conducted metabolite analysis of 4-125I-mTyrin mice. The renal handling of this compound was studied indetail because most of the 4-125I-mTyr proved to be excreted
via kidney. Renal accumulation of 4-125I-mTyr and 3-125Iiodo-␣-methyl-L-tyrosine (125I-IMT) was visualized by autoradiography. Furthermore, the carrier systems related toreabsorption of the apical membrane involved in 4-125ImTyr transport were examined using superconfluent monolayers of a porcine kidney epithelial cell line (LLC-PK1) on
plastic dishes (4,5). The results were compared with the data
for the parent Tyr and 125I-IMT. The LLC-PK1 model has
been used extensively for analyzing several functions of theproximal tubules (6 –10). In this experimental model, LLCPK1 forms an oriented monolayer with tight junctions on theplastic surface and exhibits reabsorption of electrolytes and

BIODISTRIBUTION AND EXCRETION OF 4-I-MTYR • Shikano et al.

625anesthetized with ether and killed by cardiac puncture. Radioactivity of urine and various organs was measured using an ARC1000M well-type scintillation counter (Aloka, Tokyo, Japan).

FIGURE 1.

Chemical structure of 4-125I-mTyr.

some nutrients via the microvilli of the apical membrane,
which are in contact with the uptake solution (5).
We also discuss the clinical perspectives of using 4-125ImTyr on the basis of this investigation and earlier studies(1). To the best of our knowledge, this is the first detailed
study and pharmacologic characterization of 4-I-mTyr.
MATERIALS AND METHODS
Preparation of 4-125I-mTyr
Reagent-grade chemicals were used in this investigation (Kanto
Chemical Co., Tokyo, Japan). DL-mTyr was acquired from SigmaAldrich Japan K.K. (Tokyo, Japan) and was separated by HPLC
using a chiral column (Crownpak CR(⫺), 4 ⫻ 150 mm; Daicel
Chemical Industries, Tokyo, Japan). 125I-NaI was obtained fromAmersham Pharmacia Biotech U.K. (Buckinghamshire, U.K.).
The conventional chloramine-T method was used to prepare 4-125ImTyr, as reported (2). Chloramine-T (Sigma-Aldrich Japan) at aconcentration of 1.0 ⫻ 10⫺3 mol/L in 10 ␮L 0.05 mol/L phosphatebuffer (pH 8.5) was added to a mixture of L-mTyr (100 ␮L at 1.0 ⫻10⫺3 mol/L) and carrier-free 125I-NaI (3.7–7.4 MBq) in 35 ␮L 0.4
mol/L phosphate buffer (pH 8.5). The resulting solution wasallowed to stand for 30 min, and 10 ␮L of 1.0 ⫻ 10⫺3 mol/L
sodium metabisulfite in 0.05 mol/L phosphate buffer (pH 8.5) wereadded.
Geometric isomer separation by HPLC was then performed
using a Nova-Pak C18 column (3.9 ⫻ 300 mm; Waters, Milford,
MA; eluent, 0.02 mol/L potassium acetate:ethanol ⫽ 90:1; flow
rate, 0.5 mL/min; retention time: 4-isomer, 17–20 min; I⫺, 4 –5
min; cold mTyr, 5–7 min). Labeling efficiency and radiochemical
purity were studied using silica gel thin-layer chromatography([TLC] catalog number Art. 5553; Merck, Darmstadt, Germany)with 2 solvent systems, methanol:acetic acid ⫽ 100:1 (Rf value:4-125I-mTyr, 0.50; I⫺, 0.80) and methanol:10% ammonium acetate ⫽ 10:1 (Rf value: 4-125I-mTyr, 0.55; I⫺, 0.80). Solvent exchange was performed using saline.
As references, L-14C(U)-tyrosine (L-14C-Tyr; American Radiolabeled Chemicals Co., St. Louis, MO), a labeled natural aminoacid, was used as the gold standard. 125I-IMT was prepared using
the method reported previously (11,12).
Biodistribution and Urinary Excretion in Mice
The ethical committees of all affiliated universities approved all
animal experiments in this study. Male ddY mice (n ⫽ 5; weight,
30 g) were administered 0.1 mL 4-125I-mTyr (1.11 kBq) by injection into the tail vein. For inhibition studies, probenecid (50 mg/kg
body weight) was preadministered through the tail vein 10 min
before injection of 4-125I-mTyr (13). Urine and feces were collected at various time intervals after administration. Mice were626

