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In Vivo Lipid Profiling Using ProtonMagnetic Resonance Spectroscopy
in an Experimental Liver FibrosisModel
Jerry S. Cheung, PhD, Shu Juan Fan, MSc, Darwin S. Gao, BEng, April M. Chow, PhD,Jian Yang, MD, PhD, Kwan Man, PhD, Ed X. Wu, PhD
Ac
FrS.El(KKoDeMDiXiSeKoE.
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Rationale and Objectives: The aim of this study was to characterize early hepatic lipid changes in an experimental model of liver fibrosisusing proton (1H) magnetic resonance spectroscopy (MRS) at high magnetic field in vivo.
Materials andMethods: Liver fibrosis was induced in 12 Sprague-Dawley rats by twice-weekly carbon tetrachloride (CCl4) administration
up to 4 weeks. Eight normal rats were used as controls. Single-voxel 1H MRS experiments were performed at 7 Tesla to measure signalintegrals of various lipid peaks including –CH3, (–CH2–)n, –CH2–C=C–CH2–, =C–CH2–C= and –CH=CH– at 0.9, 1.3, 2.0, 2.8, and 5.3 ppm,
respectively, and peak from choline-containing compounds (CCC) at 3.2 ppm. Total lipid, total saturated fatty acid, total unsaturated fatty
acid, total unsaturated bond, polyunsaturated bond, and CCC indices were quantified.
Results: Significant increases (P < .01) in total lipid and total saturated fatty acid indices were found in animals with CCl4-induced fibrosis
as compared with normal animals. In addition, total unsaturated bond and polyunsaturated bond indices of animals at 4 weeks after CCl4insult were significantly higher than (P < .01 and P < .05, respectively) those of normal animals and animals at 2 weeks following insult;
whereas there was only significant increase (P < .01) in total unsaturated fatty acid index in animals with 4-week CCl4 insult as comparedwith normal animals.
Conclusion: The hepatic lipid changes in CCl4-induced experimental fibrosis model were documented in vivo and longitudinally using1HMRS at 7 Tesla. The experimental findings suggested that total saturated fatty acid increase contributedmainly to the total lipid increasein animals with CCl4 insult. This study also demonstrated the potential value of high fieldMRS to resolve lipid composition and alterations in
liver fibrosis.
Key Words: Proton magnetic resonance spectroscopy (1H MRS); liver fibrosis; lipid; saturated fatty acid; unsaturated fatty acid; carbon
tetrachloride (CCl4).
ªAUR, 2011
iver fibrosis associated with chronic liver injury can progression or regression in response to treatment (3). Because
L progress to cirrhosis and ultimately hepatocellular
carcinoma (HCC) (1,2). Percutaneous liver biopsy
has been considered the standard technique for diagnosis
and staging of liver fibrosis. However, liver biopsy is highly
invasive and associated with risk of complications, limiting
its applicability in longitudinal monitoring of fibrosis
ad Radiol 2011; 18:377–383
om the Laboratory of Biomedical Imaging and Signal Processing (J.S.C.,J.F., D.S.G., A.M.C., J.Y., E.X.W.), Departments of Electrical andectronic Engineering (J.S.C., S.J.F., D.S.G., A.M.C., J.Y., E.X.W.), Surgery.M.), and Anatomy (E.X.W.), The University of Hong Kong, Pokfulam, Hongng SAR, China; Athinoula A. Martinos Center for Biomedical Imaging,partment of Radiology, Massachusetts General Hospital and Harvardedical School, Charlestown, MA 02129, USA (J.S.C); Department ofagnostic Radiology of the First Affiliated Hospital, School of Medicine of’an Jiaotong University, Xi’an, Shannxi Province, China (J.Y.). Receivedptember 12, 2010; accepted October 29, 2010. Supported by the Hongng Grant Council (GRF HKU7808/09M). Address correspondence to:X.W. e-mail: [email protected]
AUR, 2011i:10.1016/j.acra.2010.10.012
disease progression to liver cirrhosis can be prevented by early
interventions and treatments (4–6), there has been a great
interest in the development of noninvasive techniques for
early diagnosis and characterization of liver fibrosis. Proton
magnetic resonance spectroscopy (1H MRS) allows the
study of cellular biochemistry and metabolism, and provides
a noninvasive means to determine disease abnormalities and
progression in vivo and longitudinally. Liver lipid content,
which has been suggested to play an important pathogenic
role in the development of liver fibrosis and cirrhosis in
patients with chronic hepatitis C (7,8) and nonalcoholic
steatohepatitis (9–11), can be measured by 1H MRS
noninvasively. Most importantly, specific lipid changes
related to saturated and unsaturated fatty acids can be
monitored in vivo by high resolution 1H MRS.
