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Nuclear Medicine and Biology 37 (2010) 77 – 83
www.elsevier.com/locate/nucmedbio

Automatic labeling method for injectable 15 O-oxygen using
hemoglobin-containing liposome vesicles and its application
for measurement of brain oxygen consumption by PET
Vijay Narayan Tiwaria , Yasushi Kiyonoa , Masato Kobayashia , Tetsuya Moria ,Takashi Kudoa , Hidehiko Okazawaa,b , Yasuhisa Fujibayashia,b,⁎aBiomedical Imaging Research Center, University of Fukui, Fukui, Japan
Research and Education Program for Life Science, University of Fukui, Fukui, Japan
Received 22 April 2009; received in revised form 6 August 2009; accepted 8 August 2009b

Abstract
Introduction: The aim of this study was to develop an injectable 15O-O2 system using hemoglobin-containing vesicles (HbV), a type of
artificial red blood cell, and to investigate the feasibility of 15O2-labeled HbV (15O2-HbV) to measure cerebral metabolic rate of oxygen(CMRO2) in rats.
Methods: The direct bubbling method was combined with vortexing to enhance labeling efficiency of HbV with 15O-O2 gas. L-Cysteine wasadded as a reductant to protect hemoglobin molecules in HbV from oxidation at different concentrations, and labeling efficiencies were also
compared. Measurement of cerebral blood flow (CBF) and CMRO2 in five normal rats was performed using a small animal PET scanner after15
the injection of H15
O2-HbV to evaluate the precision of hemodynamic parameters quantitatively.
2 O andResults: The labeling efficiency of HbV was significantly increased when vortexing and bubbling were combined compared with the simple
bubbling method (Pb.05). The most efficient method for labeling was bubbling of 15O-O2 combined with vortexing and the addition of
2.8 mM L-cysteine in HbV solution. The mean radioactivity of 214.4±7.8 MBq/mL HbV was obtained using this method. PET scans using
15
O2-HbV and H15
2 O yielded a mean CMRO2 value of 6.8±1.4 (mL/min per 100 g) in rats with normal CBF of 51.4±7.9 (mL/min per 100 g).
Conclusion: Addition of L-cysteine to HbV and simple direct bubbling of 15O-O2 gas combined with vortexing was the most efficientmethod for preparation of 15O2-HbV. The present injectable system using 15O2-HbV was successfully utilized to measure CMRO2 in rats,
indicating that this new method could be useful for animal models to measure oxygen metabolism in the brain.
© 2010 Elsevier Inc. All rights reserved.
Keywords: Hemoglobin vesicle (HbV); Injectable 15O-O2; L-cysteine; Cerebral metabolic rate of oxygen (CMRO2); Positron emission tomography (PET)

1. Introduction
Cerebral metabolic rate of oxygen (CMRO2) is measured
in clinical practice using PET with inhalation of 15O-O2 gas.
One of the problems associated with the inhalation method isthe difficulty in handling radioactive gas, causing radioactivity exposure to the patient and hospital staff. In animal⁎ Corresponding author. Biomedical Imaging Research Center,
University of Fukui, 23-3, Matsuoka-Shimoaizuki, Eiheiji-cho, Yoshidagun, Fukui 910-1193, Japan. Tel.: +81 776 61 8170; fax: +81 776 61 8170.
E-mail addresses: tiwaridr@u-fukui.ac.jp (V.N. Tiwari),
yfuji@u-fukui.ac.jp (Y. Fujibayashi).
0969-8051/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2009.08.004

models, the inhalation method is technically very difficult toimplement. High radioactivity in the inhalation tube andmask may affect calculation of brain oxygen metabolism andappropriate scatter correction is required. In previous reportsof an injectable 15O-O2 system for small animal positronemission tomography (PET) studies, CMRO2 was measured
using rat blood and an artificial lung with reasonableaccuracy [1,2]. However, this method is not ideal as it
exposes the handler to a large amount of radioactivity andhigh blood flow rates are needed in the artificial lung forlabeling, which may result in clotting and blood component
damage as the flow tends to become turbulent at high flow
rates [3]. Moreover, an extra rat has to be sacrificed for blood

