Abnormal lipid metabolism in skeletal muscle tissue of patients with muscular dystrophy: In vitro,high-resolution NMR spectroscopy based observation in early phase of the disease

PurposeQualitative (assignment of lipid components) and quantitative (quantification of lipid components) analysis of lipid components were performed in skeletal muscle tissue of patients with muscular dystrophy in early phase of the disease as compared to control/normal subjects.MethodsProton nuclear magnetic resonance (NMR) spectroscopy based experiment was performed on the lipid extract of skeletal muscle tissue of patients with muscular dystrophy in early phase of the disease and normal individuals for the analysis of lipid components [triglycerides, phospholipids, total cholesterol and unsaturated fatty acids (arachidonic, linolenic and linoleic acid)]. Specimens of muscle tissue were obtained from patients with Duchenne muscular dystrophy (DMD) [n = 11; Age, Mean ± SD; 9.2 ± 1.4 years; all were males], Becker muscular dystrophy (BMD) [n = 12; Age, Mean ± SD; 21.4 ± 5.0 years; all were males], facioscapulohumeral muscular dystrophy (FSHD) [n = 11; Age, Mean ± SD; 23.7 ± 7.5 years; all were males] and limb girdle muscular dystrophy-2B (LGMD-2B) [n = 18; Age, Mean ± SD; 24.2 ± 4.1 years; all were males]. Muscle specimens were also obtained from [n = 30; Mean age ± SD 23.1 ± 6.0 years; all were males] normal/control subjects.ResultsAssigned lipid components in skeletal muscle tissue were triglycerides (TG), phospholipids (PL), total cholesterol (CHOL) and unsaturated fatty acids (arachidonic, linolenic and linoleic acid)]. Quantity of lipid components was observed in skeletal muscle tissue of DMD, BMD, FSHD and LGMD-2B patients as compared to control/normal subjects. TG was significantly elevated in muscle tissue of DMD, BMD and LGMD-2B patients. Increase level of CHOL was found only in muscle of DMD patients. Level of PL was found insignificant for DMD, BMD and LGMD-2B patients. Quantity of TG, PL and CHOL was unaltered in the muscle of patients with FSHD as compared to control/normal subjects. Linoleic acids were significantly reduced in muscle tissue of DMD, BMD, FSHD and LGMD-2B as compared to normal/control individuals.ConclusionsResults clearly indicate alteration of lipid metabolism in patients with muscular dystrophy in early phase of the disease. Moreover, further evaluation is required to understand whether these changes are primary or secondary to muscular dystrophy. In future, these findings may prove an additional and improved approach for the diagnosis of different forms of muscular dystrophy.     

Effect of creatine monohydrate in improving cellular energetics and muscle strength in ambulatory Duchenne muscular dystrophy patients: a randomized,placebo-controlled P MRS study

Randomized, placebo-controlled single blinded study was carried out to evaluate the effect of oral creatine supplementation on cellular energetics, manual muscle test (MMT) score and functional status in steroid-naive, ambulatory boys suffering with Duchenne muscular dystrophy (DMD; n=33). Eighteen patients received creatine monohydrate (Cr; 5 g/day for 8 weeks), while 15 received placebo (500 mg of vitamin C). Phosphorus metabolite ratios were determined from the right calf muscle of patients using phosphorus magnetic resonance spectroscopy (³¹P MRS) both prior to (baseline) and after supplementation of Cr or placebo. In addition, metabolite ratios were determined in normal calf muscle of age and sex matched controls (n=8). Significant differences in several metabolite ratios were observed between controls and DMD patients indicating a lower energy state in these patients. Analysis using analysis of covariance adjusted for age and stature showed that the mean phosphocreatine (PCr)/inorganic phosphate (Pi) ratio in patients treated with Cr (4.7; 95% CI; 3.9–5.6) was significantly higher (P=.03) compared to the placebo group (3.3; 95% CI; 2.5–4.2). The mean percentage increase in PCr/Pi ratio was also more in patients <7 years of age compared to older patients after Cr supplementation indicating variation in therapeutic effect with the age. In the placebo group, significant reduction in PCr/Pi (P=.0009), PCr/t-ATP (P=.05) and an increase in phosphodiester (PDE)/PCr ratios was observed after supplementation. Further, in the placebo group, patients <7 years showed reduction of PCr/t-ATP and Pi/t-ATP compared to older patients (>7 years), after supplementation. These results imply that the significant difference observed in PCr/Pi ratio between the Cr and the placebo groups after supplementation may be attributed to a decrease of PCr in the placebo group and an increase in PCr in the Cr group. Changes in MMT score between the two groups was significant (P=.04); however, no change in functional scale (P=.19) was observed. Parents reported subjective improvement on Cr supplementation versus worsening in placebo (P=.02). Our results indicated that Cr was well tolerated and oral Cr significantly improved the muscle PCr/Pi ratio and preserved the muscle strength in short term. However, this study provides no evidence that creatine will prove beneficial after long-term treatment, or have any positive effect on patient lifespan.     

