Nuclear Medicine and Biology 56 (2018) 21–25

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Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio

In vitro uptake and metabolism of [ 14C]acetate in rabbit atheroscleroticarteries: biological basis for atherosclerosis imaging with [ 11C]acetate
Kazuaki Yamasaki a,b, Atsushi Yamashita c, Yan Zhao d, Yoichi Shimizu e,f, Ryuichi Nishii g,h, Keiichi Kawai i,
Nagara Tamaki d, Songji Zhao a, Yujiro Asada c, Yuji Kuge e,f,⁎a

Department of Tracer Kinetics & Bioanalysis, Graduate School of Medicine, Hokkaido University, Sapporo, Japan
Department of Nutrition Management, Faculty of Health Science, Hyogo University, Kakogawa, Japan
Department of Pathology, University of Miyazaki, Miyazaki, Japand
Department of Nuclear Medicine, Graduate School of Medicine, Hokkaido University, Sapporo, Japane
Department of Integrated Molecular Imaging, Graduate School of Medicine, Hokkaido University, Sapporo, JapanfCentral Institute of Isotope Science, Hokkaido University, Sapporo, Japang
Department of Radiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japanh
Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences, Chiba, Japani
Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, Ishikawa, Japanbca r t i c l e

i n f oArticle history:
Received 12 April 2017
Received in revised form 21 August 2017
Accepted 22 August 2017
Keywords:
Atherosclerosis
Cardiovascular diseases[11/14C]Acetate
Nuclear medicine diagnosis
Nuclear imaging

a b s t r a c t
Introduction: Detection of vulnerable plaques is critically important for the selection of appropriate treatment
and/or the prevention of atherosclerosis and ensuing cardiovascular diseases. In order to clarify the utility of[11C]acetate for atherosclerosis imaging, we determined the uptake and metabolism of acetate by in vitro studies
using rabbit atherosclerotic arteries and [14C]acetate.
Methods: Rabbits were fed with a conventional (n = 5) or a 0.5% cholesterol diet (n = 6). One side of theiliac–femoral arteries was injured by a balloon catheter. Radioactivity levels in the iliac–femoral arteries were
measured after incubation in DMEM containing [1- 14C]acetate for 60 min (% dpm/mg tissue). Radioactive
components in the homogenized arteries were partitioned into aqueous, organic, and residue fractions by the
Folch method, and analyzed by thin-layer chromatography (TLC).
Results: The radioactivity level in the injured arteries of rabbits fed with the 0.5% cholesterol diet (atheroscleroticarteries) was signiﬁcantly higher than that in either the non-injured or injured arteries of rabbits fed with theconventional diet (p b 0.05) (% dpm/mg tissue: conventional diet groups; 0.022 ± 0.005 and 0.024 ± 0.007,
cholesterol diet groups; 0.029 ± 0.007 and 0.034 ± 0.005 for non-injured and injured arteries). In metabolite
analysis, most of the radioactivity was found in the aqueous fraction in each group (87.4–94.6% of total
radioactivity in the arteries), and glutamate was a dominant component (67.4–69.7% of the aqueous fractionin the arteries).
Conclusions: The level of [14C]acetate-derived radioactivity into the arteries was increased by balloon injury andthe burden of a cholesterol diet. Water-soluble metabolites were the dominant components with radioactivity inthe atherosclerotic lesions. These results provide a biological basis for imaging atherosclerotic lesions by PET
using [11C]acetate.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction
Atherosclerosis is the most common cause of cardiovasculardiseases. Therefore, it is of critical importance to detect vulnerable
plaques for the selection of appropriate treatment and/or the preventionof these diseases [1–3]. Morphological data such as those obtained bycomputed tomography techniques and/or magnetic resonance imaging⁎ Corresponding author at: Central Institute of Isotope Science, Hokkaido University,
Kita 15 Nishi 7, Kita-ku, Sapporo 060-8638, Japan.
E-mail address: kuge@ric.hokudai.ac.jp (Y. Kuge).
https://doi.org/10.1016/j.nucmedbio.2017.08.003
0969-8051/© 2017 Elsevier Inc. All rights reserved.

have been used for the diagnosis of atherosclerosis [4]. In addition tothese morphological data, nuclear medicine can provide functional
information of affected tissues, which seems to be useful for the
diagnostic evaluation of atherosclerosis and therapy selection.
In this regard, 18F-ﬂuorodeoxyglucose ([18F]FDG), a glucose analog,
is widely studied for the evaluation of atherosclerosis [5]. Highaccumulation of [ 18F]FDG is observed in vulnerable plaques owing to
the increased inﬂammation in the lesions [6,7]. However, [ 18F]FDG
uptake is largely affected by blood glucose level, which hampers thequantitative assessment of inﬂammatory states in atherosclerotic
plaques in patients with high blood glucose levels including diabetes.