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V.N. Tiwari et al. / Nuclear Medicine and Biology 37 (2010) 77–83

collection and preservation of collected blood is required.
Therefore, developing a simpler and safer method for
intravenously injectable 15O-O2 would be useful for bothanimal experiments and human studies.
Successful labeling of whole blood by bubbling 15O-O2
gas for human studies has been achieved [4]; however, direct
bubbling of 15O-O2 gas into the blood is reported to cause
foam degeneration of plasma proteins which hinders normal
functioning of red blood cells (RBCs) [1]. To prevent foam
degeneration by bubbling of 15O-O2 into the blood, one
option is to remove protein fraction from the blood by
washing the RBCs. However, it is very troublesome to wash
RBCs many times, and handling during washing may affectthe biologic function of the RBCs [5]. Oxygen loading andunloading characteristics of hemoglobin-containing vesicles(HbV) and their oxygen dissociation curves are almost
similar to that of RBCs [6–11]. HbV, a liposome containing
many hemoglobin molecules was originally developed as a
RBC alternative oxygen carrier. The synthesis processes
along with main chemical and rheological properties of HbV
were previously reported in detail [6,8,12–14]. Recently, it
was shown that HbV could combine with 15O-O2 in amanner similar to RBCs and that transfusion of HbV in ahemorrhagic shock model of rat actually improves oxygen
supply [15]. However, there were no studies that comprehensively evaluate the labeling method itself and theconsequent evaluation of CMRO2 using intravenously
injectable 15O-O2-labeled HbV (15O2-HbV). Because HbV
are smaller and more stable than normal RBCs, which could
better withstand any direct manipulation including direct
bubbling [6–10], an injectable 15O-O2 system using HbV
instead of normal RBCs is desirable. Furthermore, HbV are
free from plasma proteins, have a long storage time andrequires no preparation besides having a reduced risk ofblood-borne infections and antigen incompatibility [16].
The purpose of this study was to design and optimize an
automatic system to label HbV with 15O-O2 gas andestablish a convenient method to measure quantitativeoxygen metabolism in small animals using the single
injection of 15O2-HbV and PET. In this study, we optimized
the labeling conditions to get the maximum yield in labeled
HbV samples. To evaluate the efficacy of this 15O2-HbV invivo, CMRO2 in rats was measured using a small animal
PET scanner.

Table 1
HbV specifications
Parameters

ValuesDiameter (nm)
Lipid (g/dL)
Hemoglobin (g/dL)
P50 (torr)
pH (at 37°C)
metHb (%)
HbCO (%)

277
6.2
10.8
23
7.4
8.5
3.6

P50, oxygen affinity; metHb, methhemoglobin; HbCO, carboxyhemoglobin.

sn-glycero-3-phosphate (DPPG) at a molar ratio of 5/5/1.
Fifty-milliliter vials with 5 ml of HbV samples were used forlabeling experiments. HbV samples were prepared and kept
under anoxic conditions. All the chemicals used in this study
were of reagent grade.
2.2. 15O-O2 gas production
15