Double quantum filtered (1)H NMR spectroscopy enables quantification of lactate in muscle

In this study we address the question of quantification of muscle lactate using double quantum filtered (DQF) (1)H NMR spectroscopy where dipolar and scalar coupled spectra are acquired. For this, lactate content in muscle samples was independently determined using a conventional enzymatic assay and DQF, (1)H NMR spectroscopy. NMR quantification of lactate relied on comparison of muscle spectra with similarly acquired spectra of standard lactate solutions. Transverse relaxation, T(2), and dipolar coupling effects were investigated at two different orientations of muscle fibers relative to B(o) and at various lactate concentrations. In all cases, we found a biexponential T(2) decay of the lactate methyl signal with a long T(2) of 142 ms (+/-8 ms, n=24) and a short T(2) of 37 ms (+/-6 ms, n=24). Lactate content of muscle determined by NMR spectroscopy agreed with the results obtained from enzymatic assays of the same samples provided that T(2) effects as well as the presence of both scalar and dipolar coupling interactions of lactate in muscle were taken into account.     

Brain metabolite changes in alcoholism: An in vivo proton magnetic resonance spectroscopy (MRS) study

Image-guided, single voxel, localized proton magnetic resonance (MR) spectroscopy was performed to assess the brain metabolite changes in 10 (n = 10) alcoholic patients in the frontal lobe, cerebellum, and thalamus regions. The spectra obtained were characterized by a reduced N-acetyl-aspartate (NAA) to choline (Cho) (p < .01) and NAA to total creatine (Cr + PCr) (p < .01) ratios relative to age-matched (n = 27) controls. These decreased ratios correspond to depleted concentration of the metabolite levels such as NAA and Cho. Reduction of NAA is consistent with the neuronal loss while reduction in Cho suggests significant changes in the membrane lipids of alcoholics.     

1H NMR study of proton dynamics in (NH4)3H(SO4)2

Proton dynamics in (NH₄)₃H(SO₄)₂ has been studied by means of ¹H solid-state NMR. The ¹H magic-angle-spinning (MAS) NMR spectra were traced at room temperature (RT) and at Larmor frequency of 400.13 MHz. ¹H static NMR spectra were measured at 200.13 MHz in the range of 135–490 K. ¹H spin-lattice relaxation times, T₁, were measured at 200.13 and 19.65 MHz in the ranges of 135–490 and 153–456 K, respectively. The ¹H chemical shift for the acidic proton (14.7 ppm) indicates strong hydrogen bonds. In phase III, NH₄⁺ reorientation takes place; one type of NH₄⁺ ions reorients with an activation energy (E_a) of 14 kJ mol^− 1 and the inverse of a frequency factor (τ₀) of 0.85 × 10^− 14 s. In phase II, a very fast local and anisotropic motion of the acidic protons takes place. NH₄⁺ ions start to diffuse translationally, and no proton exchange is observed between NH₄⁺ ions and the acidic protons. In phase I, both NH₄⁺ ions and the acidic protons diffuse translationally. The acidic protons diffuse with parameters of E_a = 27 kJ mol^− 1 and τ₀ = 4.2 × 10^− 13 s. The translational diffusion of the acidic protons is responsible for the macroscopic proton conductivity, as the NH₄⁺ translational diffusion is slow and proton exchange between NH₄⁺ ions and the acidic protons is negligible.     