Carbon tetrachloride (CCl4) intoxication is a well-
characterized, reproducible and the most commonly used
experimental animal model of liver fibrosis. It has been widely
377
Figure 1. Schedule of carbon tetrachloride (CCl4) twice-weekly
administration for induction of liver fibrosis in adult Sprague-
Dawley rats, 1H proton magnetic resonance spectroscopy (MRS)experiments, and liver histology.
CHEUNG ET AL Academic Radiology, Vol 18, No 3, March 2011
studied with respect to the histological, biochemical, cellular,
andmolecular changes associated with development of fibrosis
(12,13). By interfering hepatic energy metabolism and
protein synthesis, CCl4-induced hepatotoxicity can lead to
triglyceride accumulation, mitochondrial injury, and
necrosis (14). With the increased availability of high-field
($3.0 Tesla) magnetic resonance (MR) systems for clinical
and preclinical studies, both signal-to-noise ratio (SNR) and
spectral resolution of metabolites in the MR spectra can be
improved significantly (15), allowing more accurate metabo-
lite identification and quantification and thus disease charac-
terization. Although MRS can provide insights into liver
metabolism noninvasively, detailed in vivo 1H MRS study of
liver fibrosis with high spectral resolution has been limited
(16–18). The aim of this study was to characterize early
hepatic lipid changes in the experimental CCl4-induced
liver fibrosis model by means of single-voxel 1H MRS at
high magnetic field in vivo.
MATERIALS AND METHODS
Animal Preparation
All animal experiments were approved by the institutional
animal ethics committee. Liver fibrosis was induced in male
adult Sprague-Dawley rats (220 to 260 g; n = 12) by subcuta-
neous injection of 1:1 volume mixture of CCl4 in olive oil at
a dose of 0.2 mL/100 g of body weight twice a week for 4
weeks (13,19). Intermittent administration of CCl4 has been
widely used to experimentally induce liver fibrosis in
rodents by evoking a marked infiltration of inflammatory
cells, thus mimicking the changes in chronic viral
hepatitis‑associated fibrosis in many ways (20,21). The
twice-weekly dosing can induce early stage of liver fibrosis
and established fibrosis after 2 and 4 weeks of CCl4 adminis-
tration, respectively, in rodents (13,22). This well-controlled
CCl4-induced liver fibrosis model allows the study of a homo-
geneous population of liver fibrosis. 1H MRS was performed
in the CCl4-insulted animals at 2 and 4 weeks after the start of
CCl4 administration. The animals were examined 48 hours
after last CCl4 administration to avoid acute inflammatory
effects (18). The overall schedule of the experiment for
378
animals induced with liver fibrosis is shown in Figure 1.
Normal male adult Sprague-Dawley rats (220 to 260 g;
n = 8) were used as controls.
In Vivo Liver 1H MRS Experiments
All 1H MRS experiments were performed on a 7 Tesla MR
imaging scanner with a maximum gradient of 360 mT/m
(70/16 PharmaScan, Bruker Biospin GmbH, Germany). A
60-mm inner diameter quadrature resonator was used for
both radiofrequency (RF) transmission and receiving. During
liver imaging, each animal was anesthetized with isoflurane/
air using 1.0 to 1.5% for maintenance via a nose cone with
respiratory monitoring (23,24). Body temperature was
maintained at about 36.5�C by circulating warm water in
a heating pad. Scout images were first acquired in three
orthogonal planes with a fast low angle shot sequence for
localization of a voxel or volume of interest for MRS. A 5
� 5 � 5 mm3 voxel was chosen within a homogeneous
liver parenchyma to avoid large blood vessels. First- and
second-order localized automatic shimming was first per-
formed within the voxel until a full width at half maximum
<50 Hz was achieved in the water peak. The water signal
was suppressed by variable power RF pulses with optimized
relaxation delays with bandwidth of 200 Hz. Outer volume
suppression combined with respiratory-triggered single-voxel
point-resolved spectroscopic sequence was used for acquiring
liver MR spectrum, with repetition time (TR) = two respira-
tory cycles (∼2.0 to 2.5 seconds), echo time (TE) = 15 ms,
receiver bandwidth = 4 kHz, 2048 data points, 256 averages,
and total scan time of ∼10 minutes. Note that respiratory trig-
gering was used to minimize voxel misregistration in the pres-
ence of respiratory motion, while short TE was chosen to
reduce signal loss due to T2 relaxation to improve SNR (25).
