A Preliminary Application of Magnetic Resonance ...

Journal of Analytical Science & Technology (2011) 2(1), 23-29
Article www.jastmag.org
DOI 10.5355/JAST.2011.23

A Preliminary Application of Magnetic Resonance
Spectroscopy for Quantitatively Assessing Hepatic Fat and

the Efficacy of Anti-obesity Therapy

Hyunseung Lee1, Kyeong-Hoon Jeong2, Myoung Gyu Park3,
Jongeun Kang1,4, Kwan Soo Hong1,4*

1Division of MR Research, Korea Basic Science Institute, Cheongwon 363-883, South Korea
2Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science,
Incheon 406-840, South Korea
3R&D Center, Mazence Inc, Suwon 443-813, South Korea
4Graduate School of Analytical Science and Technology, Chungnam National University,
Daejeon 305-764, South Korea

*Corresponding author:
Kwan Soo Hong ,Tel: +82-43-240-5100, Fax: +82-43-240-5119, Email: [email protected]

Running Title:
Quantitative MRS Assessment of Hepatic Fat

Abstract

Alcoholic and non-alcoholic fatty liver diseases cause insulin resistance and may develop into metabolic diseases
such as steatohepatitis or type II diabetes. Standard histopathological examinations are routinely used to measure
hepatic fat in order to assess and treat liver diseases, but this method is invasive, complicated, and time-consuming.
Here, we present a noninvasive technique, localized magnetic resonance spectroscopy (MRS), for quantitatively
measuring hepatic fat in vivo and in situ. This method allowed us to create a relatively high-resolution time series from
the same mouse. Further, it enabled us to examine the efficacy of cryptotanshinone (Ct) treatment in male mice with
non-alcoholic fatty liver disease; MRS clearly showed that mice treated with Ct experienced a dramatic reduction in
hepatic fat content compared with control mice. Thus, the localized MRS technique shows promise as a tool for in vivo
assessments of drug efficacy against liver fat diseases and for early-stage disease prevention.

Key words: Fatty liver, Obese mice, MR spectroscopy, Hepatic lipid ratio, Drug efficacy

Received for review : 17/08/2010
Published on Web: 16/11/2010
© Korea Basic Science Institute. All Rights Reserved.

24 Journal of Analytical Science & Technology (2011) 2 (1), 23-29

Introduction non-alcoholic steatohepatitis. We then correlated MRS
results with those yielded by a conventional
In addition to being a hallmark of metabolic histopathological method, oil-red O-staining, in order
syndrome, hepatic accumulation of lipids (e.g., fatty to calibrate the two techniques. Finally, we used the 1H
liver, hepatic steatosis) is also tightly correlated with MRS method to assess the efficacy of the herbal
each of the individual symptoms associated with the compound cryptotanshinone (Ct) as a possible therapy
syndrome, regardless of obesity [1-3]. Non-alcoholic against hepatic fatty liver disease.
fatty liver disease causes insulin resistance and, like
metabolic syndrome, is a predictor of both type II Materials and Methods
diabetes and advanced forms of liver diseases, including
non-alcoholic steatohepatitis and cirrhosis, as well as Ob/ob mice
cardiovascular disease [4-7]. This has important All animals were maintained in a 12:12 h light:dark
therapeutic implications, since a reduction in hepatic
steatosis may improve symptoms of other liver diseases cycle and fed ad libitum, with drinking water available at
and decrease the progression of metabolic syndrome [8, all times. Body weight change and food intake were
9]. monitored daily. Five-week-old male ob/ob mice
(B6.V-Lepob/J, n = 40; Charles River Laboratories, Japan)
The progress of liver diseases and the efficacy of liver were fed with normal chow (5 g/day; protein 18%, fat
disease therapies can decisively be assessed by 11.9%, fiber 5.2%, nitrogen-free extract 55.9%, others
quantifying hepatic fat levels. Currently, liver biopsy is 8%; OrientBio, Korea) and monitored by measuring
the only method that can be used to determine liver fat hepatic lipid ratio (HLR) with localized MR
content and pathological diagnosis for micro- or spectroscopy every week from 6 to 12 week-old. Each
macro-vesicular steatosis, inflammation, and fibrosis week, 4 mice were sacrificed after their HLRs were
[10]. The standard histopathological examination, measured, and oil red-O (ORO, Sigma-Aldrich, USA)
however, is time-consuming and involves a series of staining was performed on samples from their livers.
complex procedures for sample preparation, including Once the eight remaining mice were 12-weeks-old, they
dehydration, fixation, staining, and sectioning. Most received a 14-day treatment of either a low dose
importantly, it is almost impossible to take multiple (Ctlow-dose, n = 4) or a high dose (Cthigh-dose, n = 4) of an
sequential histopathological samples from the same anti-obesity drug (Ct) that was mixed with their normal
subject in real time. chow.