In vivo proton magnetic resonance spectroscopy (MRS) study of post polio residual paralysis (PPRP) patients

In-vivo proton MR spectroscopy carried out on post polio residual paralysis (PPRP) patients indicate that the presence or absence of intra-myocellular lipids (IMCL) is related to the severity of the paralysis. It is observed that mildly paralyzed patients are comparable (p > 0.05) with the control subjects in relation to the presence of IMCL, while moderate and severely paralysed patients are comparable (p > 0.05) in relation to the absence of IMCL. In addition, there is reduction or complete absence of creatine, carnitine and choline metabolites in severely paralyzed patients. The ability to detect noninvasively the subtle differences in in vivo, the lipid compartments of muscle may prove to be a valuable tool in understanding the pathogenesis of muscle diseases. This could open up the possibilities in designing effective rehabilitative exercise programs or development of new drug therapies.     

Chain order in filled SBR elastomers: a proton multiple-quantum NMR study

A series of cross-linked styrene-butadiene rubbers (SBR) filled with different amounts of carbon black and silica are investigated by proton multiple-quantum nuclear magnetic resonance (NMR). The method yields reliable information on residual dipolar couplings and their distribution, which in turn are related to local chain order and the effective cross-link density in these systems. Fundamental differences between the response of a linear precursor, which undergoes reptational motion, and vulcanized SBR are discussed. For the latter, it is found that the average chain order parameter as well as its distribution does not change significantly with the amount and the type of filler. This is in surprising contrast to recent results from Hahn-echo relaxometry applied to the same samples, which indicated a significant filler effect on the cross-link density.     

Probing proteins in solution by (129)Xe NMR spectroscopy

The interaction of xenon with different proteins in aqueous solution is investigated by (129)Xe NMR spectroscopy. Chemical shifts are measured in horse metmyoglobin, hen egg white lysozyme, and horse cytochrome c solutions as a function of xenon concentration. In these systems, xenon is in fast exchange between all possible environments. The results suggest that nonspecific interactions exist between xenon and the protein exteriors and the data are analyzed in term of parameters which characterize the protein surfaces. The experimental data for horse metmyoglobin are interpreted using a model in which xenon forms a 1:1 complex with the protein and the chemical shift of the complexed xenon is reported (Locci et al., Keystone Symposia "Frontiers of NMR in Molecular Biology VI", Jan. 9--15, 1999, Breckenridge, CO, Abstract E216, p. 53; Locci et al., XeMAT 2000 "Optical Polarization and Xenon NMR of Materials", June 28--30, 2000, Sestri Levante, Italy, p. 46).     

Similarity in the metabolic profile in macroscopically involved and un-involved colonic mucosa in patients with inflammatory bowel disease: an in vitro proton (H) MR spectroscopy study

Background

The histological extent of inflammatory bowel disease (IBD) is greater than that evident by colonoscopic evaluation. We hypothesized that metabolic profile in macroscopically un-involved colonic mucosa in IBD is similar to that of controls with healthy colon. We thus assessed the differences in metabolic profile in macroscopically involved and un-involved colonic mucosa of IBD patients to further substantiate the extent of disease.

Patients and Methods

Colonic mucosal biopsies were obtained and snap frozen from both the macroscopically un-involved and involved colonic mucosa of IBD patients and macroscopically normal colonic mucosa of controls and were subjected to in-vitro high-resolution proton (¹H) magnetic resonance (MR) spectroscopy and the concentrations of metabolites were determined.

Results

Thirty-two metabolites were assigned in the proton MR spectrum of colonic mucosa of IBD patients. The concentrations of amino acids (isoleucine, leucine, valine, arginine, lysine, glutamine/glutamate, alanine), membrane metabolites (choline, glycerophosphorylcholine/phosphorylcholine), glycolytic product (lactate) and short chain fatty acid (formate) were significantly lower while significantly high level of glucose were observed in the macroscopically un-involved colonic mucosa of IBD patients compared to the macroscopically normal mucosa of controls. There was no significant difference in the concentrations of metabolites in macroscopically involved and un-involved colonic mucosa of IBD patients.

Conclusions

The metabolic profile in macroscopically un-involved colonic mucosa of IBD patients is similar to that of macroscopically involved mucosa but different from colonic mucosa of controls. This suggests that even macroscopically un-involved colonic mucosa is metabolically abnormal and may explain the increase in extent of disease with time.     