Data and Statistical Analysis
The MRS data were processed using the MR spectroscopic
analysis package provided by the MR imaging vendor
(26,27). MR spectra were zero-filled to 8192 data points,
apodized with a 2-Hz exponential filter, Fourier transformed,
0th- and first-order phase corrected, and baseline corrected.
Signal integrals of lipid methyl protons (–CH3; 0.9 ppm),
methylene protons ((–CH2–)n; 1.3 ppm), allylic protons
(–CH2–C=C–CH2–; 2.0 ppm), diallylic protons (=C–CH2–
C=; 2.8 ppm), methene protons (–CH=CH–; 5.3 ppm),
and protons from choline-containing compounds (CCC;
3.2 ppm) (11,28,29) were measured by integrating areas
under peaks. Spectral noise was calculated from the standard
deviation of the last portion of spectrum from 7.3 to 11.3
ppm, in which no metabolite was observable in liver
(11,28,29). Total lipid and CCC indices were quantified by
dividing peak area of (–CH2–)n and CCC by spectral noise,
respectively, given the fact that SNR was similar among
spectra because of identical voxel size and nearly identical
hardware settings in different measurements. In addition, total
TABLE 1. Peak Area Ratios of Various Metabolite IndicesMeasured by Proton Magnetic Resonance Spectroscopy(1H MRS)
Index Peak Area Ratio Frequency
Total lipid (–CH2–)n/noise 1.3 ppm
Total saturated fatty acid 3(–CH2–)/2(–CH3) 1.3/0.9 ppm
Total unsaturated fatty
acid
3(–CH2–C=C–CH2–)/4
(–CH3)
2.0/0.9 ppm
Total unsaturated bond 3(–CH=CH–)/2(–CH3) 5.3/0.9 ppm
Polyunsaturated bond 3(=C–CH2–C=)/2(–CH3) 2.8/0.9 ppm
Choline-containing
compound (CCC)
CCC/noise 3.2 ppm
Academic Radiology, Vol 18, No 3, March 2011 1H MRS LIPID PROFILING IN LIVER FIBROSIS
saturated fatty acid, total unsaturated fatty acid, total unsaturated
bond, and polyunsaturated bond indices were estimated by
dividing peak area of (–CH2–)n, –CH2–C=
C–CH2–, –CH=CH– and =C–CH2–C= by peak area of
–CH3, respectively, and scaled to the relative number of
protons contributing to the resonance (11), as shown in Table
1. Note that the signal from (–CH2–)n and –CH2–C=
C–CH2– increases with increasing number of saturated and
unsaturated fatty acids, respectively, whereas the signal from
–CH=CH– and =C–CH2–C= increases with the percentage
of total unsaturated and polyunsaturated double bonds in the
unsaturated fatty acids, respectively (11,30). Furthermore, the
signal contribution of glutamine and glutamate at 2.2 ppm
was assumed to be negligible (11). Results were expressed as
mean � standard deviation. One-way analysis of variance
with Tukey’s multiple comparison test was employed to
compare differences in ratios of peak areas between fibrosis-
induced and control animals, with P values less than .05 consid-
ered statistically significant.
Histology
After theMR examination following 2 weeks of CCl4 admin-
istration, 4 of 12 animals were sacrificed for histological eval-
uation. Furthermore, four of the remaining eight animals
were sacrificed after MR examination after 4 weeks of CCl4insult as shown in Figure 1. One additional normal animal
was sacrificed as a control. Liver specimens were fixed in
formalin, embedded in paraffin, sectioned and examined by
light microscopy after standard hematoxylin-eosin staining
and Masson’s trichrome staining (31,32).
