Extramyocellular Lipid Research Articles

Absolute quantification of calf muscle metabolites by proton 1H-MR spectroscopy

Objectiye To measure longitudinal (T1) and transverse (T2 ) relaxation time of metabolites in m. soleus (SOL) and m. tibialis anterior TA of healthy volunteers at 3.0 T through 1H-MRS and optimize measurement protocols. Methods Altogether 24 healthy volunteers were recruited in the study. All subjects signed a letter of informed consent. After divided into 2 groups randomly by the table of random number, 1H-MRS measurements with stimulated echo acquisition mode (STEAM) sequence were undertaken in SOL and TA separately. Progressive saturation method was used for T1 measurement. Spectra with 8 different TRs (770,900,1000, 1100,1200,1500,2000 and 3000ms ) were acquired with TE=20 ms.T2 time was measured by changing TE. Altogether 8 TEs (20,30,45,60,90,135,200 and 270 ms) were used with TR = 3000 ms. Metabolites' concentration was calculated through T1 and T2 correction using water as internal reference. The t test was used for statisties. Results Altogether 22 groups of data were gained ( 12 for SOL, 10 for TA ) . T1 value of water, Creatine-CH3 ( Cr3 ), Trimethyl amonium ( TMA ),extramyocellular lipid (EMCL) and intramyocellular lipid (IMCL) in SOL were ( 1384. 0 ± 36. 9 ),( 1064. 0 ± 167.0), (964. 2 ± 144. 0 ), ( 373.0 ± 46. 8 ), ( 374. 7 ± 20. 6) ms respectively and T2 value were (26.5 ±1.2), (100.2±19.3), (149. 1 ±32.7), (81.4±5.2), (84.7±4.2) ms. InTA T1 value of water, Cr3, TMA, EMCL, and IMCL were ( 1307. 0 ± 24.4), (945.7 ± 132. 0), (968.3 ± 127. 0),(372. 7 ± 39. 2), (412. 8 ±80. 2) ms respectively and T2 value were (27. 1 ± 0. 9), (135.3 ± 18. 2 ),(62.1 ± 6. 0), ( 84. 3 ± 4. 0 ), ( 90. 7 ± 3.2 ) ms. After corrected by the calculated relaxation times, the concentrations of Cr3 in SOL and TA were (33. 1 ± 3.7) and (31.7 ± 3. 1 ) mmol/kg respectively, TMA (35.2±3.2) and (32.9 ±5.2) mmol/kg, EMCL (12.2 ±5.0) and (8.9 ±4.9) mmol/kg, IMCL (9. 0 ± 2. 4) and (3.0 ± 0. 8 ) mmoL/kg. IMCL in TA was much lower than SOL with statistical significant ( t = 8. 044, P 0. 05 ) . Conclusions Accurate relaxation time was measured at 3.0 T of the metabolites in skeletal muscles of healthy adult human. After corrected by the relaxation times, the absolute concentrations calculated were consistent with the reported results. Quantitative knowledge of muscle NMR relaxation time was a prerequisite for absolute quantification of metabolites using the 1H-MRS and also was useful for optimizing measurement protocols. Key words: Magnetic resonance spectroscopy; Muscle,skeletal; Fats

1H MRS of intramyocellular lipids in soleus muscle at 7 T: Spectral simplification by using long echo times without water suppression

The popular short echo time (1)H MR spectroscopy acquisition method for detection of intramyocellular lipids suffers from spectral overlap due to the large, broad, and asymmetric extramyocellular lipid signals, the time-consuming practice of selecting "lean" voxels for spectroscopy, and the overlap of the extramyocellular lipid signal with the creatine methyl (1)H signal at approximately 3 parts per million (ppm), commonly used as an internal standard. Using an alternative acquisition strategy, spectra with well-resolved intramyocellular lipids resonances were acquired from large volumes (10 to 15 mL) of human soleus muscle in less than 5 min by single-voxel 7-T (1)H MR spectroscopy, using an echo time of 280 ms. From the high-resolution spectra, an average intramyocellular lipid concentration of 7.7 +/- 3.5 mmol/kg muscle was found for 25 healthy subjects (male/female 17/8; age 29.4 +/- 6.6 years). Since water suppression was not required, the (1)H signals from unsaturated intracellular triglycerides at about 5.3 ppm were easily detected, which, in combination with the well-determined -(CH(2))(n)-/CH(3) intensity ratio at long echo time, enabled assessment of the composition of triglycerides in the intramyocellular lipids compartment. Long-echo single-voxel spectroscopy at 7 T offers rapid and convenient acquisition of high-resolution spectra from human soleus muscle.