Metabolism of the colonic mucosa in patients with inflammatory bowel diseases: an in vitro proton magnetic resonance spectroscopy study

Metabolism of the colonic mucosa of patients with ulcerative colitis (UC; n=31) and Crohn's disease (CD; n=26) and normal mucosa (control, n=26) was investigated using in vitro high-resolution proton magnetic resonance spectroscopy. Of the 31 UC patients, 20 were in the active phase and 11 were in the remission phase of the disease. Out of 26 CD patients, 20 were in the active phase, while 6 were in the remission phase of the disease. Twenty-nine metabolites were assigned unambiguously in the perchloric acid extract of colonic mucosa. In the active phase of UC and CD, significantly lower (P相似文献   

Natural abundance (17)O NMR spectroscopy of rat brain in vivo

Oxygen is an abundant element that is present in almost all biologically relevant molecules. NMR observation of oxygen has been relatively limited since the NMR-active isotope, oxygen-17, is only present at a 0.037% natural abundance. Furthermore, as a spin 5/2 nucleus oxygen-17 has a moderately strong quadrupole moment which leads to fairly broad resonances (T(2)=1-4 ms). However, the similarly short T(1) relaxation constants allow substantial signal averaging, whereas the large chemical shift range (>300 ppm) improves the spectral resolution of (17)O NMR. Here it is shown that high-quality, natural abundance (17)O NMR spectra can be obtained from rat brain in vivo at 11.74 T. The chemical shifts and line widths of more than 20 oxygen-containing metabolites are established and the sensitivity and potential for (17)O-enriched NMR studies are estimated.     

Differential mode delay (DMD) in graded-index multimode fiber: effect of DMD on bandwidth tuned by restricted launch conditions

The bandwidth behavior of graded-index multimode fibers (GI-MMFs) for different launching conditions is investigated to understand and characterize the effect of differential mode delay. In order to reduce the launch-power distribution the near field of a single-mode fiber is used to produce a controlled restricted launch. The baseband response is measured by observing the broadening of a narrow input pulse (time-domain measurement). The paper verifies the degradation in bandwidth due to profile distortion by scanning the spot of the single-mode fiber with a transversal offset from the center of the test sample. In addition, the impact of the launch-power distribution tuned by different spot-size diameters is demonstrated. Measurements were taken on ‘older’ 50-μm and 62.5-μm GI-MMFs as well as on laser-performance-optimized fibers more recently developed. Received: 12 November 2001 / Final version: 26 June 2002 / Published online: 25 September 2002 RID="*" ID="*"Corresponding author. Fax: +49-781/205-242, E-mail: opto@fh-offenburg.de     

Detection of taurine in biological tissues by (33)S NMR spectroscopy.

The potential of (33)S NMR spectroscopy for biochemical investigations on taurine (2-aminoethanesulfonic acid) is explored. It is demonstrated that (33)S NMR spectroscopy allows the selective and unequivocal identification of taurine in biological samples. (33)S NMR spectra of homogenated and intact tissues are reported for the first time, together with the spectrum of a living mollusc. Emphasis is placed on the importance of choosing appropriate signal processing methods to improve the quality of the (33)S NMR spectra of biological tissues.     

Sensitivity and resolution in proton solid-state NMR at intermediate deuteration levels: quantitative linewidth characterization and applications to correlation spectroscopy

We present a systematic study of proton linewidths in rigid solids as a function of sample spinning frequency and proton density, with the latter controlled by the ratio of protonated and perdeuterated model compounds. We find that the linewidth correlates more closely with the overall proton density (rho(H)) than the size of local clusters of (1)H spins. At relatively high magic-angle spinning (MAS) rates, the linewidth depends linearly upon the inverse MAS rate. In the limit of infinite spinning rate and/or zero proton concentration, the linewidth extrapolates to a non-zero value, owing to contributions from scalar couplings, chemical shift dispersion, and B(0) field inhomogeneity. The slope of this line depends on the overall concentration of unexchangeable protons in the sample and the spinning rate. At up to 30% protonation levels ( approximately 2 (1)H/100A(3)), proton detection experiments are demonstrated to have a substantial (2- to 3-fold) sensitivity gain over corresponding (13)C-detected experiments. Within this range, the absolute sensitivity increases with protonation level; the optimal compromise between sensitivity and resolution is in the range of 20-30% protonation. We illustrate the use of dilute protons for polarization transfer to and from low-gamma spins within 5A, and to be utilized as both magnetization source and detection spins. The intermediate protonation regime enhances relaxation properties, which we expect will enable new types of (1)H correlation pulse sequences to be implemented with improved resolution and sensitivity.     