RESULTS
Figure 2 shows the typical liver 1HMRS spectra from a normal
control animal and an animal assessed at 2 and 4 weeks after
start of CCl4 twice-weekly administration, with a typical
voxel placement shown in the anatomical image. It was
consistently observed that all animals with CCl4-induced
liver fibrosis exhibited substantial increase in various lipid
peaks including –CH3, (–CH2–)n, –CH2–C=C–CH2–,
–COO–CH2–, =C–CH2–C= and –CH=CH–, with similar
signals from CCC, as compared with the normal animals.
Water (H2O) signal at 4.7 ppm was effectively suppressed in
the spectra with varying degree. Figure 3 shows the total lipid,
CCC, total saturated fatty acid, total unsaturated fatty acid,
total unsaturated bond and polyunsaturated bond indices in
normal animals (n = 8) and animals with 2-week (n = 12)
and 4-week (n = 8) CCl4 twice-weekly administration. Total
lipid indices of animals at 2 weeks (3.97 � 1.57 � 104) and 4
weeks (5.66� 1.31� 104) after fibrosis induction were found
to be significantly higher than (P < .01) those of normal
control animals (0.72 � 0.17 � 104), along with significant
difference (P < .05) between 2 and 4 weeks. Meanwhile,
a similar trend was observed in total saturated fatty acid index.
Significant increase (P < .01) in total saturated fatty acid index
was found in animals with CCl4-induced liver fibrosis (8.74�1.68 and 10.42 � 1.35 for 2-week and 4-week CCl4 admin-
istration, respectively), as compared with normal animals
(5.91 � 1.23), whereas the difference between 2 and 4 weeks
was also statistically significant (P < .05). On the other hand,
there was only significant increase (P < .01) in total unsatu-
rated fatty acid index in animals with 4-week CCl4 insult as
compared with normal animals. Furthermore, total unsatu-
rated bond index of animals at 4 weeks after CCl4 insult
(1.75 � 0.30) were significantly higher than (P < .01 and P
< .05, respectively) those of normal animals (1.10 � 0.32)
and animals at 2 weeks after insult (1.32 � 0.30). Similarly,
polyunsaturated bond index of animals after 4-week CCl4administration (0.95 � 0.18) were significantly higher than
(P < .01 and P < .05, respectively) those of normal animals
(0.57 � 0.18) and animals with 2-week CCl4 insult (0.73 �0.14). No significant differences were observed in CCC
between normal and diseased animals.
Figure 4 shows the typical hematoxylin-eosin and Masson’s
trichrome staining of normal liver and livers at 2 weeks and 4
weeks after CCl4 insult. Collagen deposition was stained as
blue by Masson’s trichrome staining in fibrotic livers.
Compared with normal liver (Fig 4a), collagen deposition
and intracellular fat vacuoles were consistently observed in
livers with CCl4 insult (Fig 4b, 4c). Similar histological find-
ings were observed in all liver samples collected, and theywere
largely consistent with those from the earlier studies of CCl4-
induced liver fibrosis in rodent models (18). The histological
observations of collagen deposition in the liver samples
collected confirmed the liver fibrogenesis in the animals
studied.
DISCUSSION
As early as 2 weeks after the start of CCl4 administration, total
lipid and total saturated fatty acid indices were found to
increase substantially (P < .01) in animals with liver fibrosis
as compared with control animals (Fig 3a, 3c). Meanwhile,
no increases in total unsaturated fatty acid index were
observed (Fig 3d), suggesting that the total saturated fatty
acid increase contributed mainly to the total lipid increase
in animals with CCl4 insult. The total lipid increase in animals
379
Figure 2. Typical liver 1H protonmagnetic reso-nance spectroscopy (MRS) spectra from
a normal control animal and an animal scanned
at 2 and 4 weeks after start of carbon tetrachlo-ride (CCl4) twice-weekly administration, with
a typical voxel placement (white square) shown
in the anatomical image. Animals with CCl4-
induced liver fibrosis consistently showed mark-edly increases in various lipid peaks including
methyl protons (–CH3; 0.9 ppm), methylene
protons ((–CH2–)n; 1.3 ppm), allylic protons
(–CH2–C=C–CH2–; 2.0 ppm), a-methyleneprotons to carboxyl (–COO–CH2–; 2.2 ppm), dia-
llylic protons (=C–CH2–C=; 2.8 ppm) and meth-
ene protons (–CH=CH–; 5.3 ppm), except the
peak from choline-containing compounds(CCC; 3.2ppm). Thewater (H2O; 4.7ppm) signals
were effectively suppressed in the spectra.
