A limitation of 1H MRS for noninvasive quantitation of IMCL is the lack of high spectral resolution since the EMCL signal is separated by only ~0.2 ppm and it is about one order of magnitude larger than the IMCL signal and often asymmetric. Consequently, small voxels, careful positioning of the leg, limitation to studies of the tibialis anterior muscle, and complex data analysis schemes are needed. The current work uses a spectral acquisition strategy using long echo times combined with a large voxel STEAM sequence without water suppression. This approach contrasts to the current popular method of using smaller voxels combines with short a TE and PRESS sequence (to offset the low sensitivity of a small voxel. Note that the method of taking smaller voxels to avoid fat inclusion may not show a significantly suppression effect on EMCL). There are at least three significant benefits of using the long echo approach.
First, the IMCL –CH3
resonance rather than bulk -(CH2
- resonance can be used for assessing the IMCL concentration. The advantage of such an approach is straightforward: one does not need to assume a particular lipid composition to translate the area of the –CH3
resonance into concentration, as in the case of the -(CH2
- resonance. The long TE strategy provided the basis for such a method since the T2
of the lipid –CH3
resonance is significantly longer than the T2
of the -(CH2
- resonances (). IMCL quantification was made easier at a practical level by adopting TR = 2 s and TE = 280 ms where intensity corrections are small. From spectra collected using TE = 280 ms, and analysis of IMCL for 25 individuals shows a large variation in IMCL concentrations from 3.0 to 15.2 mmol/kg, with an average concentration of 7.7 ± 3.5 mmol / kg (). In comparison, Cui et al (35
) reported an average value of 7.7 ± 1.2 mmol/kg for soleus IMCL concentration at 4T using 1.0 mL voxels for non-obese sedentary healthy subjects (N = 7, BMI = 24.9 ± 3.8 kg/m2
, age 36.0 ± 11.7 yr). Hwang et al (22
) recently reported an average value of 8.8 ± 8.3 mmol/kg (individual values ranged from 2.8 to 33.2 mmol/kg) for a group of twelve nondiabetic subjects (age 34.0 ±15.7 yr ; 10 males, 2 females; BMI 25.6 ±3.2 kg/m). In a previous study (28
) using spectra collected for small voxels (1 mL) and a short TE (13 ms) estimated a soleus concentration of IMCL = 4.8 ± 2.2 mmol/kg for a group of nine volunteers aging from 21 – 68 y (average 44 y). The current data for healthy sedentary adults is consistent with earlier estimates of the concentration of IMCL.
A second advantage of this approach is that one can remove the contamination of the EMCL =CH-CH2-CH= signal to the total creatine –CH3 at ~3.0 ppm. Although there is no ideal internal concentration standard in skeletal muscle, the concentration of creatine is relatively constant. The long echo experiment precludes underestimation of IMCL as a result of contamination of the creatine internal standard. In addition, the long TE measurement also reduced the intensity of the EMCL -(CH2)n- resonances relative to the IMCL OOC-CH2-CH2 signal and probably other unidentified macromolecules in that chemical shift region. These spectral simplifications make automatic lineshape fitting procedures simpler.
A third advantage is that water suppression was not used. Consequently, the preparation time need to begin data collection is reduced by ~ 1 min, SAR is reduced by at least 5% (as reported by the scanner) and other metabolites with resonances near water are not attenuated (24
). The water signal in the 1
H MR spectrum thus can be explored in several different ways, for example to establish the relationship between muscle function and dehydration - an interesting yet challenging topic in athletic communities (36
The long TE used here to gain spectra resolution inevitably compromises sensitivity. One can use a larger voxel to make up the loss of sensitivity, but this may be a problem for subjects with smaller muscle mass such as children. In such cases, one may need to use smaller voxels and longer scan times. When the chemical shift displacement artifact (CSD) (32
) is not a major concern, PRESS sequence can replace STEAM to boost S/N ratio under the long TE scheme with smaller voxel. It is known that echo-based localized 1
H MR spectrum intrinsically suffers from CSD, an artifact that causes two different resonances within a given spectrum originate from two different geometric regions, shifted by a distance which is a function of the size of the localization voxel, the strength of the magnetic field, the chemical shift difference (in ppm) between these two resonances, and the pulse sequence used. PRESS gives rise to significantly larger CSD than STEAM due to the use of two 180° pulses. Additionally, TE can be reduced to enhance sensitivity or increased to further enhance resolution dependent on the actual need during the study. However, in such cases, one may have to correct the T1
difference between IMCL and reference signals due to deviation from the advantageous setting of TR = 2 s and TE = 280 ms for good spectral resolution.
