Proton nuclear magnetic resonance of human muscle extracts

High resolution proton nuclear magnetic resonance (NMR) spectra of normal and diseased human muscle extracts were recorded at 470 MHz. Resonances from lactic acid, creatine, glucose, ribose, purine and pyrimidine bases were identified. The longitudinal relaxation times of these resonances were determined to allow quantitation of muscle metabolites. With aid of a standardized reference capillary, inserted into the NMR tube containing the muscle extracts, the lactic acid and total creatine content of the extracts was determined. After 5 h of incubation at 37 degrees C, normal muscles contained on average 103 mumol lactic acid and 36 mumol creatine/173 mg of noncollagenous protein, equivalent to 1.0 g of fresh muscle. The lactic acid and creatine contents decreased slightly in scoliosis and idiopathic scoliosis and they decreased significantly in cerebral palsy. In an extract of a patient whose illness was diagnosed as 'scoliosis' no creatine was present, and in an extract of a patient with unknown diagnosis the creatine content was reduced to 2 mumol/173 mg of noncollagenous protein. The short time (1.7 sec to 6.5 min) and the small amount of tissue (300 mg) needed for an analysis add to the potential of proton NMR as a new technique for the characterization of muscular diseases.     

Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance.

31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses. The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted. Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water: (a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water; (c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP. The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2.     

Study of the conversion of GDP-mannose into GDP-fucose in Nereids: a biochemical marker of oocyte maturation

The high-resolution proton nuclear magnetic resonance spectra of carp hemoglobin have been compared to those of human normal adult hemoglobin. Carp deoxy and carbonmonoxy hemoglobins in the deoxy-type quaternary state exhibit two downfield exchangeable proton resonances as compared to four seen in human normal adult deoxyhemoglobin. This suggests that two of the hydrogen bonds present in human normal adult deoxyhemoglobin are absent or occur in very different environments in carp hemoglobin. One of the exchangeable proton resonances of carp hemoglobin, while present in the deoxy-type quaternary state of the carbonmonoxy and deoxy derivatives, is absent in the oxy-type quaternary state of both, in agreement with the assignments of these quaternary structures by other methods. The ring-current-shifted proton resonances (sensitive tertiary structural markers) of carp carbonmonoxyhemoglobin are substantially different from those of human normal adult hemoglobin. The aromatic proton resonance region of carp hemoglobin has fewer resonances than that of human normal adult hemoglobin, consistent with its much reduced histidine content. The hyperfine-shifted proximal histidyl NH-exchangeable proton resonances of carp hemoglobin suggest that during the transition from the oxy to the deoxy quaternary structure, there is a greater alteration in the heme pocket of one type of subunits (presumably the beta chain) than that in the other subunit. The present results suggest that there are differences in both tertiary and quaternary structures between carp and human normal adult hemoglobins which could contribute to the great differences in the functional properties between these two proteins.     

A proton NMR study of a DNA dumb-bell structure with hairpin loops of only two nucleotides: d(CACGTGTGTGCGTGCA).

One- and two-dimensional nuclear magnetic resonance (NMR) experiments were used to study the conformation of the DNA hexadecanucleotide d(CACGTGTGTGCGTGCA) in aqueous solution. NMR spectra were recorded for the compound in D2O and in H2O/D2O (90/10) over the temperature range 1 degree C-60 degrees C. Assignments of imino proton resonances and of non-exchangeable proton resonances (except for some H4', H5' and H5" resonances) are given. The 1H-NMR spectra indicate that below about 20 degrees C, the compound exists as a single monomolecular species. Between 20 degrees C and 55 degrees C the oligonucleotide occurs as a mixture of structures in fast exchange on the NMR time scale, except for the temperature region 30 degrees - 34 degrees C, where substantial line broadening indicates intermediate exchange; above 60 degrees C the single strand predominates. The imino proton spectra, chemical shift values, and scalar coupling and NOE data reveal that the monomeric form, which is exclusively present below 20 degrees C, consists of a structure with a B-DNA double helix region of six base pairs, both ends of which are closed by hairpin loops of only two nucleotides, giving the molecule a dumbbell-like structure: [sequence: see text].     

