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In Vivo Proton Magnetic Resonance Spectroscopy of Hepatic Fatty Acid Change: Identification of Lipid Contents with Correct and Incorrect Terminal Methyl Group in Hepatic Steatosis at Ultra High Field

Kyu-Ho Song¹, Min-Young Lee¹, Chi-Hyeon Yoo¹, Song-I Lim¹, and Bo-Young Choe¹

¹Department of Biomedical Engineering and Research Institute of Biomedical Engineering, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea

Synopsis

Magnetic resonance spectroscopy (MRS) with optimized relaxation time provides an effective means for quantifying lipid content and characterizing hepatic steatosis. The aim of this study was to quantify the difference in hepatic lipid content with metabolic changes and determine effect of diet on high-fat diet (HFD)-fed mice by measuring the main localized MRS sequence with relaxation times.

Purpose

The pathogenesis of storage disease or metabolic disorders depends on their specific effect on metabolism. Complex interactions occur between the metabolic pathways of fatty acids, glucose, and lipoproteins.^1,2Changes in the expression of metabolites, in addition to changes in saturated and unsaturated fatty acid contents following formation of abnormal metabolites, play an important pathogenic role and can be measured by proton MRS.^3,4 The aims of this study were to: (1) validate a noninvasive MRS-based procedure for quantifying differences in hepatic lipid content of the liver with respect to metabolic changes; (2) determine the possible effect of a HFD on the hepatic lipid content in mice with serum biochemistry; and (3) demonstrate that incorrect and correct relaxation times yield contradictory quantification results for liver fat tissue.

Materials and Methods

Hepatic steatosis (ten male C57BL/6N mice) was induced in the mice via HFD pellets composed of 60% fat, 20% protein, and 20% carbohydrates. Magnetic resonance imaging (MRI) and MRS analyses were performed using a horizontal 9.4 T MRI animal scanner (Biospec 94/20 USR, Bruker Biospin GmbH, Ettlingen, Germany) equipped with a 20-cm bore magnet with a 400 mT/m gradient and a four-channel receive-only array animal coil. The MRS scan parameters were as follows: standard stimulated echo acquisition mode (STEAM) sequence (repetition time [TR], 5,000 ms; mixing time [TM], 10 ms; echo time [TE], 20 ms; number of signal average, 128; acquisition data point, 2,048; acquisition bandwidth, 4,401.41 Hz; repetition time, 1; scan time, ~11 min) and multi-TEs STEAM sequence (with TE, 20–70 ms). For animal proton MRS, a voxel size of 3×3×3 mm³ (2.7 mL) was selected. Before the MRS scan, the water resonance signal in the volume of interest of the livers of HFD-fed mice was suppressed by variable pulse power and optimized relaxation delays.⁵ All the MRS data were quantified using the Linear Combination of Model Spectra (Version 6.3.1).⁶ The following correct curve-fitting equation (M(TE)=M₀e^(-TE/T₂)[cos(πJ_ETE)+b]) was used to measure the relaxation times of lipid resonance, where J_E and b are the values of the J-coupling evolution and complement number to M₀ in cases with nonzero J_E values, respectively.⁷

Results

To measure the lipid metabolite peaks, we analyzed the time points shown in Fig 1. The signal-to-noise ratios for methylene protons of the liver were as follows: 0 week, 33.2 ± 10.1; 2 weeks, 58.2 ± 8.2; 4 weeks, 70.9 ± 6.4; 7 weeks, 106.0 ± 9.2; 10 weeks, and 114.2 ± 10.9. As shown in Fig 2 and 3, the differences in relaxation times with incorrection and correction values were as follows: ~0.90 ppm (week 0: p < 0.001; week 10: p < 0.001), ~1.60 ppm (week 0: p < 0.001), ~2.03 ppm (week 0: p < 0.05), ~2.78 ppm (week 0: p < 0.001; week 10: p < 0.05), and ~5.30 ppm (week 0: p < 0.05). Figure 4 shows the total saturated fatty acid levels calculated at 2 (p < 0.05), 4 (p < 0.01), and 10 (p < 0.01). The change in the total unsaturated fatty acid was statistically significant at week 2 (p < 0.05) (week 2-10, p < 0.01) and the change in the total unsaturated bonds was statistically significant from week 4 to 10 (p < 0.05). In addition, the change in the polyunsaturated bonds was statistically significant at week 4 (p < 0.05). As shown in Fig. 5, liver weights in HFD-fed mice (1.69 ± 0.14 g) were significantly higher than those in normal-chow diet-fed mice (1.43 ± 0.10 g) as controls. Serum glucose (p < 0.05), aspartate transaminase (p < 0.001), and alanine transaminase (p < 0.001) concentrations were significantly elevated in HFD mice. Serum albumin (p < 0.05) and total bilirubin (p < 0.05) concentrations were also significantly lower in HFD mice.