O-O2 gas was produced by a 15N(p, n)15O nuclear
reaction with a 2.5% O2/N2 gas target at 18-μA proton
current accelerated by an in-house cyclotron Eclipse HP/RD(Siemens, Knoxville, TN, USA). The bombarded target gas
along with the 15O-O2 gas produced in the target box wastransported to the cyclotron line, and then, to our injectable
15
O-O2 preparation system.
2.3. Preparation of 15O-O2-labeled HbV
The labeling system mainly consisted of a 50-ml bubblingvial containing 5 ml of HbV mounted on a vortex machine.
The cyclotron line carrying target gas was connected to thevial with tubes, a three-way valve and a long needle tobubble the 15O-O2 containing target gas (Fig. 1). Waste gasline was connected to the same three-way valve and avacuum pump (KnF LAB, FTP-10 A, Asahi Techno Glass,
Funabashi, Japan) to make it a closed system. To avoid any
backflow during bubbling, the bubbling needle wasconnected to a one-way filter (Millex-FG.20 μm, Millipore
Co., Bedford, MA, USA). Pressure in the bubbling line wasmonitored with a pressure sensor (KEYENCE, Osaka,
Japan) during the entire experimental process to detect any

2. Materials and methods
2.1. HbV sample preparations
The HbV preparation was provided by Oxygenix Co. Ltd.
(Tokyo, Japan). Its properties are described in Table 1. The
synthesis and purification processes along with mainchemical and rheological properties of HbV were previously
described in detail [4,6,10–12]. Major lipid components in
HbV membrane were 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPCC), cholesterol and 1,2-dipalmitoyl-Fig. 1. Schematic diagram of the final labeling setup with lead shielding for
injectable 15O2-HbV preparation.

V.N. Tiwari et al. / Nuclear Medicine and Biology 37 (2010) 77–83

leakage in the system. The three-way valve connected to a
motor (Dynamite AX-12, Robotis motor, Tokyo, Japan)
was controlled using a computer. The labeling system was
enclosed in a lead shield box (30.5×25.5×40 cm3) with auniform thickness of 5.8 cm including the base. Using thisfinal labeling setup, HbV labeling with 15O2 gas was carriedout in the following manner. After connecting 50 ml vialwith 5 ml of HbV samples to our injectable 15O2 preparation
system, vacuuming of the target line and vial was done
while keeping the bubbling needle well above the HbV
solution. Vacuum valve was then closed, and target gas
containing 15O2 was delivered into the vial via target line
after pushing the bubbling needle inside the HbV solution.
Bubbling along with vortexing was carried out for 2 min.
Labeled HbV was then sampled using a sampling syringe
and radioactivity is measured. The flow rate of the 15O-O2
containing target gas was 200 ml/min in the target line andthe radioactivity in the target gas used for HbV labelingexperiments was 3700 MBq.
Using this system, the following tests were carried out
to optimize the preparation of 15O-O2-labeled HbV. At
first, 15O-O2 gas was bubbled into the HbV vial forlabeling experiments. To improve the radioactivity yield in
the labeled HbV, vortexing (3000 rpm) of the bubbling vial
was added for 2 min. The target gas with different
concentrations of oxygen (2.5%, 5% and 7.5%) was also
evaluated for its efficacy and oxygen saturation (%) was
measured in labeled HbV samples. Oxygen saturation in HbV
was measured using a blood gas analyzer (OPTI-1, AVL
Scientific Corporation, Roswell, USA). Finally, L-cysteine,a known non-enzymatic reductant [17], was added tobring down the methemoglobin level at different concentrations in HbV samples before labeling experiments.
Endotoxin contamination of HbV samples was determinedby the Limulus assay test [18] both before and afterlabeling experiments.
2.4. Animal PET Studies
Sprague–Dawley rats (250–300 g) were purchased fromJapan SLC Inc. (Hamamatsu, Japan). Animal studies wereconducted in accordance with institutional guideline andapproved by the Animal Care Committee at the Universityof Fukui. Rats were starved for 6 h before PET
experiments. The rats were anesthetized with the intraperitoneal injection of chloral hydrate (400 mg/kg). A small
PE-50 catheter (i.d. 0.5 mm, o.d. 0.9 mm) was then inserted
into the left femoral artery for sequential blood sampling. A
separate line using the same tube was placed in the rightfemoral vein to administer the radiotracer. PET studies
were performed using a PET scanner for small animals(SHR-41000; Hamamatsu Photonics, Hamamatsu, Japan).
The scanner acquired 213 slices covering an axial length of
160 mm with 3D mode and achieving a resolution of
approximately 2.0 mm full width at half maximum(FWHM) in the transaxial direction and 2.8 mm FWHM