In Vivo Stability
For metabolite analysis, male ddY mice (n ⫽ 5; weight, 30 g)
were injected with 0.1 mL 4-125I-mTyr (50 kBq) into the tail vein.
After 30 min, mice were anesthetized with ether and killed bycardiac puncture. An aliquot of tissue was homogenized, and the
5% trichloroacetic acid–precipitated fraction was trapped on a
GC-50 glass filter (Advantec MFS, Inc., Dublin, CA) to facilitate
measurement of the radioactivity incorporated into the protein.
Tissue supernatant was separated by silica gel TLC (catalog number Art. 5553; Merck) using the following solvent systems: methanol:acetic acid ⫽ 100:1 (Rf value: 4-125I-mTyr, 0.5; I⫺, 0.80) andmethanol:10% ammonium acetate ⫽ 10:1 (Rf value: 4-125I-mTyr,0.55; I⫺, 0.80).
In Vivo Autoradiography
For autoradiography, 0.1 mL 4-125I-mTyr or 125I-IMT (670 kBq)
was injected into the tail vein of male ddY mice (n ⫽ 4; weight,
30 g). After 5 min, mice were killed by inspiration of excess ether.
The kidney was placed in carboxymethylcellulose embedding medium (Nacalai, Kyoto, Japan) and then frozen at ⫺15°C for at least
12 h. A Handex cryostat microtome (Shiraimatsu, Osaka, Japan)
was used to cut 20-mm coronal sections from posterior to anterior.
Sections were then dried at ⫺15°C for another 24 h. Tissue slices
were left in contact with BAS-TR2040 imaging plates (Fuji Photo
Film, Kanagawa, Japan) for 24 h. Images were processed using a
BAS 2000 Bio-Imaging Analyzer (Fuji Photo Film).
In Vitro Measurement of Reabsorption
Cell line studies were performed as described by Saito et al. (5).
Briefly, LLC-PK1 cells were obtained from Dainippon Pharmaceutical Co. (Osaka, Japan; catalog number 09-1392) and were cultured on plastic dishes (Falcon; Becton Dickinson, Lincoln Park,
NJ) in Dulbecco’s modified Eagle’s medium (Sigma ChemicalCo., St. Louis, MO), supplemented with 10% fetal calf serum(Equitech-Bio Inc., Ingram, TX) without antibiotics, in an atmosphere of 5% CO2:95% air at 37°C. Subculturing was conducted
every 7 d using 0.02% ethylenediaminetetraacetic acid and 0.05%
trypsin. In this study, cells were used between the 220th and 235th
passages.
For uptake studies, LLC-PK1 cells were seeded on 60-mm
dishes at a cell density of 5 ⫻ 105 cells per dish in 5 mL complete
medium and were used on the 6th and 7th days after inoculation.
Uptake was measured in LLC-PK1 cell monolayers grown on
60-mm plastic culture dishes. The incubation medium comprised
145 mmol/L NaCl, 3 mmol/L KCl, 1 mmol/L CaCl2, 0.5 mmol/L
MgCl2, and 5 mmol/L 2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid, with a final pH of 7.4. In the Na⫹-dependence study,
NaCl was replaced with the same concentration of choline-Cl.
After removal of culture medium, each dish was washed once with
5 mL incubation medium for 10 min at 37°C. Cells were thenincubated with 2 mL incubation medium containing no-carrieradded 4-125I-mTyr or L-14C-Tyr for the specified periods at 37°C,
and in the temperature dependence study, at 4°C.
For the inhibition study, L-Tyr, D-Tyr, probenecid, and 2,4dinitrophenol (2,4-DNP) were added to give final concentrations of
5.0 mmol/L (14), 5.0 mmol/L (14), 1 mmol/L (15), and 0.1
mmol/L (16), respectively. Cells were incubated for 5 min at 37°C

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 44 • No. 4 • April 2003

with 1.11 kBq of no-carrier-added 4-125I-mTyr or L-14C-Tyr(American Radiolabeled Chemicals).
Thereafter, media were aspirated, and monolayers were rinsed
twice rapidly using 5 mL ice-cold incubation medium. Cells weresolubilized in 1.5 mL 1N NaOH, and the radioactivity of eachaliquot was counted. Radioactivity associated with solubilized
cells was determined using an ARC-1000M well-type scintillation
counter (Aloka) and an LS6500 liquid scintillation counter (Beckman Instruments, Fullerton, CA).
Statistical Analysis
Data are expressed as mean ⫾ SD of 3-5 measurements andeach experiment was performed in duplicate. Results were analyzed using the Student t test. P ⬍ 0.05 was considered statisticallysignificant.