Discussion and Conclusion

Our results indicate that methyl and methylene protons are sensitive to HFD-induced lipid accumulation in the fatty liver and reveal that the relaxation times of resonances yield significantly different quantification of lipid contents in animals. In conclusion, we suggest that for the research and clinical evaluation of disease, obtaining a STEAM sequence with T₂ correction using an ultra-high field scanner is an appropriate method for lipid content quantification. The resonances are dependent on the relaxation correction for metabolite quantification. We analyzed the lipid content (upregulated saturated and unsaturated fatty acids; downregulated polyunsaturated fatty acids) in the liver of HFD-fed mice and evaluated the lipid quantification with a sufficiently high signal-to-noise ratio.

Acknowledgements

This study was supported by grants (2012-007883) from the Mid-career Researcher Program through the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea.

References

1. Cheung JS, Fan SJ, Gao DS, et al. In vivo lipid profiling using proton magnetic resonance spectroscopy in an experimental liver fibrosis model. Acad. Radiol. 2011;18(3):377-383.

2. Cobbold JF, Patel JH, Goldin RD, et al. Hepatic lipid profiling in chronic hepatitis C: an in vitro and in vivo proton magnetic resonance spectroscopy study. J. Hepatol. 2010;52(1):16-24.

3. Bugianesi E, Leone N, Vanni E, et al. Expanding the natural history of nonalcoholic steatohepatitis: from cryptogenic cirrhosis to hepatocellular carcinoma. Gastroenterology. 2002;123(1):134-140.

4. Chabanova E, Bille DS, Thisted E, et al. 1H MRS assessment of hepatic steatosis in overweight children and adolescents: comparison between 3T and open 1T MR-systems. Abdom. Imaging. 2013;38(2):315-319.

5. Tkáč I, Starčuk Z, Choi IY, et al. In vivo 1H NMR spectroscopy of rat brain at 1 ms echo time. Magn. Reson. Med 1999;41(EPFL-ARTICLE-177519):649-656.

6. Provencher SW. Automatic quantitation of localized in vivo 1H spectra with LCModel. NMR Biomed 2001;14(4):260-264.

7. Yahya A, Tessier AG, Fallone BG. Effect of J‐coupling on lipid composition determination with localized proton magnetic resonance spectroscopy at 9.4 T. J. Magn. Reson. Imaging. 2011;34(6):1388-1396.

Figures

Fig 1. The anatomical image shows a voxel (red box) in the liver. Representative in vivo proton magnetic resonance spectra acquired with short echo time of STEAM in the liver region are shown (a, methyl protons (-CH₃; ~0.90 ppm, dashed red box); b, methylene protons ((-CH₂-)_n); c, β-methylene resonance to the carboxyl group (-CH₂-CH₂-CO-); d, allylic protons (-CH₂-C=C-CH₂-); e, α-methylene resonance to the carboxyl group (-CH₂-CH₂-CO-); f, diallylic protons (=C-CH₂-C=); g, methane protons (-CH=CH-)). Panels show original raw data and residue (black line), fitted spectra (blue line) with baseline from (A) 0 week or (B) 10 weeks.

Fig 2. The mean areas of the resonances (~0.90 ppm, ~1.60 ppm, ~2.03 ppm, ~2.25 ppm, ~2.78 ppm, and ~5.30 ppm) fit on mono exponential curve (baseline [0 week]; black dash line) and enhanced exponential curve (baseline [0 week]; black solid line) with error bar.

Fig 3. The mean areas of the resonances (~0.90 ppm, ~1.60 ppm, ~2.03 ppm, ~2.25 ppm, ~2.78 ppm, and ~5.30 ppm) fit on mono exponential curve (10 weeks; black dash line) and enhanced exponential curve (10 weeks; black solid line) with error bar.

Fig 4. Indices of (A) total lipid, (B) total saturated fatty acid, (C) total unsaturated fatty acid, (D) total unsaturated bond, and (E) polyunsaturated bond in the liver of high-fat diet-fed mice for the stimulated echo acquisition mode sequence. The asterisks (*, **, and ***) indicate the statistical significance at p < 0.05, p < 0.01, and p < 0.001, respectively. All statistical analyses were conducted using SPSS. For baseline (week 0) to week 10 data, metabolic changes and body weight changes with diet intake were analyzed by one-way repeated measures analysis of variance with Bonferroni post-hoc comparisons.

Fig 5. Changes in body weight (A) and intake of HFD pellets (B) of mice fed a HFD. (C) Representative images of the liver from the NCD-fed and HFD-fed mice. The liver from the HFD-fed mice appeared to have a fatty liver-like appearance. (D) Representative images of liver sections stained with typical H&E (original magnification ×100; normal hepatocyte, yellow arrow; macro vesicular with fatty change, blue arrow; micro vesicular with fatty change, red arrow). (E) The liver weights were determined at necropsy. The asterisks (*, **, and ***) indicate statistical significance at p < 0.05, p < 0.01, and p < 0.001, respectively.

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)

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