Echo planar correlated spectroscopic imaging: Implementation and pilot evaluation in human calf in vivo

Exploiting the speed benefits of echo-planar imaging (EPI), the echo-planar spectroscopic imaging (EPSI) sequence facilitates recording of one spectral and two to three spatial dimensions faster than the conventional magnetic resonance spectroscopic imaging (MRSI). A novel four dimensional (4D) echo-planar correlated spectroscopic imaging (EP-COSI) was implemented on a whole body 3 T MRI scanner combining two spectral with two spatial encodings. Similar to EPSI, the EP-COSI sequence used a bipolar spatial read-out train facilitating simultaneous spatial and spectral encoding, and the conventional phase and spectral encodings for the other spatial and indirect spectral dimensions, respectively. Multiple 2D correlated spectroscopy (COSY) spectra were recorded over the spatially resolved volume of interest (VOI) localized by a train of three slice-selective radiofrequency (RF) pulses (90°-180°-90°). After the initial optimization using phantom solutions, the EP-COSI data were recorded from the lower leg of eight healthy volunteers including one endurance trained volunteer. Pilot results showed acceptable spatial and spectral quality achievable using the EP-COSI sequence. There was a detectable separation of cross peaks arising from the skeletal muscle intramyocellular lipids (IMCLs) and extramyocellular lipids (EMCLs) saturated and unsaturated pools. Residual dipolar interaction between the N-methylene and N-methyl protons of creatine/phosphocreatine (Cr/PCr) was also observed in the tibialis anterior region.

Relationship between Insulin Sensitivity and Muscle Lipids may Differ with Muscle Group and Ethnicity

Intramyocellular lipid (IMCL) has been inversely associated with insulin sensitivity in some, but not all, studies. This study utilized fast, high-resolution, magnetic resonance spectroscopic imaging (MRSI) to: investigate relationships between muscle lipids (IMCL and extramyocellular lipid (EMCL)) and insulin sensitivity in muscles of varying oxidative capacity, explore ethnic differences in these relationships, and determine whether a eucaloric, low-fat dietary intervention would reduce IMCL and increase insulin sensitivity. Subjects were 30 healthy, African-American (AA; n=14) and European-American (EA; n=16) males, BMI 26.49 (±5.57) kg/m(2), age 21.80 (±7.84) yrs. Soleus and tibialis anterior muscle lipids were quantified using MRSI. Insulin sensitivity was assessed via intravenous glucose tolerance test. A 2-week, eucaloric, low-fat diet intervention was conducted in a sub-group (n=12) subjects with assessments at baseline and post-intervention. Neither IMCL nor EMCL levels differed between ethnicities. In the total group, and within EA (but not AA), both tibialis anterior IMCL and EMCL were inversely associated with insulin sensitivity (P<0.05 for both); soleus muscle lipids were not associated with insulin sensitivity. Soleus, but not tibialis anterior, IMCL declined in both ethnic groups (average 25.3%; p<0.01) following dietary intervention; insulin sensitivity was unchanged. Results suggest that an association of muscle lipids with insulin sensitivity may be influenced by the oxidative capacity of the muscle group studied and may vary with ethnicity.

In Vivo Assessment of Mouse Hindleg Intramyocellular Lipids by 1H-MR Spectroscopy

(1)H-magnetic resonance spectroscopy ((1)H-MRS) has proved to be the sole in vivo technique able to measure intramyocellular lipids (IMCL) in both humans and animals. Mouse models are now widely used for physiologic studies and drug discovery. However, IMCL assessment using (1)H-MRS is hindered in this animal model by the small muscle size and strong contamination from the extramyocellular lipid (EMCL) signal. The objective of this study was to the use of (1)H-MRS for IMCL quantification in mice at different ages. Noninvasive IMCL quantification was performed at 7 T in tibialis anterior (TA) muscles of healthy male C57/BL6 mice (n = 9; age, 13.6 +/- 1 months), db/db mice (n = 4), and their C57BL/KSJ control littermates (n = 4) at 7 and 17 weeks of age. The IMCL content of diabetic mice TA was significantly higher than their littermates (2.41 +/- 0.5 vs. 1.21 +/- 0.35, P < .01). An age effect was observed, with TA IMCL levels being lower in older than younger control mice, but increasing between 7 and 17 weeks in the db/db mice. The feasibility of (1)H-MRS spectroscopy was demonstrated in mice muscle, despite its small size, and used to assess IMCL content in db/db mice.