Magnetic resonance imaging (MRI) and proton On average, daily Ct intake was 200 ± 20 and 400 ±
magnetic resonance spectroscopy (1H MRS) allow 45 mg/kg for the low- and high-dose treatments,
relatively high-resolution, non-invasive time-series respectively. Age-matched normal lean male mice were
investigations in the same mouse. 1H MRS has been also included for comparison. HLRs of the ob/ob mice
used to perform in vivo quantitative analyses of were determined by MRS at days 0, 6, and 13 during the
water-soluble bio-molecules for assessing brain[11-13] Ct administration and the mice were sacrificed at day 18
and muscle [14-16] metabolites, and for diagnosing after a final MRS analysis. All protocols were approved
cancers [17, 18] and liver diseases [19-21]. Recently, 1H by the Institutional Review Committee on animal care
MRS has been used to diagnose liver diseases, (KBSI-AEC1010).
particularly steatohepatitis [22-24]. However, there have
not yet been any studies correlating results of MRS Measurement of hepatic lipid content using 1H MRS
techniques with those of conventional histopathological Animals were anesthetized with inhalational
and bio-molecular methods. Because 1H MRS is
non-invasive, it could offer an alternative to isoflurane (5% induction, 1.5% maintenance) in a
histopathological and bio-molecular tests, if it can be O2­N2O (3:7) mixture, and body temperature was
shown to provide reliable in vivo quantitative maintained at 37°C by running warm water ducts into the
assessments of hepatic lipid contents. animal bed. A quadrature birdcage RF resonator (Bruker,
Germany) with an inner diameter of 72 mm
In this study, we used the 1H MRS method to
non-invasively measure hepatic fat in ob/ob mice with

Hyunseung Lee et al. 25

was connected to a 4.7 T animal MR scanner (Biospec Statistical analysis
47/40, Bruker) for signal transmission and reception. Paired and unpaired Student’s t-tests were used to
T1-weighted MRI images were obtained to position
voxels inside the liver using the following parameters: assess differences in the experimental datasets.
repetition time (TR) = 665 ms; echo time (TE) = 14 ms, Correlations between the two data sets were assessed
slice thickness = 1 mm; field of view (FOV) = 3.5 × 3.5 using one-way ANOVAs, followed by post-hoc Turkey’s
cm2; matrix size = 256 × 256; average number = 2. tests for multiple comparisons. The analyses were
Respiratory-gated localized 1H-MR spectra were performed with Origin software (version 6.0; MicroCal,
acquired from the left and right lateral lobes of the liver LLC, Northampton, MA). Significance was defined as P
using a point-resolved spectroscopy sequence (PRESS) < 0.05. All data are presented as mean ± SD.
where TR = 2500 ms, TE = 144 ms, voxel size = 3 × 3 × 3
mm3, and average number = 128. The water peak (4.7 Results and Discussion
ppm) was used as an internal reference against which to
register fat peaks; there was only a single water peak at Figure 1A is a representative axial slice MR image
4.7 ppm and all others were from lipids. Peak from the abdomen of an ob/ob mouse; it shows
assignments were accomplished using methods subcutaneous fat (white), liver (grey), and the spinal cord
previously reported elsewhere [25, 26] and resonance (one of relatively black). MR spectroscopy was
intensities were measured by Gaussian curve-fitting. performed on regions located two or three voxels away
The hepatic lipid ratio (HLR) was defined and from large vessels inside the liver (Fig. 1A; FOV = 3.5 ×
measured using the following formula: HLR = 100 × 3.5 cm2). Similar spectra were obtained from the left and
[integral value of all lipidpeaks]/[integral value of all right lobes, indicating that liver fat is diffusive and
peaks]. homogeneous (Figs. 1B and 1C).

