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Comparing omega-3 content in adipose tissue of mice and rats fed a high omega-3 diet with MRS at 9.4T1Department of Oncology, University of Alberta, Edmonton, AB, Canada, 2Department of Medical Physics, Cross Cancer Institute, Edmonton, AB, Canada, 3Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada
Synopsis
Omega-3 (ω-3) fat intake is important to disease and reflected in adipose tissue composition. Optimized PRESS (Point RESolved Spectroscopy) at 9.4T is used to quantify relative ω-3 fat content in adipose tissue of mice and rats fed a 16% ω-3 diet and in a control diet mouse. The actual ω-3 fat content in the mice was determined using gas chromatography and compared to that obtained using optimized MRS (R2 = 0.98). On average, the % ω-3 content measured with MRS in rat adipose tissue was approximately 39% higher than that in mouse.
Introduction
Dietary omega-3 (ω-3) fat correlates inversely with disease, including obesity 1,2, breast cancer 3,4, osteoporosis 5,6 and diabetes 1,2. Magnetic Resonance Spectroscopy (MRS) can assess fat composition in vivo 7-9; however, ω-3 quantification is usually neglected because of its lower concentration (≈1% ) 10 and because its quantification is challenging. The ω-3 methyl resonance (≈0.98ppm) overlaps the non-ω-3 methyl resonance (≈0.9ppm) 11 with standard short TE (echo time) techniques, even at high field strengths 12. Measuring ω-3 fat content is desirable since levels can change with disease and in feeding trials 13-19. Feeding trials can explore correlations between pathology and dietary intakes that are ingested by people or diets meant to alleviate or prevent disease 1,2,4-6,20. Dietary impacts can be evaluated in animal trials 8,19. The presented work measures relative ω-3 fat content obtained using an optimal PRESS (Point RESolved Spectroscopy) TE of 109ms at 9.4T in-vivo in adipose tissue of mice and rats fed a high ω-3 fat diet and in a mouse fed a control diet.Methods
At 9.4T, a PRESS TE of 109ms minimizes signal from the side peaks of the ω-3 and non-ω-3 methyl resonance triplets, resolving the two peaks. Three mice and two rats were fed a high fat diet (20% weight/weight fat) with 16% ω-3 fat. A control mouse was fed a standard chow diet (6% weight/weight fat), with ≈6 % ω-3 fat 21. Animals were scanned with a 21.5 cm diameter bore 9.4T MRI scanner (mice at about 5 months of age and rats at about 3 months). A 3x3x3 mm3 voxel (mice) or 5x5x5 mm3 (rats) was positioned in the abdominal visceral adipose tissue (Figure 1) and spectra were acquired using the following parameters: repetition time (TR) of 5 s for mice and 3 s for rats, 2048 complex points, 8-step phase cycling, 10,000 Hz sampling frequency, and 32 averages. PRESS spectra from all animals were acquired with a short TE of 25 ms (TE1 = 15 ms, TE2 = 10 ms), and with the optimized TE of 109 ms (TE1 = 15 ms, TE2 = 94 ms). Peak areas of the ω-3 methyl (0.94 – 1.0 ppm) and the non-ω-3 methyl (0.86 – 0.93 ppm) were measured from long TE spectra and a % ω-3 fat content was calculated as the ω-3 methyl area divided by the sum of the ω-3 and non-ω-3 methyl peak areas. Mice were euthanized immediately after scan completion and their abdominal visceral fat was collected for gas chromatography (GC) analysis to determine % ω-3 content.Results
Figure 2(a) displays the spectra acquired from the ω-3 fat diet mice and the control mouse with the short-TE PRESS sequence. Only one mouse in the ω-3 diet group exhibited an ω-3 methyl shoulder in its short-TE spectrum; the ω-3 and non-ω-3 methyl peaks were completely overlapped in all the other mice. Figure 2(b) displays the long-TE methyl spectra from each of the mice. Figure 3 displays the correlation between % ω-3 content using GC and long-TE MRS for the four mice; R2 = 0.98. The % ω-3 content in the mice computed from GC were on average 5.7 ± 0.9% for the ω-3 diet mice and 2.4% for the control mouse. No ω-3 MRS signal was detectable in the control mouse using the short TE; however, ω-3 signal was observed using the long-TE with a signal to noise ratio of ≈26 for the ω-3 resonance. The % ω-3 content obtained using long TE MRS was 20.6 % and 13.5 % in the two rats. Figure 4 displays the PRESS spectra acquired from the rats using the short-TE and the optimized long TE.Discussion
Spectral overlap between the ω-3 and non-ω-3 methyl resonances renders it challenging to quantify ω-3 with MRS using standard short- TE methods, even at 9.4T (as shown in Figures 2(a) and 4(a)). Using PRESS with a TE of 109 ms resolves the two methyl peaks (Figures 2(b) and 4(b)). Figure 3 demonstrates the efficacy of the long TE PRESS sequence for relative in-vivo quantification of ω-3 content at 9.4T, detecting small variations in mice fed the same high ω-3 fat diet. MRS measures of ω-3 content are larger than those obtained from GC, likely due to J-coupling effects and differences in T2 relaxation between the ω-3 and non-ω-3 protons. Gas chromatography analysis of the control mouse resulted in about 2.4% ω-3 fat content. This level was observable using the long TE PRESS sequence. The % ω-3 content obtained by long-TE MRS in the two rats was on average 17.1% compared to an average of 12.3% obtained in the three mice. This could indicate that rats may store more ω-3 in their adipose tissue; however, more animals need to be studied to confirm the preliminary findings.Conclusion
PRESS with a TE of 109 ms at 9.4T was employed to estimate relative ω-3 fat content in adipose tissue of mice and rats fed a high ω-3 diet. Rat adipose tissue ω-3 content was higher. The method can be used to study correlations between adipose tissue ω-3 fat content and disease or diet in animal models.Acknowledgements
Grant and student funding from the Natural Sciences and Engineering Research Council of Canada are gratefully acknowledged.References
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