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K. Yamasaki et al. / Nuclear Medicine and Biology 56 (2018) 21–25

Therefore, the development of new diagnostic agents showing accumulation mechanisms different from that of [ 18F]FDG is strongly required.
Our previous study using metabolomics analysis revealed that thelevels of metabolites such as citrate, fumarate, and malate in the TCA
cycle were signiﬁcantly higher in the injured arteries of rabbits fed
with a cholesterol diet than in either the non-injured or injured arteriesof rabbits fed with a conventional diet [8]. These results indicate thatradiolabeled substrates of the TCA cycle may be utilized for atherosclerosis imaging. From the viewpoint of nuclear medicine, [ 11C]acetate
may be a potential candidate for this purpose, because acetate is metabolized to acetyl-coenzyme A (CoA) and enters the TCA cycle. Indeed,[ 11C]acetate has been used for the evaluation of TCA cycle activity inthe myocardium [9]. Recently, Derlin et al. have shown the potential
utility of [ 11C]acetate for atherosclerosis imaging by clinical research[10]. However, the accumulation mechanisms of [ 11C]acetate including
its metabolites in atherosclerotic lesions remain to be investigated indetail. Although a few previous studies indicated that the incorporationof [ 14C]acetate into lipid-soluble metabolites such as triglyceride andcholesterol ester was increased in rabbit atherosclerotic lesions owing
to enhanced lipid synthesis [11,12], there have been no studies of thecontribution of TCA cycle metabolites to the accumulation ofradiolabeled acetate in atherosclerotic lesions.
Accordingly, in order to provide a biological basis for atherosclerosis
imaging with [ 11C]acetate and clarify its utility, we determined theuptake and metabolism of acetate by in vitro studies using rabbit
atherosclerotic arteries and [ 14C]acetate.

PBS, and homogenized on ice. Radioactivity in the homogenized arteries
was counted using a liquid scintillation counter (LSC-6100; ALOKA Co.,Tokyo, Japan).
2.5. Metabolite analysis in arteries
Metabolite analysis was performed using the non-injured or injured
iliac–femoral arteries as described in our previous study [13]. The radioactive components in the homogenized arteries were partitioned into
the aqueous, organic, and residue fractions by the Folch method [14],and then radioactivity was measured. The radioactive components inthe aqueous layer were then analyzed by thin-layer chromatography(TLC) (silica gel 60; Merck, Darmstadt, Germany) using a solvent system
consisting of ethanol: 28% aqueous ammonia (3:1). The TLC plates were
exposed to imaging plates, and the exposed plates were analyzed using
an image analyzer (FLA-7000; Fuji Photo Film Co., Tokyo, Japan).
Radioactive [1- 14C]acetate (unchanged compound), acetyl-CoA,glutamate, and aspartate on the TLC plates were identiﬁed bycomparing with corresponding standards.
2.6. Data analysis

2. Materials and methods

All statistical analyses were performed using Stat View version 5.0(SAS Institute, Inc.). All values are expressed as mean ± standard
deviation (SD). In the evaluation of [1- 14C]acetate uptake (Fig. 2A
and Supplemental Table 1) and metabolite analysis (Fig. 3 andSupplemental Table 2), the Mann–Whitney U test was used. p b 0.05
was considered to indicate a statistically signiﬁcant difference.

2.1. Radiopharmaceutical agents and standards

3. Results[1- 14C]Acetic acid (37 MBq/ml) was purchased from Perkin Elmer(Boston, MA) and dissolved in Dulbecco's modiﬁed Eagle's medium(DMEM). Authentic standards, acetyl coenzyme A, L-glutamic acid,and L-aspartic acid were purchased from Wako Pure Chemical Inc.
(Osaka Japan).