Orientation of lipid strands in the extracellular compartment of muscle: Effect on quantitation of intramyocellular lipids

Single-voxel (1)H NMR spectra from gastrocnemius and soleus muscle were acquired in healthy volunteers at 7T with the objective of measuring the concentration of intramyocellular lipid [IMCL] (note: throughout this article, square brackets indicate concentration). However, significant asymmetry in the resonance assigned to the methylene protons (-CH(2)-)(n) in extramyocellular lipids (EMCL) interfered with fitting the spectra. Since muscle fibers in these tissues are generally not parallel to B(0), the influence of variable orientation in strands of extracellular fat was examined using a mathematical model. Modest variation in orientation produced asymmetric lineshapes that were qualitatively similar to typical observations at 7T. Analysis of simulated spectra by fitting with a Voigt function overestimated [IMCL]/[EMCL] except when EMCL fibers were nearly parallel to B(0). Estimates of [IMCL]/[EMCL] were improved by including variations in fiber orientation in the lineshape analysis (fiber orientation modeling, or FOM). Calculated [IMCL] using FOM, 4.8 +/- 2.2 mmol/kg wet weight, was lower compared to most previous reports in soleus.

Hydrochlorothiazide, but not Candesartan, Aggravates Insulin Resistance and Causes Visceral and Hepatic Fat Accumulation

Treatment with angiotensin II receptor blockers is associated with lower risk for the development of type 2 diabetes mellitus compared with thiazide diuretics. The Mechanisms for the Diabetes Preventing Effect of Candesartan Study addressed insulin action and secretion and body fat distribution after treatment with candesartan, hydrochlorothiazide, and placebo. Twenty-six nondiabetic, abdominally obese, hypertensive patients were included in a multicenter 3-way crossover trial, and 22 completers (by predefined criteria; 10 men and 12 women) were included in the analyses. They underwent 12-week treatment periods with candesartan (C; 16 to 32 mg), hydrochlorothiazide (H; 25 to 50 mg), and placebo (P), respectively, and the treatment order was randomly assigned and double blinded. Intravenous glucose tolerance tests and euglycemic hyperinsulinemic (56 mU/m(2) per minute) clamps were performed. Intrahepatic and intramyocellular and extramyocellular lipid content and subcutaneous and visceral abdominal adipose tissue were measured using proton magnetic resonance spectroscopy and MRI. Insulin sensitivity (M-value) was reduced following H versus C and P (6.07+/-2.05, 6.63+/-2.04, and 6.90+/-2.10 mg/kg of body weight per minute, mean+/-SD; P<or=0.01). Liver fat content was higher (P<0.05) following H than both P and C. The subcutaneous to visceral abdominal adipose tissue ratio was reduced following H versus C and P (P<0.01). Glycosylated hemoglobin, alanine aminotransferase, aspartate aminotransferase, and high-sensitivity C-reactive protein levels were higher (P<0.05) after H, but not C, versus P. There were no changes in body fat, intramyocellular lipid, extramyocellular lipid, or first-phase insulin secretion. Blood pressure was reduced similarly by C and H versus P. In conclusion, visceral fat redistribution, liver fat accumulation, low-grade inflammation, and aggravated insulin resistance were demonstrated after hydrochlorothiazide but not candesartan treatment. These findings can partly explain the diabetogenic potential of thiazides.