TABLE 2
Biodistribution of 125I-IMT in Male ddY Mice
Biodistribution
Blood*
Brain
Pancreas
Liver
StomachIntestine
Kidney
Heart

5 min

15 min30 min6.70 ⫾ 1.44
0.58 ⫾ 0.12
41.48 ⫾ 5.16
4.63 ⫾ 1.29
4.31 ⫾ 1.66
5.08 ⫾ 1.21
102.62 ⫾ 0.93
5.67 ⫾ 2.16

3.12 ⫾ 0.43
0.47 ⫾ 0.08
25.84 ⫾ 9.50
2.00 ⫾ 0.15
4.81 ⫾ 1.39
2.49 ⫾ 0.65
75.34 ⫾ 15.74
3.10 ⫾ 0.41

1.47 ⫾ 0.08
1.05 ⫾ 0.13
9.18 ⫾ 1.89
0.93 ⫾ 0.03
2.93 ⫾ 0.28
0.91 ⫾ 0.06
24.05 ⫾ 6.04
1.53 ⫾ 0.08*%ID/mL blood (n ⫽ 5 mice).
All data except blood are expressed as %ID/g tissue, mean ⫾ 1 SD.

RESULTS
Preparation of 4-125I-mTyr

In Vivo Stability

Labeling of L-mTyr yielded 4-125I-mTyr with labelingefficiency exceeding 80%. After purification, no-carrieradded 4-125I-mTyr with radiochemical purities exceeding
95% was obtained.

Metabolite analysis revealed that intact free amino acidaccounted for ⬎98.11% ⫾ 1.21% of the radioactivity inmouse kidney and ⬎96.29% ⫾ 3.10% in mouse urine.
Protein incorporation of 4-125I-mTyr was not observed inmouse kidney. Furthermore, 4-125I-mTyr displayed high stability against deiodination because free iodine comprised
only 0.17% ⫾ 0.18% of radioactivity in the kidney and3.50% ⫾ 1.59% in urine.

Biodistribution and Urinary Excretion in Mice

Table 1 summarizes the biodistribution of 4-125I-mTyr inhealthy mice. The observed accumulation of 4-125I-mTyr inthe pancreas was higher than that in the kidney. Accumulation of 4-123I-mTyr in mouse kidney cortex was approximately 80% lower than that of 125I-IMT in terms of percentage injected dose/gram of tissue (%ID/g tissue) (Tables 1
and 2; Fig. 2B).
As shown in Figure 2A, rapid clearance of 4-125I-mTyrfrom the blood was observed. Although the clearance of
4-125I-mTyr was faster than that of 125I-IMT in all organs
studied, organ-to-blood ratios were similar with the exception of the kidney.
Urinary excretion of 4-125I-mTyr was rapid and delayedby probenecid administration (Fig. 3). These results demonstrate that secretion of 4-125I-mTyr is competitively inhibited by probenecid.

TABLE 1
Biodistribution of 4-125I-mTyr in Male ddY Mice
Biodistribution
Blood*
Brain
Pancreas
Liver
StomachIntestine
KidneyHeart
Lung
Spleen

5 min

15 min30 min3.72 ⫾ 0.40
0.61 ⫾ 0.16
25.44 ⫾ 1.85
4.31 ⫾ 0.51
1.24 ⫾ 0.22
2.26 ⫾ 0.29
23.77 ⫾ 4.89
2.51 ⫾ 0.33
3.76 ⫾ 0.52
4.04 ⫾ 0.64

1.56 ⫾ 0.36
0.47 ⫾ 0.07
14.03 ⫾ 3.69
1.80 ⫾ 0.42
1.05 ⫾ 0.37
1.03 ⫾ 0.26
12.89 ⫾ 9.53
1.42 ⫾ 0.34
1.90 ⫾ 0.41
2.35 ⫾ 1.05

0.45 ⫾ 0.09
0.32 ⫾ 0.08
2.00 ⫾ 0.57
0.64 ⫾ 0.16
2.82 ⫾ 2.27
0.56 ⫾ 0.06
4.24 ⫾ 1.92
0.92 ⫾ 0.36
0.74 ⫾ 0.14
0.54 ⫾ 0.17*%ID/mL blood (n ⫽ 5 mice).
All data except blood are expressed as %ID/g tissue, mean ⫾ 1 SD.