Incorporation and utilization of [3-13C]lactate and [1,2-13C]acetate by rat skeletal muscle.

Skeletal muscle can utilize many different substrates, and traditional methodologies allow only indirect discrimination between oxidative and nonoxidative uptake of substrate, possibly with contamination by metabolism of other internal organs. Our goal was to apply 1H- and 13C-nuclear magnetic resonance spectroscopy to monitor the patterns of [3-13C]lactate and [1,2-13C]acetate (model of simple carbohydrates and fats, respectively) utilization in resting vs. contracting muscle extracts of the isolated perfused rat hindquarter. Total metabolite concentrations were measured by using NADH-linked fluorometric assays. Fractional oxidation of [3-13C]lactate was unchanged by contraction despite vascular endogenous lactate accumulation. Although label accumulated in several citric acid cycle (CAC) intermediates, contraction did not increase the concentration of CAC intermediates in any muscle extracts. We conclude that 1) the isolated rat hindquarter is a viable, well-controlled model for measuring skeletal muscle 13C-labeled substrate utilization; 2) lactate is readily oxidized even during contractile activity; 3) entry and exit from the CAC, via oxidative and nonoxidative pathways, is a component of normal muscle metabolism and function; and 4) there are possible differences between gastrocnemius and soleus muscles in utilization of nonoxidative pathways.     

In Vitro Metabolomic Approach to Hippocampal Neurodegeneration Induced by Trimethyltin

Search for indicators of neurodegenerative disorders is a hot topic where much research remains to be done. Our aim was to determine proton nuclear magnetic resonance (¹H-NMR) spectra of brain metabolites in the trimethyltin (TMT) model of neurodegeneration. Male Wistar rats were subjected to TMT or saline and were sacrificed on day 3 or 24 after administration. ¹H-NMR spectrum was measured on the 600 MHz Varian VNMRS spectrometer in nano-probe in the volume of 40 μl of hippocampal extracts. TMT administration resulted in reduction of the hippocampal weight on day 24. Of the sixteen identified metabolite spectra, decreased aspartate and increased glutamine contents were observed in the initial asymptomatic stage of neurodegeneration on day 3 in hippocampal extracts of TMT exposed rats compared to sham animals. Increased myo-inositol content was observed on day 24. The presented data provide further knowledge about this experimental model and putative indicators of neuronal damage.     

Proton magnetic resonance spectroscopy of leech muscle and nervous system

1. Proton nuclear magnetic resonance spectroscopy (1H NMR) was used to measure the major intracellular metabolites in perchloric acid extracts of the Macrobdella decora muscle and nervous systems and the Oryctolagus cuniculus cerebrum. 2. Acetate, alanine, choline, glutamate, inositol, and lactate were assigned in the spectrum of leech ventral cord, leech muscle, and rabbit cerebrum. 3. Hirudonine and propionate were clearly observed only in the spectrum of leech muscle. 4. Creatine, N-acetyl aspartate, gamma aminobutyric acid, aspartate, and taurine, distinctive components of spectra of the mammalian cerebrum, were not seen in the invertebrate spectra. 5. 1H NMR spectroscopy provides a simple and rapid means of characterizing the major organic metabolites found in leech muscle and nervous tissues.     

1H-NMR studies of the intracellular water of skeletal muscle fibers under various physiological conditions.

Intracellular water of frog skeletal muscle fibers has been studied under various physiological conditions by use of the 1H-nuclear magnetic resonance (NMR) technique. 1H-NMR spectra of muscle fibers had single peaks derived from the intracellular water. The spectra changed in a characteristic fashion when the fiber axis was aligned at various angles relative to the magnetic field of the NMR magnet. Further, the relaxation rates of the 1H-NMR spectra changed depending on the water content of muscle fibers, and in association with contraction and rigor formation of muscle fibers. The obtained results indicate that the intracellular water of muscle fibers is structured and aligned along the myofilaments, and further that the state of the intracellular water changes with physiological conditions.     

