Synopsis

The idea of defining a biomarker to assess the progression of Nonalcoholic Fatty Liver Disease (NAFLD) by using Magnetic Resonance Spectroscopy (MRS) emerges due to the need to find a way to replace biopsy with a non-invasive method. The purpose of this study is to investigate the composition of the liver fatty acids based in the metabolite signals in MRS in NAFLD mice fed with a western-diet at 3 time-point during the progression of the disease. Our findings showed significant changes in the diallylic (2.8 ppm), olefinic (5.3ppm), allilic (2.0 ppm) and bulk methylene (1.3 ppm) peaks during the progression of the NAFLD by using high-resolution MRS.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of intracellular fatty acids in the liver. The only method to confirm the stage of this disease is the biopsy, but it is invasive and risky for the patients. Currently, the methods for NAFLD diagnosis are based in the total amount estimation of fat infiltration in the liver, but they do not consider the differences in its composition, which has been proposed as a better method to discriminate between this group of diseases¹. The idea of defining a classifier by using magnetic resonance spectroscopy (MRS) emerges due to the need to find a way to replace biopsy with a non-invasive method that can classify NAFLD based on the chemical structure of fatty acids (FA) stored in the liver.

The purpose of this study is to investigate and compare the fatty acids composition in NAFLD mice liver based in the metabolites signals in MRS at 3 time-point during the progression of the disease.

Methods

We fed a group of C57BL/6 male mice with western diet for 4 weeks (n=6), 10 weeks (n=6) and 24 weeks (n=6). We also fed a control group with a chow diet (n=6). A portion of the liver was used for histology analysis and the remaining liver was used to extract their fatty acids methyl esters (FAMEs) using a protocol adapted by Folch et al ².

Those FAMEs were analyzed by using a 9.4T MRS and gas chromatography with mass spectrometer (GC-MS). The spectroscopy pulse programming was the Zg30. The acquisition parameters were: spectral width 8012.820 Hz, relaxation delay 1 s, number of scans 16, flip angle 30 to avoid T1 relaxation effects and total acquisition time 48.72 s.

All experiments were approved by the scientific ethics committee for the care of animals of the Catholic University of Chile (Number: 170614002).

Results

Mice fed with western diet increased their weights from 28.3 ± 2.7 grams (4 weeks-diet) to 46.1 ± 2.4 grams (24 weeks-diet), p=0.002. The amount of fat stored in the hepatocytes during the diet intervention increase from 5.8 ± 2.1 % (4 weeks-diet) to 23.6 ± 2.5 % (24 weeks-diet), p<0.0001. Histological analysis confirmed the progression of fat liver content.

We identified 12 main fatty acids using the CG-MS, however 5 of them had less than 1% of participation, therefore we only considered 7 FA for the analysis. Three of the FA changed significantly between all the 4 groups (control, 4 weeks-diet, 10 weeks-diet and 24 weeks-diet) and 3 of them changed between 3 of the 4 groups (Fig 1). By grouping them into the main categories (PUFA, MUFA, SFA) (Fig 2): PUFA liver content decreased with the progression of the disease from 40.5% in controls to 4.4% at 24 weeks (p<0.05). In contrast, MUFA liver content increased from 26.8% in controls to 67.8% at 24 weeks (p<0.05).

We have identified seven metabolite peaks in the MRS spectra that correspond to the FAs and one peak corresponding to the methyl ester. We calculated the area under the curve (AUC) of all the metabolites using MetreNova V10 software³ (Fig 3). Four of them (diallylic, olefinic, allilic and bulk methylene) showed significant differences between control, 4 weeks-diet, 10 weeks-diet and 24 weeks-diet, according to the Mann-Whitney test (Fig 4).The terminal ester, methyl terminal, alfa and beta methylene should be present in all FA in only one quantity, therefore we shouldn’t expect any significant differences (Fig 5).

Discussion

The progression of the NAFLD showed an increase in the total liver FA concentration, as expected; however, individual and subgroups of fatty acids showed a different behavior. The PUFA concentration decreased, and the MUFA concentration increased with the progression of the disease.

The peak corresponding to FA with two or more double bonds, the diallylic (2.8 ppm), also decreased in good agreement with the PUFA results. The peak olefinic (5.3ppm), allilic (2.0 ppm) and bulk methylene (1.3 ppm) are somehow related to this change in PUFA and MUFA also, because they are related to double bonds (Fig. 5).

Conclusion

Acknowledgements

References

[1] Ahmed, M. World J Hepatol 2015 June 18; 7(11): 1450-1459.

[2] Folch et al. The Jour Bio Chem. 226(1) ,1957.

[3] Ren et al. Jour Lip Res 49(1), 2008.