79

in the axial direction [19]. Each anesthetized rat was placedin the supine position on the scanner bed, and the limbs
were fixed using surgical tape. The optimal cranial position
was determined using a laser beam on a scanner. Before
emission scans, a transmission scan was performed for
60 min using 68Ge/68Ga.
To measure cerebral blood flow (CBF), a PET scan
was conducted with intravenous administration of H15
2 O(37 MBq, 0.5 ml) in the femoral vein using an automaticsyringe pump (pump 11; Harvard Apparatus, Holliston,
MA, USA) with a constant rate for 30 s. A 6-min
dynamic PET scan in 3D mode was initiated at the H15
2 O
injection using list-mode acquisition. Repeated arterial
blood sampling in 20–40 μl was performed manually
every 5 s for the first minute, every 15 s for the next 2 minand every 30 s for the rest of the scan time with a total of
24 blood samples, which was the similar sampling method
to that in the previous report [1]. The radioactivity in the
blood samples thus obtained was immediately measured
with a well scintillation counter (ARC 380, Aloka, Tokyo,
Japan). The scintillation counter was carefully crosscalibrated with the PET scanner before the PET scans
using a small cylinder pool phantom and 18F− solution.
Fifteen minutes after the H15
2 O-PET scan, CMRO 2
measurement was performed with 37 MBq of 15O2-HbV(1 ml). The injection rate for tracer, PET scan protocol andblood sampling were the same as the H15
2 O PET study.
15
O2-HbV was injected intravenously at a constant rate for
60 s. Blood gas data were analyzed after each PET scan of15
O2-HbV for various physiological andH15
2 O andhematological parameters. Rat body temperature wasmaintained throughout the experimental process. The listmode data acquired for initial 3 min were reconstructedusing a Fourier rebinning algorithm. After rebinning thedata, 3D images were reconstructed using a 2D filtered
back-projection algorithm and a Hanning filter (2.8 mm)with time frames of 5 s ×12, 10 s ×6, 20 s ×3. Decay for15
O activity was corrected to the start of the PET scan.
2.5. Calculation of CBF and CMRO215
H15
O2 -HbV were coregistered, and using H15
2 O and
2 O
images, five regions of interest (ROIs) on the whole brain weredrawn at the middle level of the brain. Same set of ROIs were15
applied in both H15
2 O and O2 -HbV images. The time activity
curve (TAC) of the whole brain was obtained by averaging
ROI data from several slices. The CBF values (ml/min per 100g) were calculated by the autoradiographic method [20,21]. A
partition coefficient of 0.8 was applied for calculations [1,2].
The CMRO2 values (ml/min per 100 g) were calculated
based on two-compartment analysis [22]. Rate constants ofK1 and k2 for the two-compartment model were determinedby nonlinear least squares fits for the following equation(Eq. 1) using TAC from dynamic brain images and arterial
input function.

CO ðt Þ = K1 AO ðt Þ  e−k2t + V0 AO ðt Þ;

ð1Þ

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V.N. Tiwari et al. / Nuclear Medicine and Biology 37 (2010) 77–83

where CO(t) and AO(t) are the radioactivity in brain tissue
and arterial blood, V0 is the vascular volume and ⊗represents convolution. The initial 3-min dynamic PET
data and input function were used for the fitting. Finally,
CMRO2 was calculated as a product of K1 and the totalarterial oxygen content (tO2c) as shown in Eq. 2.
ð2Þ

CMRO2 = K1 tO2 c:

The advantage of this one-step method by Ohta et al. [22]is that CMRO2 can be calculated without independent
estimation of oxygen extraction fraction (OEF) because K1
can be defined as CBF×OEF.
2.6. Data analysis and statistics
Results are expressed as the mean±S.D. For statistical
data analysis, one-way analyses of variance (ANOVAs) withpost hoc Scheffe's test were applied for multiple comparisons. A Student t test was applied for two groupcomparisons. Blood gas data in the PET studies werecompared at two time points using paired t test. A P value ofless than .05 was considered statistically significant.
3. Results

Fig. 2. Radioactivity of 15O2-HbV after L-cysteine was added in HbV
samples at different concentrations of 0, 2.0, 2.8, 3.0 and 4.0 mM. Values are
mean±S.D. (n=5 for each condition). ⁎Pb.001 different from all otherconditions by one-way ANOVA with post hoc Scheffe's test.

preparation were determined to be 5 ml of HbV containing
2.8 mM L-cysteine and a bubbling time of 2 min withvortexing. Using this method, an average 15 O2-HbV
activity of 214.4±7.8 MBq/mL was achieved (n=5).
Endotoxin contamination of HbV samples before and afterlabeling experiments was found to be below 0.2 EU/mL,
which was within permissible limits.
3.2. Animal PET studies

3.1. 15O-O2-labeled HbVUsing bubbling-only method, the radioactivity obtainedin 15 O2-labeled HbV was 28.9±9.6 MBq/mL, which
increased to 79.2±11.8 MBq/mL when bubbling was
combined with vortexing. The difference in the labelingefficiency of HbV between the two methods was significant(Pb.05, Student t test, n=5). The results for the effect ofdifferent oxygen concentrations in the target gas forlabeling are summarized in Table 2. No significant
difference was found in oxygen saturation of HbV atdifferent oxygen concentrations.
In order to further increase the efficiency of radio
labeling, the effect of L-cysteine was estimated. Fig. 2
shows a graph of radioactivity yields in 15O2-HbV withdifferent concentrations of L-cysteine. Radioactivity of 15O2HbV was significantly higher in HbV samples containing Lcysteine at concentrations of 2.8, 3.0 and 4.0 mM comparedwith 2.0 mM (Pb.05), but no significant difference wasobserved in the radioactivity yield between 2.8, 3.0 and
4.0 mM (ANOVA with post hoc Scheffe's test). Fromthese results, the optimum conditions for 15 O2-HbV

Fig. 3 shows the TACs of arterial blood and brain tissue
after injection of around 37 MBq of 15O2-HbV. Radioactivity in the arterial blood peaked at about 70 s after injection,and brain tissue radioactivity reached a plateau after agradual increase. Table 3 shows blood gas parameters
15
O2-HbV PET scans. Arterial
observed during H15
2 O andblood gas data were not significantly different in the two
conditions. Moreover, administration of small volume (1 ml)of 15O2-HbV is unlikely to alter the hematological andrheological properties of blood. The mean tO2c value in fiverats studied was 0.22±0.01 during 15O2-HbV scans. The
mean values of CBF and CMRO2 obtained from five rats

Table 2Oxygen saturation at different oxygen concentrations (n=3)
Oxygen concentration (%)

O2 saturation in HbV (%)

2.5
5
7.5

77.7±1.778.0±2.377.8±2.0

Values are mean±S.D. The O2 saturation was not significantly different
among different conditions (P=.99, ANOVA).

Fig. 3. TACs of arterial blood (●) and brain tissue (□) after the injection of
15
O-O2-labeled HbV (n=5) via femoral vein. Rats were anesthetized withchoral hydrate (400 mg/kg, ip). The injected dose and volume of labeled
HbV were around 37 MBq and 1 mL, respectively.