The advantages of collecting long TE spectra at 7T can be primarily attributed to the shorter T2 of the EMCL protons, the relaxation mechanism of which remains unclear. The phantom studies show that the proton linewidths of oils in capillary tubes is not orientation-dependent () yet the appearance of asymmetric “EMCL” signal in the short T2 spectrum (see ) of an oil soaked Kleenex oriented in agar suggests that EMCL signals in human muscle may also be broadened by magnetic susceptibility anisotropy (MSA) in a magnetically heterogeneous environment. In fact, the increased EMCL chemical shift dispersion at higher magnetic field strength was thought to be one of the drawbacks of going to higher field. However, the phantom study demonstrated that the triglyceride molecules trapped in the tightly-rolled Kleenex cylinder (“EMCL”) do appear to have shorter T2 than the oil molecules in droplets (“IMCL”) due to more restricted motion. Based on MRI, a significant percentage of EMCL is in fairly large depots, one may not consider that such lipid stores constitute a motion-restricted environment for the fatty acid and triglyceride molecules. This implies that the improved resolution at long TE is due to the attenuation of EMCL component with much short T2 and dispersed chemical shift.
If the same strategy for resolving EMCL and IMCL signals of skeletal muscle fats also works as well in insulin-resistant patients as illustrated here for healthy subjects, then one should be able to use this technique to confirm the early work on the correlation between insulin resistance and elevated IMCL and to clarify the issue of the “metabolic paradox” (11
). This in turn may help position 1
H MRS of skeletal muscle fat as a useful diagnostic clinical tool. The long TE strategy may also provide new venues for studies of lipid metabolism in skeletal muscle using 1
H MRS. For instance, the IMCL and EMCL - (CH2
signal intensity ratios, which can only be reliably obtained using a long TE, is a index worthy of further exploration. It is anticipated that this index will be sensitive to fatty acid composition, since the three most abundant fatty acids in human tissue have very different -(CH2
ratios: palmitic acid 9.3 (= 14 × 2/3), oleic acid 6.7 (= 10 × 2/3) and linoleic acid 4. 7 (= 7 × 2/3). Given that skeletal muscle is an essential tissue for whole-body energy metabolism, including insulin-stimulated glucose uptake and fatty acid oxidation, the feasibility and non-invasiveness of the long TE 1
H MRS in determining the fatty acid composition of fat droplets in myocytes may profoundly impact future muscle metabolism studies. Evidence derived from skeletal muscle biopsies and blood samples indicates that the fatty acid composition of skeletal muscle phospholipids is related to peripheral insulin sensitivity and obesity in several human populations (38
). One may use this index to study factors that influence deposition and mobilization of fats within and between individuals. (39
In conclusion, the current 1H MRS study at 7T demonstrated that the use of long echo times (TE), together with large voxel sizes without water-suppression greatly enhances the resolution of skeletal muscle EMCL and IMCL and thereby allows a more reliable quantitative measure of IMCL content. The long TE strategy also has the advantage of lower SAR, large sampling volume, short scan time and inclusion of water in the spectra. The successful application of this long TE strategy may provide new opportunities to explore the fat composition and metabolism in skeletal muscle and to better characterize metabolic disorders in diseases such as obesity, insulin resistance and diabetes.