Distinct patterns of fat metabolism in skeletal muscle of normal-weight, overweight, and obese humans

The link between body weight, lipid metabolism, and health risks is poorly understood and difficult to study. Magnetic resonance spectroscopy (MRS) permits noninvasive investigation of lipid metabolism. We extended existing two-dimensional MRS techniques to permit quantification of intra- and extramyocellular lipid (IMCL and EMCL, respectively) compartments and their degree of unsaturation in human subjects and correlated these results with body mass index (BMI). Using muscle creatine for normalization, we observed a statistically significant (P < 0.01) increase in the IMCL-to-creatine ratio with BMI (n = 8 subjects per group): 5.9 +/- 1.7 at BMI < 25, 10.9 +/- 1.82 at 25 < BMI < 30, and 13.1 +/- 0.87 at BMI > 30. Similarly, the degree of IMCL unsaturation decreased significantly (P < 0.01) with BMI: 1.51 +/- 0.08 at BMI < 25, 1.30 +/- 0.11 at 25 < BMI < 30, and 0.90 +/- 0.14 at BMI > 30. We conclude that important aspects of lipid metabolism can be evaluated by two-dimensional MRS and propose that degree of unsaturation measured noninvasively may serve as a biomarker for lipid metabolic defects associated with obesity.

Non-invasive assessment of lower extremity muscle composition after incomplete spinal cord injury

Cross-sectional study. (1) To quantify intramyocellular lipid (IMCL) content of the soleus muscle. (2) To assess the T(2) relaxation rates in the lower extremity skeletal muscles in persons with incomplete spinal cord injury (SCI). Academic Institution, Florida. Eight subjects (42+/-10 years old; 70+/-12 kg; 176+/-10 cm) with chronic (17+/-9 months post injury) motor SCI (C4-T12; ASIA C or D) and eight matched healthy controls were tested. Localized unsuppressed proton spectroscopy (H-MRS) was performed to estimate total lipid content and individual lipid components; IMCL and extramyocellular lipid (EMCL) from the soleus muscle. T(2)-weighted imaging of lower extremity muscles yielded muscle T(2) rates. The IMCL content of the soleus muscle was 3.3 times higher in the patient group as compared to controls (P=0.002; 0.0401 (0.0234-0.0849) versus 0.0123 (0.0090-0.0175)). Similarly, EMCL measures were 4.5 times higher as compared to the controls (P=0.002). Significant differences were observed in the T(2) relaxation times of the soleus and gastrocnemius muscles (P<0.05). The increased levels of IMCL might interfere with the glucose uptake in skeletal muscle; potentially predisposing persons with incomplete SCI to the development of peripheral insulin resistance. Marked elevations in the T(2) relaxation times of the locomotor muscles are reflective of an altered muscle composition.

Gender Differences in Musculoskeletal Lipid Metabolism as Assessed by Localized Two-Dimensional Correlation Spectroscopy

Gender differences in lipid metabolism are poorly understood and difficult to study using conventional approaches. Magnetic resonance spectroscopy (MRS) permits non-invasive investigation of lipid metabolism. We employed novel two-dimensional MRS techniques to quantify intramyocellular (IMCL) and extramyocellular (EMCL) lipid compartments and their degree of unsaturation in normal weight adult male and female subjects. Using muscle creatine (Cr) for normalization, a statistically significant (p < 0.05) increase in IMCL/Cr (7.8 ± 1.6) and EMCL/Cr (22.5 ± 3.6) for female subjects was observed (n=8), as compared to IMCL/Cr (5.9 ± 1.7) and EMCL/Cr (18.4 ± 2.64) for male subjects. The degree of unsaturation within IMCL and EMCL was lower in female subjects, 1.3 ± 0.075 and 1.04 ± 0.06, respectively, as compared to that observed in males (n=8), 1.5 ± 0.08 and 1.12 ± 0.03, respectively (p < 0.05 male vs female for both comparisons). We conclude that certain salient gender differences in lipid metabolism can be assessed noninvasively by advanced MRS approaches.