Figure 3. (a) Total lipid, (b) choline-containingcompounds (CCC), (c) total saturated fatty
acid, (d) total unsaturated fatty acid, (e) totalunsaturated bond, and (f) polyunsaturated
bond indices in normal animals and animals
with 2-week and 4-week CCl4 twice-weekly
administration. One-way analysis of variance(ANOVA) with Tukey’s multiple comparison test
was performed with **P < 0.01, *P < 0.05 and
n.s. for insignificance.
CHEUNG ET AL Academic Radiology, Vol 18, No 3, March 2011
with CCl4-induced liver fibrosis was likely due to fatty
infiltration/fatty changes in hepatocytes (Fig 4). During toxic
CCl4 insult, hepatocytes are incapable of synthesizing
380
lipoproteins that are needed for removing triglycerides in
the cytoplasm as a result of the destruction of microsomal
proteins by lipid peroxidation (18,33,34), leading to
Figure 4. Typical hematoxylin-eosin (H&E) staining (400�; left column) and Masson’s trichrome staining (200� and 40�; middle and rightcolumn, respectively) of normal liver (a), and livers subjected to2-week (b)and4-week (c) carbon tetrachloride (CCl4) twice-weeklyadministration.
Collagen deposition (green arrows), fat vacuoles (blue arrows), and cell necrosis/apoptosis (black arrows) were observed in the insulted livers.
Academic Radiology, Vol 18, No 3, March 2011 1H MRS LIPID PROFILING IN LIVER FIBROSIS
increased triglyceride accumulation (35). It is noteworthy that1H MRS can provide valuable information on lipid composi-
tion, which cannot be revealed by histological analysis. The
total saturated fatty acid increase in liver may reflect the
lipid-induced cell toxicity, which has been suggested to be
related with activated apoptosis induced by saturated fatty
acids (9,10,36). In nonalcoholic fatty liver disease,
endoplasmic reticulum stress associated with increased
saturated fatty acids in the liver have been shown to
promote liver injury and partly contribute to the disease
progression from simple steatosis to steatohepatitis (9,10).
Nonetheless, the potential toxic effect of increased saturated
fatty acids in fibrotic livers has yet to be determined.
Although there was no significant change in total unsatu-
rated fatty acid index at week 4 as compared with week 2 after
CCl4 administration, significant increase (P < .05) in total
unsaturated bond index was observed (Fig 3e). These findings
suggested that without a substantial increase in amount of
unsaturated fatty acids, more unsaturated double bonds were
formed within the unsaturated fatty acids at 4 weeks after
the start of fibrosis induction. As such, significant increase in
polyunsaturated bond index (P < .05) was observed (Fig 3f)
as expected. Therefore, the amount of polyunsaturated fatty
acids was likely to increase at the expense of monounsaturated
fatty acids in the animals with 4-week CCl4 insult. Such
increase in degree of polyunsaturation has been observed
and ascribed to increased necrosis/apoptosis in various exper-
imental models (11,37,38), which was also observed in the
current study (Fig 4). Because CCC has been considered to
represent the important constituents in phospholipid metabo-
lism of cell membranes (39), similar CCC levels observed in
the normal and CCl4-insulted animals were largely expected
in the early stage of liver fibrosis because of the similar rate
of cell turnover of hepatocytes prior to the development of
cirrhosis (40).
In vivo phosphorus-31 (31P) MRS has been found prom-
ising in evaluating the cellular turnover and liver energy status
(41–43), but it cannot measure the hepatic lipid content and is
not readily available onmost clinicalMR imaging scanners. By
contrast, in vivo 1H MRS is feasible on most standard MR
381
CHEUNG ET AL Academic Radiology, Vol 18, No 3, March 2011
imaging scanners, where high-resolution anatomical images
and functional information such as perfusion can be acquired
in the same imaging session. 1H MRS has been used to
investigate focal liver lesions and found to be useful in
characterizing HCC in humans (44–46) and animal models
(37,47–49) by providing valuable metabolic information.