3.1. Histological ﬁndings

2.2. Animals
Animal care and all experimental procedures were performed withthe approval of the Animal Care Committees of Hokkaido University
and Miyazaki University. Eleven male Japanese white rabbits were fed
with a conventional (n = 5) or a 0.5% cholesterol diet (n = 6) andthen arterial lesions were induced in the one side of iliac–femoral
arteries using a balloon catheter. Three weeks later, the rabbits were
subjected to [1-14C]acetate uptake assay [8].
2.3. Immunohistochemical analysis
The iliac–femoral arteries were ﬁxed in 4% paraformaldehyde for
12 h at 4 °C and embedded in parafﬁn. Sections (3 μm thick) were
stained with hematoxylin and eosin and immunohistochemicallyassessed using antibodies against rabbit macrophages (RAM11; DAKO,Glostrup, Denmark) and smooth muscle actin (1A4; DAKO), a marker
of smooth muscle cells (SMCs).
2.4. [1-14C]Acetate uptake assay
Rabbits fasted for 4 h were sacriﬁced by exsanguination under deep
pentobarbital anesthesia (60 mg/kg) after the intravenous administration of heparin (500 U/kg). Then, the rabbits were perfused with
50 ml of 0.01 mol/l PBS, followed by 50 ml of DMEM, and their iliac–
femoral arteries were rapidly removed and weighed. The arteries
were preincubated in DMEM containing 10% fetal bovine serum (FBS)and 37 kBq of [1- 14C]acetate was added. After 60 min of incubation,
DMEM was removed and the arteries were rinsed three times with

Fig. 1 shows representative histological images obtained three
weeks after the balloon injury of the iliac–femoral arteries of rabbits
fed with the conventional or 0.5% cholesterol diet. The injured arteriesof rabbits fed with the conventional diet showed neointimal formation
composed of SMCs. On the other hand, the injured arteries of rabbits
fed with the 0.5% cholesterol diet showed the development of an
expansive lesion composed of prominently accumulated macrophages
and proliferated SMCs. On the other hand, no vascular lesion was
induced in the non-injured arteries of the cholesterol- or conventional
diet-fed rabbits. It is known that the plaque prone to rupture, avulnerable plaque, is characterized by macrophage- and lipid-rich core
and thin ﬁbrous cap. In contrast, a stable plaque has rich SMCs. Thus,in this study, we deﬁned the injured iliac–femoral arteries of rabbits
fed with the 0.5% cholesterol diet as atherosclerotic arteries thatmimic vulnerable plaques.
3.2. [1-14C]Acetate uptake assay
Fig. 2A and Supplemental Table 1 show the radioactivity levels in theiliac–femoral arteries. The radioactivity level in the atheroscleroticarteries was signiﬁcantly higher than that in either the non-injured
or injured arteries of rabbits fed with the conventional diet. The radioactivity levels in both non-injured and injured arteries were relatively
higher in the 0.5% cholesterol diet group than in the conventional dietgroup. Although the radioactivity level in the conventional diet group
was hardly affected by balloon injury; that in the 0.5% cholesterol dietgroup was slightly increased.
3.3. Metabolite analysis in the arteries
The radioactive components in the iliac–femoral arteries werepartitioned into the aqueous, organic, and residue fractions by the
Folch method (Fig. 2B and Supplemental Table 1). Most of theK. Yamasaki et al. / Nuclear Medicine and Biology 56 (2018) 21–25

23

Fig. 1. Representative immunohistochemical ﬁndings of iliac–femoral arteries 3 weeks after balloon injury in rabbits fed with conventional or 0.5% cholesterol diet. The arterial crosssections were stained with hematoxylin eosin (HE) and antibodies for rabbit macrophage and smooth muscle cells (SMCs). The injured artery in a rabbit fed with the conventional
diet shows neointimal formation (N) composed of SMCs. The injured artery in a rabbit fed with the 0.5% cholesterol diet shows the inﬁltration and accumulation of macrophage (Mϕ)
and the proliferation of SMCs with expansive remodeling.