SARCOPENIA IN FRAIL ELDERLY OBESE PARTICIPANTS

Purpose/Hypothesis: To quantify skeletal muscle lipid accumulation in sarcopenic skeletal muscle of obese older adults with mild to moderate physical frailty and to assess its impact on strength and function. Subjects: Eighteen physically-frail, obese elderly participants (13 women, 5 men; mean age=69 ± 4, BMI=36.7 ± 5,PPT= 29 ± 2). Materials/Methods: Intra-myocellular (IMCL) and extra-myocellular (EMCL) lipid content in the soleus (SOL) and medial gastrocnemius (MG) muscles were quantified by non-invasive magnetic resonance spectroscopy (1H MRS) using a 3T whole-body imaging system. Thigh and calf muscle and fat volumes were quantified using magnetic resonance imaging. Muscle strength (peak torque) of the lower extremities was quantified using a Biodex Isokinetic dynamometer. The nine item physical performance test (PPT) was used to provide an objective assessment of physical function. T-tests were used to compared IMCL and EMCL values between muscles. Pearson correlation coefficients were calculated to assess impact of lipid content on strength and function. Results: IMCL content was 2.9% ± 1.5 and 3.5% ± 1.8 in the MG and SOL, respectively. EMCL content was 12.4% ± 8.4 and 9.2% ± 8.1 in the MG and SOL, respectively. IMCL and EMCL contents in the predominately slow twitch SOL were not significantly different (p > .05) compared to MG. MG IMCL content correlates negatively with LE strength (r = −0.16 to −0.42) and function (r = −0.46), whereas EMCL accumulation in both LE muscles (predominately MG) was inversely associated with LE strength. EMCL accumulation in the SOL negatively impacts function (r = −0.42). Conclusions: Obese older adults with mild to moderate physical frailty have an excessive amount of IMCL present in MG and SOL. Excess lipid accumulation in predominately type II muscle fibers negatively impacts strength and function to a greater degree than excess lipid accumulation in slow twitch fibers. Clinical Relevance: Skeletal muscle lipid content is elevated in elderly obese individuals. Increased lipid accumulation in the muscle with aging may be a critical component of sarcopenia. Further studies aimed at improving our understanding of the role of therapeutic interventions for ameliorating impairments in sarcopenia will assist physical therapists in the design of optimal exercise prescriptions to improve physical function. Supported by: New Investigator Fellowship Training Initiative (NIFTI), Foundation for Physical Therapy & NIH R01 AG 025501.

Implementation and validation of localized constant‐time correlated spectroscopy (LCT‐COSY) on a clinical 3T MRI scanner for investigation of muscle metabolism

To implement and evaluate a novel single-volume two-dimensional localized constant-time-based correlated spectroscopy (2D LCT-COSY) sequence on a clinical 3T MR scanner. This sequence exhibits homonuclear decoupling along the F1 dimension, leading to improved spectral resolution compared to that of non-constant-time localized correlated spectroscopy (L-COSY). A GE 3T MR scanner equipped with a quadrature transmit and receive extremity coil was used in this study. The 2D LCT-COSY sequence was programmed using General Electric's EPIC compiler. Simulations for a two-spin 1/2 system were performed using GAMMA libraries to evaluate the theoretical performance of the sequences, and were also compared with corresponding phantom experiments using trans-cinnamic acid. Finally, spectra were acquired from the soleus muscle of healthy volunteers in order to evaluate performance in vivo. Simulations and experimental results confirmed the improved spectral resolution of LCT-COSY over L-COSY, as well as its homonuclear decoupling performance. The behavior of resonance amplitudes as a function of evolution time in the experiment also was appropriately reflected by the simulation. Corresponding results were obtained for the in vivo muscle spectra, in which separation of overlapping olefinic and allylic methylene protons from the intra- and extramyocellular lipids (IMCL and EMCL, respectively) was achieved. Simulations and experimental results in vitro and in vivo demonstrate the strengths of LCT-COSY. This technique can be implemented on systems of any field strength, and has the potential to separate overlapping metabolites in tissue when employed on high-field clinical MRI scanners equipped for proton spectroscopy.