In Vivo Autoradiography

Localization of 4-125I-mTyr and 125I-IMT in the kidney iscompared in Figure 4. At 5 min after administration, 125IIMT displayed substantial accumulation in the renal cortex,
whereas 4-125I-mTyr showed only moderate accumulation.
Most of the excreted 4-125I-mTyr was observed in the medulla. Probenecid inhibited the cortical accumulation of
125I-IMT and 4-125I-mTyr.
In Vitro Measurement of Reabsorption

Time course analysis of 4-125I-mTyr uptake into LLC-PK1
cell monolayers shows that steady-state levels were reached
25 min after starting incubation (data not shown). An incubation time of 5 min was chosen for the inhibition study.
The results of inhibition studies of 4-125I-mTyr, 125I-IMT,and L-14C-Tyr uptake are shown in Figure 5. Significant

FIGURE 2. Biodistribution of 4-125I-mTyr compared with thatof 125I-IMT in mice. (A) Blood clearance. (B) Kidney radioactivity.
Each bar represents mean ⫾ SD of 5 animals.

BIODISTRIBUTION AND EXCRETION OF 4-I-MTYR • Shikano et al.

627

inhibition of 4-125I-mTyr uptake as well as inhibition of
125I-IMT and L-14C-Tyr uptake was noted with L-Tyr (P ⬍0.01). When compared with L-Tyr, D-Tyr had a smaller inhibitory effect on 4-125I-mTyr, 125I-IMT, and 14C-Tyr uptake.
Replacing sodium with choline slightly reduced 4-125I-mTyrand L-14C-Tyr transport into LLC-PK1 cell monolayers.
Administration of 2,4-DNP slightly enhanced accumulation of 4-125I-mTyr and L-14C-Tyr in LLC-PK1 cell monolayers. However, 2,4-DNP remarkably enhanced the accumulation of 125I-IMT.
As seen in Figure 5, transport of 4-125I-mTyr into LLCPK1 cell monolayers predominantly (87%) used the Na⫹independent system, with minor (13%) use of the Na⫹dependent system. Incubation at 4°C significantly decreased
accumulation of 4-125I-mTyr, L-14C-Tyr, and 125I-IMT (P ⬍0.01). These compounds behaved similarly in inhibition
studies.
DISCUSSIONFIGURE 3. Urinary excretion of 4-125I-mTyr and effects ofprobenecid administration in mice. Control compared with probenecid administration. Each bar represents mean ⫾ SD of 5
animals. *P ⬍ 0.05; **P ⬍ 0.005.

Changes in amino acid transport are frequently observedin conditions such as cancer (17), neuropsychiatric diseases,
and substance abuse (18). Some of the tyrosine derivatives
used in positron emission CT (PET), such as O-(2-18F-

FIGURE 4. Comparison of 4-125I-mTyr (A)and 125I-IMT (B) accumulation in mouse kidney with or without probenecid administration. (Left) Photograph. (Center) Superimposed. (Right) Autoradiograph.

628

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 44 • No. 4 • April 2003

FIGURE 5. Effects of inhibitors on accumulation of 4-125I-mTyr (A) L-14C(U)-Tyr (B), and 125I-IMT (C) in LLC-PK1 cell monolayers.
Each column represents mean ⫾ SD of 4 monolayers. *P ⬍ 0.01; **P ⬍ 0.05.

fluoroethyl)-L-tyrosine (18F-FET) and 3-18F-fluoro-␣-methylL-tyrosine (18F-FMT), and those used in SPECT, such as
123I-IMT, are reportedly transported into cells via the neutralamino acid transport system (19 –21). Of these compounds,
123I-IMT has been successfully applied clinically for brain
tumor imaging using SPECT (22); it has also been applied
as an imaging agent to study brain and pancreas function(11,12).
In an attempt to develop a new amino acid transport
marker, we opted to use SPECT, which is widely conductedwith radioactive iodine. Because the phenol group of mTyrprovided a suitable site for labeling, direct electrophilic
radioiodination was performed for preparation of 4-125ImTyr. High labeling efficiency and specific activity of
4-125I-mTyr (no carrier added) can be obtained with thechloramine-T method. Radioactive iodine has the advantageof availability, low cost, and relatively long half-life. 4-125ImTyr in saline was stable for 3 wk at 4°C.
The radioiodinated compound must be resistant to rapid
breakdown by general metabolic pathways (23) because acomplicated metabolic fate of a radioiodinated compound
requires a complicated kinetic model for quantitative study.
Furthermore, stability against deiodination is essential toprevent internal irradiation of organs that accumulate iodine, such as the thyroid and stomach. On the basis of the
hypothesis that position 4 of the phenol hydroxyl groupplays an important role in the substrate recognition of deiodinase, which reacts with 3-iodo-L-Tyr and 3,5-diiodo-LTyr, we hoped to increased resistance to iodination and to
decrease renal accumulation by changing the position of thehydroxyl group of L-Tyr without ␣-methylation, as in 125IIMT. Furthermore, we hoped that 4-125I-mTyr would retain
affinity for system L transport. The results of metabolic
analysis of this study successfully demonstrated the highmetabolic stability of 4-125I-mTyr, which is comparable to125I-IMT (21). 4-125I-mTyr was not a substrate for protein
synthesis or deiodination. In the brain, no interaction with