V.N. Tiwari et al. / Nuclear Medicine and Biology 37 (2010) 77–83
Table 3Arterial blood gas data before H2O15 and 15O2-HbV PET scans (n=5)

pH
PCO2 (mm Hg)
PO2 (mm Hg)
Hct (%)O2Sat (%)
Hb (g/dl)

After H2O15

After 15O2-HbV

Difference

7.3±0.1
47.1±7.777.1±12.5
0.6±0.1
93.5±2.8
20.1±1.5

7.2±0.1
41.1±5.4
79.5±20.1
0.51±0.1
92.9±4.4
20.0±0.6

n.s.
n.s.
n.s.
n.s.
n.s.
n.s.

Values are means±S.D. n.s., not significant (paired t test).
15
using H15
O2-HbV PET were 51.4±7.9 (mL/min per
2 O and
100 g) and 6.8±1.4 (mL/min per 100 g), respectively. Mean
OEF values obtained in this study were 0.61±0.16. Fig. 4
shows typical PET images acquired for 3 min after the
15
O2-HbV. The images
injection of 37 MBq of H15
2 O andclearly show the presence of radioactivity in the brain region
which demonstrates that 15O-O2 is carried by HbV in vivo.

4. Discussion
Since we wanted to develop a simple system for
injectable 15HbV preparation, a direct bubbling method
was selected for labeling. To increase the radioactivity yieldin the labeled HbV, various modifications were made to thedirect bubbling method to achieve the optimal labelingefficiency. The binding efficiency of 15 O2-HbV wasincreased when simultaneous bubbling and vortexing were81

performed in HbV samples containing L-cysteine. Effects ofdifferent concentrations of L-cysteine in HbV on labelingefficacy were compared and the radioactivity of 15O2-HbV
was maximal at 2.8 mM and higher. The greater labelingefficacy by adding L-cysteine could be attributed to its
ability to suppress methemoglobin formation, which isincapable of binding with oxygen, as reported previously[23]. A physiologic sigmoid curve of the hemoglobin–
oxygen reaction indicates that the absorption fraction ofoxygen in RBCs is greater at a higher partial pressure ofoxygen [24]. However, contrary to our expectations, thebinding efficiency of HbV did not increase even after
changing the partial pressure of oxygen in the target gas.
This result can be attributed to the carrier effect [1]. The
hemoglobin molecules in HbV are considered to besaturated even at a low concentration of oxygen, and it
seems an unimportant factor in deciding the labelingefficiency of HbV. Pressure in the vial was also monitored
throughout the bubbling process to achieve the range of 120
to 130 kPa at the maximum, which is close to atmosphericpressure. Although pressure does not seem to affect thehemoglobin binding with oxygen at these values [25],monitoring the experimental conditions during bubbling was
helpful to check possible leakage in the system.
HbV is useful to prepare injectable 15O-O2 because of its
smaller size and stability with less wear and tear in vivo,
which seemed appropriate for direct bubbling [9,26]. It was
shown that a fraction of HbV can bind with 15O2 moleculesin vivo after inhalation of 15O2 gas when it is injected in an

15
Fig. 4. Small animal PET images of a rat obtained for 3 min after the injection of 37 MBq of H15
2 O and O2-HbV. Coronal (A), sagittal (B) and axial (C) views ofa rat brain PET images showing substantial 15O2 uptake in the brain.

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V.N. Tiwari et al. / Nuclear Medicine and Biology 37 (2010) 77–83