Measuring muscle lipids with 1H-MR spectroscopy

Fat is a key element of normal skeletal muscle. It is stored inside muscle cells as droplets called intramyocellular lipids (IMCL), or in adipocytes scattered between muscle cells, called extramyocellular lipids (EMCL). Since the 1980s, IMCL droplets have been of particular interest because they were noted to be in contact with mitochondria when examined under electron microscopy. This observation led to the concept that IMCL served as nearby fuel during prolonged exercise [1]. The focus on IMCL, however, intensified after a link with the etiology of diabetes was uncovered. In 1997, 38 Pima Indians—a group of Native Americans living in central and southern Arizona—participated in a study on diabetes [2]. Pima Indians have a marked predisposition to developing Type 2 diabetes and are less sensitive to insulin action. Such a condition, called insulin resistance, generally precedes the clinical development of Type 2 diabetes by 10 to 20 years [3]. Biopsies from the vastus lateralis muscle of these participants showed increased amounts of lipids that strongly correlated with their insulin resistance. This finding pioneered further research that established a connection between IMCL accumulation and insulin resistance. Recent studies indicate that IMCL may act indirectly: increased sub-products of IMCL, such as fatty acyl-CoA and diacylglycerol, hamper signals from insulin that promote transport of glucose into the cell [4]. Interestingly, increased IMCL is not always a herald of disease. Elite athletes show high levels of IMCL and are very insulin-sensitive. This paradox is explained by the fact that muscles of highly trained persons have enhanced fatburning (oxidative) capacity, being very efficient at lipid utilization [4]. Sedentary and obese individuals, on the other hand, have decreased muscle oxidative capacity in which IMCL accumulation may be harmful. Such observations relied on several techniques to measure muscle lipids. Biochemical analysis, histochemistry, and electron microscopy represent methods that directly assess lipid content. Yet they require biopsy, which limits multiple measures of the same area and application in children. On the other hand, non-invasive methods such as computerized tomography and magnetic resonance (MR) imaging have been optimized for muscle lipid quantification. However, only proton magnetic resonance spectroscopy (1H-MRS) succeeded in discriminating the IMCL and EMCL compartments. An important principle behind 1H-MRS is that protons resonate at different frequencies depending on the molecules they are linked to. For example, methylene protons in lipids compared with methyl protons in creatine resonate differently by about 1.5 ppm (ppm is a measure of frequency independent of magnetic field strength). MR spectroscopy data is displayed as a graphic where frequency is along the x-axis, allowing identification of metabolites. Peak intensity is plotted on the y-axis. The area under a peak corresponds to its concentration, which is measured by software that compares one peak with another (relative quantitation), or compares peaks with in vitro data (absolute quantitation). Several important metabolites can be identified in the proton spectrum of muscle, including: IMCL (0.9 and 1.3 ppm), EMCL (1.1 and 1.5 ppm), creatine (2.8, 3.0, and 3.2 ppm), trimethylamines (3.2 ppm), and an assumed taurine signal (3.5 ppm; Fig. 1) [5]. A signal of acetylcarnitine can be seen at 2.13 ppm, immediately after strenuous exercise. These data can be gathered by most clinical MR scanners, using pulse sequences that measure metabolites from very specific areas while ignoring the surrounding tissues. The most popular pulse sequence for IMCL measurement is single-voxel short echo time point-resolved spectroscopy (PRESS). Using this technique, Schick et al. [6] and later Boesch et al. [5] demonstrated that IMCL and EMCL showed slightly different frequencies due to contrasting spatial arrangements in muscle. While IMCL droplets are roughly spherical, EMCL are within sheets of adipocytes, causing each compartment to experience the magnetic field differentSkeletal Radiol (2007) 36:607–608 DOI 10.1007/s00256-006-0252-8

Quantification of intramyocellular lipids in obese subjects using spectroscopic imaging with high spatial resolution

Quantification of intramyocellular lipids (IMCL) in obese subjects by single-voxel spectroscopy (SVS) or conventional spectroscopic imaging (SI) often fails due to overlap of IMCL spectral lines by extramyocellular lipids (EMCL), and signal contamination from subcutaneous fat and bone marrow. This study demonstrates that these problems can be solved by high-resolution SI with 128 phase-encoding steps and a read gradient during acquisition. The small voxels obtained in this way facilitated differentiation between EMCL and IMCL. This method offers the possibility of studying different muscle groups and the variation of lipids within one muscle.

Investigation of muscle lipid metabolism by localized one‐ and two‐dimensional MRS techniques using a clinical 3T MRI/MRS scanner

To demonstrate the feasibility of estimating the relative intra- and extramyocellular lipid (IMCL and EMCL) pool magnitudes and calculating the degree of lipid unsaturation within soleus muscle using single-voxel localized one- and two-dimensional (1D and 2D) MR spectroscopy (MRS). Localized 1D point resolved spectroscopy (PRESS) and 2D correlation spectroscopy (L-COSY) were performed in identical locations in the soleus muscle of 10 healthy subjects. A GE 3-T MRI/MRS scanner and a quadrature extremity transmit/receive coil was used. The 1D and 2D MR spectra were used to compute IMCL/creatine (Cr) and EMCL/Cr ratios. In addition to cross peaks between the methyl and methylene protons in the high-field region, the 2D spectra showed cross peaks due to J-coupling between allylic, diallylic methylene pro- tons, and olefinic protons. The cross-peak volume ratios also provided a measure of double bonds, suggesting that this ratio can be used to assess unsaturation within IMCL and EMCL lipid pools. We have demonstrated the feasibility of detecting 2D cross peaks between different groups of IMCL and EMCL, including the unsaturated protons within these two lipids pools. This protocol may be easily extended to study the lipids present in other tissues.