DOPA decarboxylase was observed during NSD-1015 inhibition studies (3). We believed that the hydroxy group at
position 3 and the iodine at position 4 would result in themetabolic stability of 4-125I-mTyr (Fig. 1) (1).
Three hours after administration, ⬎70% and ⬍5% of
intact compound was detected in the urine and feces, respectively. 4-125I-mTyr is predominantly excreted via theurinary tract, and hepatobiliary clearance does not appear tobe substantially involved in excretion. Metabolic stability
may be 1 factor contributing to the rapid renal excretion of
the compound.
In the biodistribution study of 4-125I-mTyr (Table 1),
rapid blood clearance, maximum tissue uptake, and prolonged retention in the organs were found, and this iscomparable to the biodistribution of other amino acid analogs, such as 18F-FET, 18F-FMT, and 123I-IMT (19 –21). The
higher accumulation in the pancreas is consistent with thecharacteristics of amino acid biodistribution.
The main difference between the biodistribution of 4-125ImTyr and 125I-IMT was the higher accumulation of 125I-IMT
in all organs studied (Tables 1 and 2). In particular, theaccumulation of 125I-IMT in kidney was about 5-fold higher
than that of 4-125I-mTyr (Fig. 2B). Another ␣-methylated
Tyr analog, 18F-FMT, also exhibits high accumulation in therenal cortex (24). We propose that ␣-methylated Tyr analogs possess a higher affinity for accumulative renal organic
anion secretion systems than non-␣-methylated Tyr analogs, such as 18F-FET (19) or 4-125I-mTyr. This is supportedby the fact that probenecid, which is an organic anion
secretion inhibitor in the kidney (13,25), markedly inhibits
accumulative secretion of the ␣-methylated Tyr analog 125IIMT in the cortex of proximal tubule S2-like areas (Figs. 3
and 4). The actions of probenecid are largely confined toinhibition of organic anion transport across epithelial barriers (13). This is most important for the renal tubule, inwhich tubular secretion of many drugs and drug metabolites
is inhibited (13). Furthermore, plasma protein binding of

BIODISTRIBUTION AND EXCRETION OF 4-I-MTYR • Shikano et al.

629both compounds was approximately 50% in our studies withrat (data not shown). Considering the results of urinary
excretion inhibition analysis with probenecid, as shown in
Figure 3, these data seem quite reasonable on the basis of
the inhibited level of excretion. These results suggest different mechanisms of renal handling for 4-125I-mTyr and
125I-IMT, and ␣-methylation could be the reason for thisdifference.
The results of recent studies have raised questions as tothe nature of systems that mediate the membrane transportof Tyr analogs, such as 125I-IMT, in cells of the renal cortex(26). After glomerular filtration, natural amino acids generally undergo intensive reabsorption and most natural aminoacids are resorbed by S1 to S3 cells, and thus few aminoacids are excreted (27). Figure 5 illustrates 1 possible solution to the above problem. In this experimental model,
LLC-PK1 exhibits reabsorption, because LLC-PK1 comprises polar cells, with the apical side of monolayers contacting medium and the basolateral membrane on the wall of
the culture dish (5). Figure 5 shows that reabsorption of both
4-125I-mTyr and L-Tyr was mediated by Na⫹-independent aswell as Na⫹-dependent systems. Decreased accumulation of
4-125I-mTyr with incubation at 4°C suggests temperaturedependent carrier-mediated mechanisms for 4-125I-mTyr reabsorption. Probenecid exerted no effects on 4-125I-mTyrreabsorption.
Structural requirements for aromatic amino acid secretion
and reabsorption in canines were investigated by Williams
and Hang (28,29) using a series of Tyr analogs. It was
concluded that the L-amino acids undergo secretion andreabsorption and that the L-configuration and amino groupare required for reabsorption, whereas ring hydroxylation
exerts little effect on resorptive transport (29). Results of
excretion studies using probenecid in mice and similar
studies in LLC-PK1 provide evidence of secretion and reabsorption of 4-125I-mTyr. The results of our study agreewith the above reports.
Several amino acid transport systems are known to mediate neutral amino acid transport in the kidney. Systems B0
and B0,⫹ are Na⫹-dependent neutral amino acid transportsystems, predominantly involved in the reabsorption of aromatic amino acids in the apical membrane of proximal
tubule cells (30 –32). System b0,⫹ is a Na⫹-independenttransport system in the apical membrane of proximal tubule
cells (33). This transport system is predominantly involvedin the reabsorption of neutral and basic amino acids, including aromatic amino acids. The majority of A and ASC,Na⫹-dependent neutral amino acid transport systems exist
on the basal side of the membrane (14). System L is aNa⫹-independent major nutrient transport system responsible for large neutral amino acids, including several essential
amino acids (34). System L is exclusively located in thebasolateral membranes (14). In addition to these transportsystems, probenecid-competitive organic anion transportsystems are believed to mediate 125I-IMT as well as 4-125ImTyr.