Detection by proton nuclear magnetic resonance of elevated lactate concentration in serums from patients with malignant tumors.

Proton nuclear magnetic resonance (1H-NMR) spectra of the serum specimens from patients with malignant tumors were compared with those from presumably healthy persons. We found that 87% of serum specimens from the patients yielded a common proton signal, which was ascribed to the methyl protons of lactic acid; whereas only 9% of serum specimens from the healthy persons tested gave this signal. On the basis of these results we concluded that the lactate level in the serum can be used as a criterion for the diagnosis of cancer in humans and that the determination of lactate concentration in the serum is easily performed by means of 1H-NMR.     

Magnetic resonance spectroscopy and metabolism. Applications of proton and 13C NMR to the study of glutamate metabolism in cultured glial cells and human brain in vivo.

Nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of cells from the central nervous system both in vitro on perchloric acid extracts obtained either from cultured tumoral cells (C6 rat glioma) or rat astrocytes in primary culture, and in vivo within the human brain. Analysis of carbon 13 NMR spectra of perchloric acid extracts prepared from cultured cells in the presence of NMR [1-13C] glucose as substrate allowed determination of the glutamate and glutamine enrichments in both normal and tumoral cells. Preliminary results indicated large changes in the metabolism of these amino acids (and also of aspartate and alanine) in the C6 cell as compared to its normal counterpart. Localized proton NMR spectra of the human brain in vivo were obtained at 1.5 T, in order to evaluate the content of various metabolites, including glutamate, in peritumoral edema from a selected volume of 2 x 2 x 2 cm3. N-acetyl aspartate, glutamate, phosphocreatine, creatine, choline and inositol derivative resonances were observed in 15 min spectra. N-acetyl-aspartate was found to be at a lower level in contrast to glutamate which was detected at a higher level in the injured area as compared to the contralateral unaffected side.     

Single syllable tongue motion analysis using tagged cine MRI

The complicated muscle activity of the human tongue and the resultant surface shapes can give us important clues about speech motor control and pathological tongue motion. This study uses tagged magnetic resonance imaging to provide a 2D surface deformation analysis of the tongue, as well as a 4D compression–expansion analysis, during utterances of four different syllables (/ba/, /ta/, /sha/ and /ga/). All speech tasks were performed several times to confirm the repeatability of the motion analysis. The results showed that the tongue has unique motion patterns for utterances of different syllables, and these differences, which may not be observed by a simple surface analysis, can be examined thoroughly by a 4D motion model-based analysis of the tongue muscles.     

Structure of methylmenaquinone-7 isolated fromAlteromonas putrefaciens IAM 12079

The structure of methylmenaquinone-7, a major component of methylmenaquinones isolated fromAlteromonas putrefaciens IAM 12079, was determined by comparing the proton nuclear magnetic resonance (¹H-NMR) spectrum of its chromenyl derivative with those of menachromenyl acetate prepared from menaquinone-7 and phyllochromenyl acetate. Based on the numbering system used for naphthopyrane nucleus, proton signals of the 7- and 10-position for chromenyl derivatives were assigned from the signals on the¹H-NMR spectra phyllochromenyl acetate and phyllochromenol. As a result, the chemical structure of methylmenaquinone-7 was determined as 2,8-dimethyl-3-farnesylgeranygeranyl-1,4-naphthoquinone.     

High-resolution proton nuclear magnetic resonance characterization of seminolipid from bovine spermatozoa

The high-resolution one- and two-dimensional proton nuclear magnetic resonance (1H-NMR) characterization of seminolipid from bovine spermatozoa is presented. The 1H-NMR data was confirmed by gas-liquid chromatography-mass spectrometric analysis of the partially methylated alditol acetates of the sugar unit, mild alkaline methanolysis of the glyceryl ester, mobility on normal phase and diphasic thin-layer chromatography (HPTLC), and fast atom bombardment mass spectrometry (FAB-MS). The structure of the molecule corresponds to 1-O-hexadecyl-2-O-hexadecanoyl-3-O-beta-D-(3'-sulfo)-galactopyranosyl- sn-glycerol.     