Histopathological and biochemical studies Each spectrum consisted of approximately 10 peaks,
After the MRI and MRS experiments, mice were which were separated and assigned by line-fitting (Fig. 2).

anesthetized by an intramuscular injection of 100 mg/kg Figure 1. Representative MR data from the liver of an ob/ob
ketamine and 10 mg/kg xylazine. About 1 ml of blood mouse. (A) MR image obtained with a T1-weighted spin-echo
was collected by cardiac puncture, then only centrifuged pulse sequence. White indicates fat deposits, gray indicates
serum was stored at -80oC. After a transcardial perfusion liver tissue, and black indicates the spinal cord region; the red
of PBS, the livers were removed and embedded in boxes indicate the regions on which MR spectroscopy was
optimal cutting temperature (OCT) compound (Sakura performed. (B, C) MR spectra and PRESS sequences from two
Finetek, Torrance, CA) and cut into 7-μm-thick sections. different regions of mouse liver. More than 8 peaks are shown;
OCT-embedded cryosections were treated with 60% all peaks are assigned from liver fat except for the line at 4.7
isopropanol, stained with 0.18% oil red-O in 60% ppm. Spectra from the left and right lobes of the liver are
isopropanol for about 10 min, and then washed with tap nearly identical.
and distilled water. Sections were counter-stained with
hematoxylin and mounted in aqueous solution. After this
staining protocol, the slices were observed under a
microscope (IX81, Olympus, Japan). Fat vacuoles in the
liver were identified by red color droplets in the
ORO-stained images. Twelve ORO-stained images from
each mouse liver were obtained and averaged, and the
area of red color droplets was measured by ImageJ (1.43u,
NIH, USA) software. Serum triglyceride (TG) and
cholesterol (Chol) were measured with commercial kits
(Wako, Japan) by a clinical analyzer (7020; Hitachi,
Japan).

26 Journal of Analytical Science & Technology (2011) 2 (1), 23-29

ratio (HLR) derived from the measured spectrum in
Figure 2 was HLR = 59%.

As shown in Figure 3, MR images and spectra from the
livers of mice with different body weights are clearly
distinguishable. Obese mice (Fig. 3A; body weight = 41
g) had brighter livers than leaner mice (Fig. 3B; body
weight = 30 g); this corresponded with the intensities of
the accompanying MR spectra lipid peaks in Figures 3C
(HLR = 55%) and 3D (HLR = 17%), respectively. HLR
was highly correlated with age over the duration of the
MRS measurements made for this study (e.g., from 6 to
14 weeks of age; r2 = 0.982, P < 0.0001; Figure 3E).

Figure 2. Localized 1H MR spectrum obtained from the liver Figure 4. Correlation between hepatic lipid ratio (HLR) and
of an ob/ob mouse. (A) Raw spectrum. (B) Deconvolution with oil red O-stained area. Representative examples of MR spectra
10 fitted peaks (dotted blue lines) and summed line (red line). (A-C) and oil red O (ORO)-stained images (D-F) from three
(C) Difference after (A) is subtracted from the fitted curve in different ob/ob mice. Red color droplets in the ORO-stained
(B). Ten NMR lines were assigned and separated with images indicate fat vacuoles in the mouse livers. (G)
Gaussian functions, then integrated so that fat content could Correlation between the mean ORO-stained area in each
be measured for each peak. Numerals represent the integrated mouse and the hepatic lipid ratio (HLR) value calculated from
values at the resonance frequencies (shown in parentheses). localized MR spectra (R2 = 0.98, P < 0.001).
The integral of all lipid peaks was 648, compared to 450 for the
water peak; this yielded an HLR of 59%.