However, only limited 1H MRS studies have been
performed to investigate liver fibrosis in both humans and
animals (16–18). Note that our study used the highest field
strength to study liver fibrosis compared with previous
studies (16–18). The advantages of performing 1H MRS at
high magnetic field include better SNR and increased
spectral resolution (15), thus providing high-quality spectra
in acceptable scan times. In this regard, 1H MRS at 7 Tesla
in this study can provide precise biochemical information in
liver noninvasively that can be used for characterizing and
monitoring of liver diseases including liver fibrosis. It is worth-
while to note that it is relatively difficult to accurately resolve
and quantify several lipid signals other than (–CH2–)n peak
because of limited spectral resolution and poor SNR at low
field strengths in previous studies (16,18). In contrast, our
current study provided more detailed information about
lipid composition by resolving various lipid peaks (eg,
–CH2–C=C–CH2–, =C–CH2–C= and –CH=CH–) in
fibrotic livers at 7 Tesla. Note that the separation between
methene (5.3 ppm) and water (4.7 ppm) peaks is 180 Hz at
7 Tesla, thus the methene proton peak was minimally
affected by the water suppression RF pulses with bandwidth
of 200 Hz. In addition, the effects of motion on liver MRS
were largely reduced using respiratory gating in the current
study, providing water-suppressed MR spectra with high
quality (Fig 2). It is worth noting that although fat-selective
MR imaging using Dixon methods can provide quantitative
information about hepatic lipid content, these techniques
are only sensitive to one specific lipid proton species (ie, meth-
ylene protons [3.4 ppm apart from water]). In contrast, 1H
MRS can resolve and quantify various lipid proton species
so as to provide insights into lipid composition.
Ratio measurements using spectral noise as internal refer-
ence were employed for quantifying total lipid and CCC
levels in the current study. Note that spectral noise could be
affected by a number of factors such as local sensitivity of
coil and acquisition protocol. Such factors were expected to
be similar among measurements in this study because of the
use of volume RF coil, identical hardware settings and acqui-
sition protocol, yielding similar noise levels in the spectra of
different measurements (Fig 2). The use of lipid peak as
internal reference (16) was avoided, because the amount of
lipid increased markedly in the CCl4-induced liver fibrosis
model (Fig 2). Unsuppressed water signal was not chosen as
internal reference either because the water content may be
affected by pathological conditions (46). Previous human
studies reported inconsistent observations on the lipid levels
in chronic hepatitis (16,17), probably due to varying types
of virus-induced hepatitis investigated. Our results showed
an increase in total lipid levels, consistent with other MR
382
studies in CCl4-induced fibrosis model (18,35). However,
changes in saturated fatty acids, unsaturated fatty acids and
unsaturated double bonds were not investigated in those
studies. Our MRS data at high field allowed better
discrimination and quantification of the –CH2–C=C–CH2–,
–CH=CH– and =C–CH2–C= lipid peaks, thus total
unsaturated fatty acid, total unsaturated bond and
polyunsaturated bond indices can be measured. Moreover,
the elevated total saturated fatty acid measured by 1H MRS
in the current study may suggest its role in the development
of liver fibrosis (9,10). One potential limitation of the
present study is lack of sham controls receiving olive oil
alone. As subcutaneous injection of CCl4 with olive oil is
a well-established protocol to induce liver fibrosis in rodents
(13), effects of olive oil were not examined in the current
study. Nonetheless, previous studies showed that hepatic lipid
changes are associated with metabolic alterations due to intox-
ication of CCl4 (50), whereas no evidences of steatosis
induced by olive oil have been found.
In conclusion, the lipid changes related to saturated and
unsaturated fatty acids in CCl4-induced experimental fibrosis
model were documented in vivo and longitudinally using 1H
MRS at 7 Tesla. Our experimental results demonstrated that1H MRS at high-field was useful in detecting and character-
izing various hepatic lipid alterations as early as 2 weeks from
the start of induction of liver fibrosis in the animal model. 1H
MRSmay be valuable in detecting fatty changes at early phase
in human liver fibrosis prior to the development of cirrhosis,
and potentially useful in determining treatment strategies and
evaluating therapeutic outcomes.
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