animal [11]. Moreover, to avoid foam generation when using
normal RBCs, the plasma protein fraction has to be separated
from whole blood by repeated centrifuging and washing of
the RBC fraction, although repeated washing of the RBC
fraction may affect their biologic function [5]. Magata et al.
[1] reported an injectable 15O-O2 system using an artificiallung and rat blood for labeling. This method requires a
specific system called CUPID to concentrate 15O-O2 in thetarget gas because high-radioactivity gas was administeredinto the artificial lung along with the previously collected rat
blood. Therefore, this method requires another animal to besacrificed for blood collection, and blood clots have to becontinuously removed from the collected blood before
administration. The target gas and blood have to be
circulated at a very high flow rate in the artificial lung toachieve good labeling efficiency. Our HbV-based injectable
15
O-O2 system does not require expensive equipment such as
CUPID and an artificial lung, and has no need for prior bloodcollection from another rat. It requires shorter time to label
HbV (2 min) and less radioactivity exposure since the system
is semiautomatic and well shielded. Radioactivity thus
obtained using the final labeling conditions was greaterthan 200 MBq/mL HbV on average, which is sufficient
activity for animal experiments. Ter-Pogossian et al. [4]reported a yield of 37 MBq/mL of blood when direct
bubbling of 15O-O2 gas was done for 6 min in whole blood
though radioactivity of 15O-O2 gas used for labeling blood
was not mentioned. Awasthi et al. [13] reported thatradioactivity in HbV by the direct labeling trial with 15O2
was only 14.8 MBq/mL of HbV, and thus, the present study
is the first report where sufficient radioactivity in the form of
injectable 15O2-HbV is achieved for carrying out brain
oxygen metabolism studies in rats.
CBF values obtained from five rats using the autoradiographic method were consistent with previous reports [1,27],
indicating the state of cerebral circulation was in the normal
range under anesthesia. CBF values obtained in this study arealso not very different from the previous report [2] wheresame type of anesthesia was used. Since the radioactivity
used in measurement of CBF and CMRO2 was 37 MBq
because of limitations due to count rate performance of the
PET scanner and slow injection rate, it was prudent to use
3-min scan duration for better image quality and contrast.
For calculation of CBF, 2–3 min scan duration is used inanimal studies and the values obtained are identical [1]. In
the 15O2-HbV experiments, the tracer was administeredwith a slow bolus injection and CMRO2 was calculatedusing two-compartment analysis [22]. CMRO2 values inrats obtained from this 15O2-HbV injection method wereslightly higher compared with previous report using thesame anesthesia [2], although difference in anesthesia may
provide a larger difference in CMRO2 as observed in aprevious study [1]. They used Mintun's method, whichcalculates CMRO2 by the combination of CBF, CBV andO2 images [28]. We used Ohta's method which is reported
to show a tendency to overestimate oxygen consumption,

probably due to the effect of radioactivity in the veins andignoring recirculation of 15O-water [29]. However, thissimple method with a single injection of 15O2-HbV isuseful for measuring CMRO2 in small animals because it
requires shorter time and the procedure is easier to carryout. Regions of interest were placed on the whole brain
region in this study to obtain global values. Local CMRO2
values could be evaluated if PET images are coregisteredwith MRI images to detect a precise location of changes inregional CMRO2. Moreover, it is also expected that local
CMRO2 values can be estimated with a higher-resolution
animal PET system.
HbV, a cellular type of hemoglobin-based oxygen carriers(HBOCs), is still in preclinical trial stage and has not yet
approved by FDA for human use. Noncellular types of
HBOCs are known to produce vasoconstriction due tounbound hemoglobin, but there have been no reports
involving HbV which suggests any kind of undesirable
effects. Therefore, we believe it can be of potential use in
humans though we have no experimental evidence as yet to
substantiate our point.
5. Conclusion
Our results show that intravenously injectable 15O2-HbV
could be prepared to carry out quantitative animal studies
that successfully calculated CMRO2 in rats. A simple direct
bubbling method combined with vortexing and addition of
L-cysteine was effective to improve radioactivity yield in
15
O2-HbV. This simpler and safer injectable 15 O-O2
method will enhance basic researches in various fields
and has potential for human use.
Acknowledgments
The authors thank Shingo Kasamatsu, Toshinao Nakakoji
and other staff of the Biomedical Imaging Research Center,
University of Fukui, for their technical and clinical support.
This study was partly funded by a Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science(17209040) and 21st century COE Program (Medical
Science) and Research and Development Project Aimed at
Economic Revitalization (Leading Project) from MEXT.
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