Recently, lipid quantification using proton magnetic resonance spectroscopy (¹H-MRS) has been used to diagnose diseases such as obesity, cirrhosis, and hepatitis.¹ Typically, spectra from 0.9 ppm to 5.3 ppm obtained from in vivo ¹H-MRS of the liver tissue show various lipid peaks as well as choline-containing compounds.² Increased spectral resolution and signal-to-noise ratio (SNR) in high field strength scanners have been demonstrated in previous studies.³ An increase in SNR shortens the total acquisition time and increases spectral resolution, thus improving quantification of metabolites and relaxation time.⁴ For minimizing signal loss, localized point-resolved spectroscopy (PRESS) and short echo time (TE) produce better SNR of metabolite peaks and their T₂ relaxation times rather than stimulated echo acquisition mode (STEAM) sequence with a long TE.⁵ However, PRESS and STEAM in liver fat have shown different quantification results. The objective of this study is to compare lipid contents using the PRESS and STEAM sequences to assess lipid resonances in the liver, using in vivo high-resolution spectra at 9.4 T.In vivo PRESS and STEAM Male C57BL/6N (n=10) mice were housed in standard plastic cages with ad libitum access to water with weight matched and were included in this study. Fatty liver disease was induced with a high fat diet and comprised pellets composed of 60% fat, 20% protein, and 20% carbohydrate (Rodent Diet with 60% Kcal% fat, D12492, Research Diets, New Brunswick, NJ). The examinations were performed on a Bruker 9.4T scanner. After scout imaging, T1 weighted images (in Fig. 1) was acquired in three orthogonal planes before localization of volume of interest (VOI). To avoid large blood vessels, a voxel (0.3×0.3×0.3 cm³) was placed in a homogeneous parenchyma of the liver. For this, we used PRESS (repetition time [TR]/TE=3500/20 ms; number of signal averages [NSA]=128; acquisition data points=2048) and STEAM (TR/TM/TE=3500/10/20 ms; NSA=128; acquisition data points=2048). Lipid relaxations in HF diet mice were estimated at a fixed TR of 5000 ms, and TEs of 20–70 ms. The water suppression of each VOI was achieved using variable pulse power and optimized relaxation delays applied before the scan. For lipid content measurement, total lipid ((-CH₂-)_n/noise), saturated fatty acid (3(-CH₂-)/2(-CH₃)), total unsaturated fatty acid (3(-CH₂-C=C-CH₂-)/4(-CH₃)), total unsaturated bond index (3(-CH=CH-)/2(-CH₃)), and polyunsaturated bond index (3(=C-CH₂-C=)/2(-CH₃)) were quantified by separating each peak area of (­CH₂­)_n, ­CH₂­-C=C-­CH₂­, =C-­CH_­2­-C=, and ­CH=CH­ by ­CH₃. –CH₃ peak was used as an internal chemical shift reference. Total lipid was measured by dividing (-CH­₂-)_n peak area by spectral noise that was acquired with the standard deviation of peak area (from 7.3 to 11.3 ppm) at which no lipid metabolites was observable in the liver. All spectra acquired were processed using the Advanced Method for Accurate, Robust, and Efficient Spectral fitting algorithm including in the Java-based Magnetic Resonance User Interface software package. Before fitting, apodization of the spectra in HF diet mice with baseline was performed at 3Hz. Preprocessing was performed using an automatic frequency shift and the Hankel-Lanczos Singular Value Decomposition filter with subtraction of water peak. To improve SNR, all measurable lipid peaks were fitted with single Gaussian line shape. All statistical analysis was conducted with the statistical package for the social sciences (SPSS version 21, IBM Corp. Armonk, NY, USA) including the independent t-test to determine metabolic changes and to establish significant difference.Fig. 1 compares water and lipid peaks. Fig. 2A to E compares relative lipid contents in the studied liver between the PRESS and STEAM sequences. In Fig. 2, the total lipid and total saturated fatty acid were differently changed in HF diet mice that underwent PRESS compared with those that underwent STEAM (total lipid, p<0.05; total saturated fatty acid, p<0.001). Compared with PRESS, STEAM showed different total unsaturated fatty acid value (p<0.001). Compared with PRESS, STEAM showed different total unsaturated and poly unsaturated bonds (total unsaturated bond, p<0.001; poly unsaturated bond, p<0.001).Theoretically, STEAM produces more accurate results than PRESS. In addition, a relaxation description of the lipid molecule for quantification is quite complex and difficult to describe analytically.⁶ The relaxation behavior of lipid resonances may lead to errors in signal quantification.⁶ T₂ relaxation may create errors in quantitative analysis of lipid composition. Because all lipid resonances in the liver showed a J-coupling effect and different sensitivities in J-coupling, at 9.4 T, CH₃ at 0.9 ppm was strongly coupled at the adjacent (-CH₂-)_n protons.⁵ In conclusion, due to stronger J-coupling effects on the PRESS sequence, the accurate estimation could not be obtained. STEAM is less sensitive to J-coupling and gives a theoretically more accurate estimate at 9.4T.