Figure 3. Comparison of MR images and localized MR spectra In order to verify that the MRS method could be used
from mice with different body weights. (A, B) MR images from in mice of a variety of ages, we conducted HLRs and
obese (41 g) and lean (30 g) mice, respectively. (C, D) Localized corresponding histochemical analyses in 6 to
MR spectra corresponding to the MR images in (A) and (B), 14-week-old ob/ob mice. The intensity of lipid peaks in
respectively. The MR image of the heavier mouse (A) is the localized MR spectra (Figs. 4A-4C) corresponded to
brighter than that of the leaner mouse (B), which corresponds the number of fat vacuoles visualized by the ORO staining
with the signal intensities of the lipid peaks in the MR spectra (Figs. 4D-4F), regardless of mouse age. In fact, the
(C, HLR = 55%; D, HLR = 17%). (E) Correlation between number of red-stained fat vacuoles in the ORO-stained
HLR and age in 6- 14-week-old mice (r2 = 0.982, P < 0.0001). images was highly correlated with the HLR value
calculated from the localized MR spectra (R2 = 0.98, P <
All peaks (except for the water peak at 4.7 ppm) were 0.001; Fig. 4G).
caused by liver lipid content, which allowed lipids to be
quantitatively differentiated from water. The hepatic lipid When the HLR measurement method was used to
comparatively assay, with serum Chol and TG findings,
for the effect of cryptotanshinone (Ct) as an
anti-obesity treatment, significant differences were
found between the Ct-treated and vehicle-treated control
mice (P < 0.01 for HLR and serum Chol; P < 0.05 for
serum TG), but not between high and low dose Ct mice
(n= 4 each; Fig. 5). There was good accordance in the

changes of HLR, serum Chol, and serum TG.

Hyunseung Lee et al. 27

Figure 5. Effects of Ct on hepatic lipid content and serum There were significant changes in HLR, serum Chol
biomarkers of ob/ob mice. The MRS method detected and TG between mice groups of vehicle- and
significant differences in hepatic lipid ratio (HLR) between Ct-treatment for two weeks, but we measured no
control mice and mice receiving the cryptotanshinone (Ct) changes between low and high doses of Ct. For the
treatment, which is in good agreement with the changes in measurement of Ct dose dependence in HLR, serum Chol,
serum cholesterol (Chol) and serum triglyceride (TG). There serum TG, and other biomarkers, we need a further study
were no significant differences between mice receiving the high in the Ct dose range of < 200 ± 20 mg/kg.
(Cthigh dose, 400 mg/kg) and low (Ctlow dose, 200 mg/kg) Ct doses. *,
P < 0.01; **, P < 0.05. Additional future work should investigate correlations
between HLR and components of the AMP-activated
kinase (AMPK) and sirtuin 1 (SIRT1) pathways, which
regulate hepatic lipid metabolism in healthy mice, but
are inhibited in individuals with alcoholic fatty liver
disease [8, 31-34]. Specifically, further studies should
measure circulating levels of glutamate pyruvate
transaminase, lactate dehydrogenase, blood urea nitrogen,
hepatic TG. Finally, it would also be informative to
perform a quantitative RT-PCR study examining the
expression of genes related to lipogenesis and fatty acid
oxidation

There are some potential limitations to this study. First, Conclusions
density and size of red droplets (fat vacuoles) in the
ORO-stained images were not homogeneous across the Cumulatively, our data indicate that the hepatic lipid
20-image sample size (2 images × 10 slices). Thus, ratio, as measured by the noninvasive MRS method, is a
ImageJ was used to make the color image promising new tool for obtaining a time series of
black-and-white, and the droplets were selected and quantitative in vivo hepatic fat measurements from the
extracted with two threshold points in gray-scale. This same animal; this avoids the large number of animal
image-processing procedure may have added sacrifices required at every experimental time point in
unintentional error. In the measured HLR range lower conventional histochemical studies, and is also a more
than 52%, there was a correlation between HLR and economical alternative to existing methods. Further, this
ORO-stained area. Another potential drawback of our technique can be used even in the early stages of disease,
methodology is the summing of all MRS peaks (except when it can be used to prevent further deterioration and
for the water peak at 4.7 ppm) when defining hepatic to determine appropriate treatment protocols (e.g.,
fat. In order to make a more detailed comparison evaluating whether, when, and what kind of treatment
between HLR and histopathological/biochemical should be performed), as well as measuring the efficacy
findings, it would have been better to analyze each of treatments once they are under way.
different metabolite separately (e.g., hepatic
triglycerides, TGs, and total lipid extracts converted to Acknowledgements
fatty acid methyl esters, FAMEs) [27-30]. This would
have enabled us to separately evaluate the intrinsic This work was supported in part by a grant from the
information provided by the resonance frequency of Korea Basic Science Institute (T30403), and by the
each separate metabolite, thus providing more precise Ministry of Education, Science and Technology
results. In the future, it will be necessary to conduct a (PUA019).
systematic study comparing all lipid peaks, one by one,

with ORO-stained area, hepatic TG, and FAMEs.

There were significant changes in HLR, serum Chol

28 Journal of Analytical Science & Technology (2011) 2 (1), 23-29

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