Effects of vitamin B<Subscript>12</Subscript> on murine embryonic dexamethasone-induced cleft palates using <Superscript>1</Superscript>H-NMR-based metabonomic analysis

Metabonomics using proton nuclear magnetic resonance (¹H-NMR) spectroscopy and multivariate data analysis of blood plasma samples can be used to characterize metabolic differences between healthy and diseased organisms, which can reveal important information about the causes of the disease. Here we evaluated whether the ¹H-NMR-based metabonomic method can detect differences in blood plasma between healthy pregnant mice (outbred C57BL/6J strain) and pregnant mice injected with dexamethasone (Dex) to induce cleft palate. Both groups were injected with vitamin B₁₂. We found some metabolic differences from the “outliers” among the mice, indicating that vitamin B₁₂ protected against Dex-induced Cleft lip with or without palate (CLP) formation.     

The value of enzyme histochemical techniques in the classification of fibre types of human skeletal muscle

Summary In the present investigation the results of a lead salt technique and two calcium salt techniques for the demonstration of the activity of myosin adenosine triphosphatase in sections of both normal and pathological human skeletal muscle specimens are compared. It was seen that the histochemical results obtained by the different techniques are similar, especially with regard to the identification of fibre-types.It can be clearly stated, that the alkaline phosphatase activity present in muscle fibres of diseased skeletal muscles revealed only a very slight activity with the substrate ATP, so the alkaline phosphatase activity in general did not disturb the reliability of the different myosin ATPase techniques.Moreover it was found that the presence of the mitochondrial Ca²⁺-ion activated ATPase with a high pH-optimum in muscle fibres did not give rise to faulty results.From studies with dinitrophenol it can be concluded that this substance activates the myosin ATPase present in type I fibres especially.     

Early metabolic changes measured by 1H MRS in healthy and dystrophic muscle after injury

Skeletal muscle injury is often assessed by clinical findings (history, pain, tenderness, strength loss), by imaging, or by invasive techniques. The purpose of this work was to determine if in vivo proton magnetic resonance spectroscopy ((1)H MRS) could reveal metabolic changes in murine skeletal muscle after contraction-induced injury. We compared findings in the tibialis anterior muscle from both healthy wild-type (WT) muscles (C57BL/10 mice) and dystrophic (mdx mice) muscles (an animal model for human Duchenne muscular dystrophy) before and after contraction-induced injury. A mild in vivo eccentric injury protocol was used due to the high susceptibility of mdx muscles to injury. As expected, mdx mice sustained a greater loss of force (81%) after injury compared with WT (42%). In the uninjured muscles, choline (Cho) levels were 47% lower in the mdx muscles compared with WT muscles. In mdx mice, taurine levels decreased 17%, and Cho levels increased 25% in injured muscles compared with uninjured mdx muscles. Intramyocellular lipids and total muscle lipid levels increased significantly after injury but only in WT. The increase in lipid was confirmed using a permeable lipophilic fluorescence dye. In summary, loss of torque after injury was associated with alterations in muscle metabolite levels that may contribute to the overall injury response in mdx mice. These results show that it is possible to obtain meaningful in vivo (1)H MRS regarding skeletal muscle injury.     

Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics

Although a wide range of risk factors for coronary heart disease have been identified from population studies, these measures, singly or in combination, are insufficiently powerful to provide a reliable, noninvasive diagnosis of the presence of coronary heart disease. Here we show that pattern-recognition techniques applied to proton nuclear magnetic resonance (1H-NMR) spectra of human serum can correctly diagnose not only the presence, but also the severity, of coronary heart disease. Application of supervised partial least squares-discriminant analysis to orthogonal signal-corrected data sets allows >90% of subjects with stenosis of all three major coronary vessels to be distinguished from subjects with angiographically normal coronary arteries, with a specificity of >90%. Our studies show for the first time a technique capable of providing an accurate, noninvasive and rapid diagnosis of coronary heart disease that can be used clinically, either in population screening or to allow effective targeting of treatments such as statins.     