630

An interesting finding of this in vitro inhibition study in
LLC-PK1 is that 2,4-DNP, an inhibitor of energy-rich phosphate generation (26), caused a significant increase in 125IIMT uptake. Some of the energy-independent amino acidtransport systems that exchange intracellular amino acids
for extracellular amino acids in the apical side of the membrane of the proximal tubule cells are known (30). With theexistence of an energy-dependent secretion system comprising energy-independent influx of 125I-IMT and energy-dependent efflux, this phenomenon is well explained. 2,4DNP could cause an increase in 125I-IMT uptake byinhibiting efflux, and influx cannot be inhibited. However,
2,4-DNP caused a slight and statistically insignificant increase in 4-125I-mTyr and L-14C-Tyr uptake.
Although few natural amino acids are excreted, rapid
excretion of 4-125I-mTyr and 125I-IMT via the urinary tracts
was observed. The affinity of the parent Tyr to varioustransport systems, and metabolic pathways, may be changedby altering the structural heterogeneity of artificial aminoacids through ␣-methylation and iodination of the benzene
ring.
These results indicate several potential clinical applications 4-125I-mTyr. In the biodistribution study, brain uptakeof 4-125I-mTyr was comparable to that of 125I-IMT, suggesting the possibility of brain function imaging. A possible use
in pancreas imaging was also indicated because of therelatively high uptake. The properties of 4-125I-mTyr, including rapid clearance from blood, rapid urinary excretion,and relatively low accumulation in kidney, offer advantages
for performing abdominal tumor SPECT of areas around thekidney when compared with ␣-methylated Tyr analogs. A
prerequisite for an improved amino acid, such as 4-125ImTyr, must be that tumor uptake is at least as high but
preferably higher than that of 123I-IMT. Further investigation is needed to clarify these points and to develop 4-125ImTyr as an amino acid transport marker for SPECT.
CONCLUSION

The artificial amino acid 4-125I-mTyr demonstrated highmetabolic stability, rapid blood clearance, rapid urinary
excretion, and biodistribution similar to that of 125I-IMT.
We believe that 4-125I-mTyr is a competitive substrate of
L-Tyr reabsorption. However, 4-125I-mTyr possesses different pharmacologic features than 125I-IMT, particularly inrenal handling. 4-125I-mTyr may potentially be applied as a
new amino acid transport marker. Further investigation isneeded to develop 4-125I-mTyr as an amino acid transport
marker for SPECT and to clarify its behavior in each target
organ and tissue.
ACKNOWLEDGMENTS

The authors thank Shyu-ichi Nakajima, Toshio Miyamoto, and Yuri Aizawa (Ibaraki Prefectural University) for
their technical assistance. This work was supported by Grantsin-Aid for Scientific Research (10770451, 14770498, and

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 44 • No. 4 • April 2003

13557075) from the Ministry of Education, Science, Sports
and Culture of Japan and the Japan Society for the Promotionof Science. Financial support was also provided from the
Ibaraki Prefectural University Project Research (grants
9808-3, 0118-1, and 0220-1) and Ibaraki Prefectural University Grants-in-Aid for the Encouragement of Young Scientists 2001 and 2002.
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