Diagnostic Value of Proton MR Spectroscopy in Peripheral Arterial Occlusive Disease: A Prospective Evaluation

The objective of the present study was to determine the detectability of metabolic alterations in patients with peripheral arterial occlusive disease (PAOD) using proton MR spectroscopy (hydrogen-1 MR spectroscopy). Twenty-seven people were included in this study: 10 patients with PAOD and a pain-free walking distance of less than 200 m served as the patient group and 17 young healthy subjects served as a control group. Hydrogen-1 MR spectroscopy was performed on a 1.5-T scanner using an extremity coil and a point-resolved spectroscopy (PRESS) sequence (TR/TE, 1,500/30; 256 repetitions). For the patient group, a voxel was localized in the gastrocnemius muscle of the diseased leg. The data were processed using standard 1H MR spectroscopy tools. The identification of resonances detected on all MR spectra was made: intramyocellular lipids at 1.2 ppm, extramyocellular lipids at 1.6 ppm, lactate at 4.1 ppm, glucose with two main peaks at 3.4 and 3.8 ppm, choline at 3.2 ppm, and creatine at 3.0 and 3.9 ppm. To avoid operator bias, three spectral intensities were measured after correcting baseline and phase of MR spectra each time. The creatine signal was used as an internal reference; thus, all spectra were scaled relative to creatine. We compared the resultant intensity ratios between the two groups using the Mann-Whitney U test. The lactate-creatine quotient was higher in the patient group, with a ratio of 1.6, than in the control group, with a ratio of 0.6. The glutamate-creatine ratio was higher in the patient group than in the control group (1.3 vs 0.8, respectively). All other ratios were higher in the control group. The best ratio for differentiating between healthy subjects and patients with PAOD was the glucose-lactate ratio. The patient group had a glucose-lactate quotient of 5.4, whereas the control group had a glucose-lactate quotient of 21.5 (p = 0.001). Proton MR spectroscopy has the potential to allow identification of patients who have PAOD on the basis of altered muscle metabolism.

Intramyocellular lipid quantification from 1H long echo time spectra at 1.5 and 3 T by means of the LCModel technique

To introduce a method of independent determination of CH2 and CH3 components of intramyocellular lipids (IMCLs) by using long TE for spectra measurement and LCModel for spectra evaluation, to test this technique in controls and insulin-resistant subjects, and to compare results at 1.5 and 3 T. Eight healthy volunteers and 11 patients with type 2 diabetes mellitus underwent measurement using a 1.5-T MR scanner; six healthy volunteers were measured using a 3-T MR scanner. Spectra from the tibialis anterior muscle were acquired by using a point resolved spectroscopy (PRESS) sequence with the following parameters: TR/TE/ACQ = 2000 msec/270 msec/256. Spectra were processed by LCModel 6.1 software with two types of adopted basis-set. Spectra with good separation of both CH2 and CH3 components of IMCL and extramyocellular lipids (EMCLs) were obtained and the LCModel routine was successfully applied. The reproducibility comparison (N= 7 at 1.5 T vs. N = 5 at 3 T) showed that better results can be obtained at higher B0 values. The comparison of the healthy and insulin-resistant subjects proved that both IMCL_CH2/Cr and IMCL_CH3/Cr ratios significantly differ. Long TE spectroscopy of the human muscle with IMCL quantification using the LCModel technique can detect changes in IMCL levels as well as help in the study of fatty acyl chain composition. Using a higher field strength increased the intra-individual reproducibility by approximately 150%.