15N NMR of glycine metabolism in liver,muscle and kidney of rats using a new pulsed overhauser technique (POE)

Following intravenous injections of [¹⁵N]glycine (97 at.% ¹⁵N, 400 mg in 1.5 ml isotonic saline) to Lewis rats several organs were excised. The time courses of the ¹⁵N NMR spectral (40.55 MHz) were measured at 37°C after cold (4°C) or warm (37°C) storage. The application of the Overhauser POE technique yielded signal enhancement by a factor of -2.7 and -1.3 in sceletal and heart muscle, respectively, at 37°C, but no enhancement in liver and kidney.

In the time course of liver measurements at 37°C, intermediate metabolic products of glycine were observed, such as serine, glutamine, alanine, and tryptophane.     

In vivo (31)P MRS study of skeletal muscle metabolism in patients with postpolio residual paralysis

The muscle metabolism of at-rest patients with varying degrees of postpolio residual paralysis (PPRP) was studied and compared with that of controls using in vivo phosphorus magnetic resonance spectroscopy. The phosphocreatine (PCr)/inorganic phosphate (Pi) and PCr/adenosine triphosphate ratios were lower in patients than in controls. Reduction in PCr/Pi suggests abnormalities in oxidative phosphorylation. A significant increase was observed in the phosphomonoester/PCr ratio in patients, indicating the accumulation of intermediary compounds of the glycolytic pathway. Furthermore, the phosphodiester/PCr ratio was also significantly increased in patients. In general, the observed changes in metabolite ratios were found to be related to the degree of residual paralysis, suggesting that metabolic changes are secondary to chronic neurogenic processes. These metabolic alterations appear to be the possible cause of energy deficit and underlying muscle fatigue in PPRP patients. The present results provide an insight into the metabolic impairment and degree of muscle damage in patients with PPRP.     

Initial oxidation of the Mg(0001) surface,an electron spectroscopy study

The initial oxidation of Mg(0001) has been studied using AES (Auger electron spectroscopy), LEED (low energy electron diffraction), and EELS (electron energy loss spectroscopy). The oxidation proceeds through different stages; first oxygen atoms are incorporated to chemisorption sites below the top layer magnesium. This chemisorption phase is followed by the formation of an oxide layer. The oxide layer covers the Mg surface after an oxygen exposure of ～ 10 L O₂. After this exposure the bulk-like MgO formation slowly increases the oxide thickness. The oxide layer formed for exposures up to ≤ 10 L O₂ gives rise to a diffuse LEED pattern of the same symmetry as the original “clean” LEED pattern; the possibility of an epitaxial oxide formation at this stage is discussed.     

Water and alcohol(s): what's the difference? A proton NMR and DFT study of hetero‐association with pyridine

Hetero‐association of water and some simple aliphatic alcohols with pyridine in benzene has been studied by ¹H nuclear magnetic resonance (NMR) spectroscopy at very low donor concentration, where self‐association is negligible. Association constants for the formation of 1:1 and 2:1 pyridine:water complexes can then be determined without recourse to ad hoc computer programmes. That for association of a second pyridine with water is about 10 times lower than for the first. Reaction parameters for the first association with water are very similar to those for the alcohols, whereas the reaction enthalpy for the second association is somewhat smaller. The chemical shift of the OH protons and the H? C? O? H coupling constants for alcohols at high dilution in benzene are almost identical with gas‐phase data. The change in chemical shift upon association with pyridine correlates with the free energy of the reaction. Quantum mechanical calculations [BPE0 functional, 6‐311+G(d,p) basis set and a polarized continuum model of the solvent (IEFPCM)] have been run on complexes of pyridine with water, both 1:1 and 2:1, and with four alcohols. Calculated reaction enthalpies are in qualitative and, in some cases, almost quantitative agreement with the experimental data. The association constants for 1:1 complexation of pyridine with alcohols follow a rough Taft correlation in terms of polar substituent constants. Substituent size, even in the case of very bulky groups, seems to be unimportant. Copyright © 2008 John Wiley & Sons, Ltd.