radioactivity was partitioned into the aqueous fraction (92.6 and 91.7%
for non-injured and injured arteries of the conventional diet group, and94.6 and 87.4% for non-injured and injured arteries of the 0.5%cholesterol diet group, respectively). The radioactivity in the aqueous
fraction of rabbits fed with the 0.5% cholesterol diet was signiﬁcantly
higher than that in either the non-injured or injured arteries of rabbits
fed with the conventional diet. On the other hand, the percentage ofradioactivity in the organic fraction to total radioactivity was negligible.
The radioactivity in the organic fraction was signiﬁcantly higher in
the atherosclerotic arteries than in other groups of arteries (Fig. 2B
and Supplemental Table 1).
Radioactive acetyl-CoA, aspartate, glutamate, and unchanged
compound were determined in the aqueous fraction (Fig. 3 andSupplemental Table 2). Glutamate fraction was a dominant radioactive
component of the aqueous fraction (67.4–69.7% of the aqueous

fraction). The glutamate fraction was signiﬁcantly higher in theatherosclerotic arteries than in the non-injured arteries of rabbits fed
with the conventional diet. No marked difference was observed in thepercentage of radioactivity in each fraction to total radioactivity in the
aqueous fraction among the four groups of arteries, including theglutamate fraction (Fig. 3 and Supplemental Table 2).
4. Discussion[ 11/14C]Acetate is rapidly transported into cells through themonocarboxylic acid transporters (MCTs) and then metabolized to[ 11/14C]acetyl-CoA by acetyl-CoA synthetase. The generated [ 11/14C]
acetyl-CoA condenses with oxaloacetate to form citrate, which thenenters the TCA cycle as well as form substrates for lipid synthesis[15,16]. Glutamate and aspartate are synthesized from α-ketoglutarate

Fig. 2. Results of (A) [1-14C]acetate uptake assay and (B) the Folch method. Data represent mean ± SD (DPM/mg tissue, n = 5 for conventional diet group and n = 6 for 0.5% cholesterol
diet group). *p b 0.05.

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K. Yamasaki et al. / Nuclear Medicine and Biology 56 (2018) 21–25

Fig. 3. Metabolite analysis of aqueous fraction. Data represent mean (% of radioactivity ineach fraction to total radioactivity, n = 5 for conventional diet group and n = 6 for 0.5%cholesterol diet group). *p b 0.05.

and from oxaloacetate, respectively, in the TCA cycle (Fig. 4). We
considered that our results are relevant to this pathway. Furthermore,
our previous metabolomics analysis showed that the level of glutamate
as a polarized metabolite was highest in the arteries, and it further
increases in the atherosclerotic arteries (data not shown). Thus, thepresent results of our study using [ 14C]acetate are consistent with ourprevious metabolomics analysis. On the other hand, balloon injury and/or the burden of the cholesterol diet did not affect the ratio of radioactivityof each fraction in the water-soluble metabolite (Supplemental Table 2),
indicating that enzymes in the TCA cycle are not largely involved in theincreased accumulation of [ 14C]acetate-derived radioactivity in theatherosclerotic arteries.
MCTs, an acetate transporter, are widely distributed in many cells
including macrophages and SMCs [17,18]. Particularly, expression
level of MCTs in macrophages is enhanced by various factors such as
lipopolysaccharide (LPS) stimulation [18,19]. Thus, the accumulationof acetate-derived radioactivity may reﬂect the expression levels of
MCTs and/or metabolic status of the arteries including subsequent
TCA cycle related enzyme.
As for the metabolic aspects of [ 14C]acetate in atherosclerosis,
Charles and Howard showed that the in vitro [ 14C]acetate-derived
radioactivity was higher in the atherosclerotic rabbit aorta than in
the control aorta, owing to the enhanced metabolic conversion tocholesterol ester and triglyceride [11]. The enhanced [ 14C]acetate-