Muscle‐associated Triglyceride Measured by Computed Tomography and Magnetic Resonance Spectroscopy

Muscle triglyceride can be assessed in vivo using computed tomography (CT) and 1H magnetic resonance spectroscopy (MRS), two techniques that are based on entirely different biophysical principles. Little is known, however, about the cross-correlation between these techniques and their test-retest reliability. We compared mean muscle attenuation (MA) in soleus and tibialis anterior (TA) muscles measured by CT with intra- and extramyocellular lipids (IMCL and EMCL, respectively) measured by MRS in 51 volunteers (26 to 72 years of age, BMI = 25.5 to 39.3 kg/m2). MA of midthighs was also measured in a subset (n = 19). Test-retest measurements were performed by CT (n = 6) and MRS (n = 10) in separate sets of volunteers. MA of soleus was significantly associated with IMCL (r = -0.64) and EMCL, which by multiple regression analysis was explained mostly by IMCL (p < 0.001) rather than EMCL (beta = -0.010, p = 0.94). Muscle triglyceride was lower in TA than in soleus, and MA of TA was significantly correlated with EMCL (r = -0.40) but not IMCL (r = -0.16). By CT, MA of midthighs was correlated with MA in soleus (r = 0.40, p = 0.07) and whole calf (r = 0.62, p < 0.05). Finally, both MA and IMCL were highly reliable in soleus (coefficient of variation = < 2% and 6.7%, respectively) and less reliable in TA (4% and 10%, respectively). These results support the use of both CT and MRS as reliable methods for assessing skeletal muscle lipid.

Distribution of intramyocellular lipids in human calf muscles as determined by MR spectroscopic imaging.

In this study the distribution of intramyocellular lipids (IMCL) in human calf muscles was determined by 1H-MR spectroscopic imaging (MRSI) measurements. An obstacle for MRSI measurements in the calf, including different muscles, is the inevitable inclusion of regions with high concentrations of extramyocellular lipids (EMCL). This can lead to signal bleeding and consequently to unpredictable overlaps of IMCL resonances with EMCL in voxels of interest. The results of this study show that signal bleeding from EMCL can be substantially reduced in voxels from calf muscles by the application of a lipid extrapolation (LE) procedure (Haupt et al., Magn Reson Med 1996;35:678). The spectra of all voxels located within muscle tissue were fitted, and the metabolite values were assigned to one of 10 different muscles based on image segmentation. Significant IMCL differences between some muscles were obtained, with high values in m. soleus and two to three times lower values in the tibialis anterior, tibialis posterior, and gastrocnemius muscles. In addition to gross differences between muscles, significant intersubject differences were observed in both IMCL content and distribution over different muscles. A significant correlation between fiber orientation (obtained from orientation-dependent dipolar coupling of creatine and taurine resonances) and IMCL content was found, indicating that IMCL content is directly correlated to biomechanical properties.

Rat Myocellular and Perimysial Intramuscular Triacylglycerol: A Histological Approach

There is controversy as to the use of intramuscular triacylglycerol (IMTAG) during exercise and to whether endurance training increases its utilization, despite the various methodologies used to address these questions. We used a histological-morphometrical approach to study the relative contribution of the two compartments of IMTAG storage, intramyocellular, and perimysial adipocytes, during exercise in sedentary and endurance-trained rats. After osmium impregnation, the soleus (SOL) and gastrocnemius (GAS) were studied under light and electron microscopy. IMTAG content (after Triton WR1339 treatment or not) and 14C-oleate incorporation into the muscles were studied. In GAS, training, but not exercise alone, decreased extramyocellular lipid (P < 0.001 vs sedentary), an effect not found for SOL. Both muscles presented reduced lipid inclusion number (P < 0.001) and area (P < 0.05), immediately after exercise in sedentary and trained rats. For SOL, a greater number (P < 0.001 vs sedentary) of inclusions was found 24 h after exercise in trained rats. Triton WR1339 treatment decreased IMTAG content 12 h after exercise in SOL (but not in GAS), in sedentary (33%), and trained rats (52%). The multi-analytical approach adopted allowed the discernment between the IMTAG compartments and provided evidence for an effect of training upon storage of lipid in perimysial adipocytes in rat gastrocnemius, as well as clearly showed that the IMTAG mobilized during submaximal exercise in sedentary and trained rats derives from intramyocellular lipid, both in SOL and GAS. Moreover, the reposition of these stores 12 h after exercise was shown to be different in GAS and SOL, as plasma triacylglycerol clearly contributed to the process only in the latter, possibly reflecting the differences in lipoprotein lipase activity in the muscles reported by others.