Proton nuclear magnetic resonance studies of hemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKee Rocks (beta145HC2 Tyr replaced by term): an assignment for an important tertiary structural probe in hemoglobin

High-resolution proton nuclear magnetic resonance studies of deoxyhemoglobins Osler (beta145HC2 Tyr replaced by Asp) and McKees Rocks (beta 145HC2 Tyr replaced by term) indicate that these hemoglobins are predominately in the oxy quaternary structure in 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl) methane buffer at pH 7. Upon the addition of inositol hexaphosphate, the proton nuclear magnetic resonance spectra of these hemoglobins become similar to those characteristic of a hemoglobin molecule in the deoxy quaternary structure. The exchangeable proton resonance which is found at -6.4 ppm from H2O in the spectrum of normal human adult deoxyhemoglobin is absent in the spectra of these two mutant hemoglobins. Consequently we believe the hydrogen bond between the hydroxyl group of tyrosine-beta145HC2 and the carboxyl oxygen of valine-beta98FG5 gives rise to this resonance. This assignment allows us to use the -6.4ppm resonance as an important tertiary structural probe in the investigation of the cooperative oxygenation of hemoglobin.     

MG-63 human osteosarcoma cells grown in monolayer and as three-dimensional tumor spheroids present a different metabolic profile: a (1)H NMR study

High resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy was used to determine if the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as small (about 50-80 microm in diameter), three-dimensional tumor spheroids with no hypoxic center has different metabolic characteristics. Consequently, the (1)H NMR spectra were obtained from both types of cultures and then compared. The results indicate that the type of cellular spatial array determines specific changes in MG-63 cells. In particular, small but significant differences in lactate and alanine indicating a perturbation in energy metabolism were observed in the two cell models. In addition, although variations in CH(2) and CH(3) groups were also seen, it is not possible at this time to establish if lipid metabolism is truly different in cells and spheroids.     

Noninvasive measurement of temperature and fractional dissociation of imidazole in human lower leg muscles using 1H-nuclear magnetic resonance spectroscopy.

The temperature change of the fractional dissociation of imidazole (alpha-imidazole) in resting human lower leg muscles was measured noninvasively using (1)H-nuclear magnetic resonance spectroscopy at 3.0 and 1.5 T on five normal male volunteers aged 30.6 +/- 10.4 yr (mean +/- SD). Using (1)H-nuclear magnetic resonance spectroscopy, water, carnosine, and creatine in the muscles could be simultaneously analyzed. Carnosine contains imidazole protons. The chemical shifts of water and carnosine imidazole protons relative to creatine could be used for estimating temperatures and alpha-imidazole, respectively. Using the chemical shift, the values of temperature in gastrocnemius (Gast) and soleus muscles at ambient temperature (21-25 degrees C) were estimated to be 35.5 +/- 0.5 and 37.4 +/- 0.6 degrees C (means +/- SE), respectively (significantly different; P < 0.01). The estimated values of alpha-imidazole in these muscles were 0.620 +/- 0.007 and 0.630 +/- 0.013 (means +/- SE), respectively (not significant). Alternation of the surface temperature of the lower leg from 40 to 10 degrees C significantly changed the temperature in Gast (P < 0.0001) from 38.1 +/- 0.5 to 28.0 +/- 1.2 degrees C, and the alpha-imidazole in Gast decreased from 0.631 +/- 0.003 to 0.580 +/- 0.011 (P < 0.05). However, the values of alpha-imidazole and the temperature in soleus muscles were not significantly affected by this maneuver. These results indicate that the alpha-imidazole in Gast changed significantly with alternation in muscle temperature (r = 0.877, P < 0.00001), and its change was estimated to be 0.0058/ degrees C.