derived radioactivity and metabolic conversion to lipid-soluble metabolites in the atherosclerotic arteries were also observed in our present
study, indicating an agreement with the results reported by Charles
and Howard. On the other hand, our results clearly showed that the contribution of the lipid-soluble metabolites to total radioactivity was
small, and water-soluble metabolites largely contribute to [ 14C]acetate-derived radioactivity in the atherosclerotic lesions. Unfortunately,previous studies [11,12] did not evaluate the conversion of [ 14C]acetate
to water-soluble metabolites, including TCA cycle intermediates andglutamate. Thus, our results are the ﬁrst to demonstrate that the increased levels of the water-soluble metabolites are an important factor
contributing to the increased uptake level of acetate in atherosclerotic
lesions. It should be noted, however, that the relative change was thelargest in the organic fractions (Fig. 2B and Supplemental Table 1).
The results clearly indicate the importance of changes in the lipid metabolism in atherosclerotic lesion progression.
Derlin et al. observed the high accumulation level of [ 11C]acetatederived radioactivity in the site of predilection for atherosclerosis in
human clinical trials. They suggested that enhanced fatty acid synthasefunction induced by atherosclerosis was a major factor involved in theincreased accumulation level [10]. Our results indicate that expression/activity levels of MCTs and subsequent TCA cycle related enzymes,
in addition to the enhanced fatty acid synthase function, should beconsidered to interpret images with [ 11C]acetate in atherosclerotic
lesions. The present evidences would provide important biologicalbasis to clarify the potentials of atherosclerosis imaging with [ 11C]acetate and understanding of acetate metabolism, although further clinicaland basic studies, including those concerning the roles of MCTs and TCA
cycle related enzymes on atherosclerotic lesion progression, arestrongly required. In vivo imaging studies in human and experimental
animals are also needed to clarify the potentials of atherosclerosis
imaging with [11C]acetate.
The radioactivity level was relatively higher in the non-injured arteries of rabbits fed with the 0.5% cholesterol diet than in either the noninjured or injured arteries of rabbits fed with the conventional diet(Fig. 2A). The non-injured and injured arteries were obtained from thesame rabbit in our experiment. It seems that the radioactivity level is increased by hyperlipidemia and/or mild intimal injury induced by theburden of the cholesterol diet, even in the non-injured arteries of rabbits
fed with the 0.5% cholesterol diet. Thus, our data showed that the increased uptake level of [ 11C]acetate-derived radioactivity may be seen
not only in the advanced and/or vulnerable lesions but also in theearly lesions as in the case of [ 18F]FDG [20], which should be taken
into account in the diagnosis of atherosclerosis.

Fig. 4. Potential metabolic pathways of [11/14C]acetate for atherosclerosis imaging.

K. Yamasaki et al. / Nuclear Medicine and Biology 56 (2018) 21–25

In this study, the coefﬁcient of variation was relatively large.
Individual differences in lesion formation may cause the variations.
Inhomogeneous lesion formation was observed even in the individual
artery, which also seems to cause the variations. In order to solve thisproblem and to further understand the cells involved in the increasein [ 14C]acetate accumulation and metabolism, autoradiographic studies
with [ 11/14C]acetate and/or immunohistochemical analysis are required.
It is also necessary to carry out in vivo imaging using [ 11C]acetate to
conﬁrm the results observed in our in vitro study and the lesion toblood radioactivity ratio. Thereby, we should clarify the clinicalimportance of incorporation of acetate into water-soluble metabolites
in the atherosclerotic arteries.
Until now, a number of reports have not mentioned the watersoluble metabolites of [ 11/14C]acetate in atherosclerotic lesions. Our
results would provide useful information on the metabolic aspects of[ 11C]acetate in atherosclerotic lesions.
5. Conclusions
The level of [ 14C]acetate-derived radioactivity into the artery wasincreased by balloon injury and the burden of a cholesterol diet.
The water-soluble metabolites of [ 14C]acetate were the dominantradioactive components in the atherosclerotic lesions. These results
provide the biological basis for imaging atherosclerotic lesions by PET
using [ 11C]acetate.
Conﬂict of Interest Statement
The authors declare that they have no conﬂict of interest.
Ethical approval
All applicable institutional and/or national guidelines for the careand use of animals were followed.
Acknowledgements
This study was performed through the Creation of Innovation
Centers for Advanced Interdisciplinary Research Areas Program,
Ministry of Education, Culture, Sports, Science and Technology,
Japan, and also supported by JSPS KAKENHI (Grant Numbers
26293268, 15K15440, and 16H05163) and the Intramural ResearchFund(25-4-3)for Cardiovascular Diseases of the National Cerebral andCardiovascular Center.

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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.nucmedbio.2017.08.003.
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