Application of high-field 1H-NMR spectroscopy for the study of perifused amphibian and excised mammalian muscles

Arús, Carlos; Bárány, Michael

doi:10.1016/0167-4889(86)90177-1

Cited by 42 publications

(23 citation statements)

References 35 publications

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“…The new peak cannot be from the H-8 proton of ATP because it resonates too far downfield. The peak is in the approximate position of the H-2 proton of ATP, which was observed in resting whole muscle by Arú s and Barany (14). Since the H-2 peak is observable using an FID sequence but not using an echo sequence at TE ϭ 24 ms, the T 2 of the H-2 resonance has to be on the order of 5 ms or less in resting muscle.…”

Section: Both Peaks Are Carnosine C-2mentioning

confidence: 69%

“…If this compartmentation is physical (such as two muscle fiber types), these two peaks should collapse into one in muscle homogenates. Based on Arú s and Barany's work (14), a total of four peaks would be seen in this region of the spectrum: one imino proton from the imidazole ring of histidine, the H-2 and H-8 protons of ATP and ADP, and the carnosine C-2 proton. Indeed, in homogenates from exercised frog gastrocnemii, Shen (28) identified these four peaks in the 8.0 -8.6 ppm region of the 1 H spectrum.…”

Section: Compartmentation Results From Pools Of Oxidative and Glycolymentioning

confidence: 97%

“…To address this issue, it is useful to start by examining the spectra published by Yoshizaki et al (8) and Arú s and Barany (14). Using selective saturation of the water peak and a pulse-and-acquire sequence, Yoshizaki et al (8) observed three peaks in the 8.0 -9.0 ppm region of the 1 H spectrum of the cytosolic fraction of muscle homogenates: the carnosine C-2 peak and two peaks from ATP, both downfield of the C-2 peak.…”

Section: Both Peaks Are Carnosine C-2mentioning

confidence: 99%

“…Several authors have taken advantage of this pH sensitivity to measure the pH I of muscle in vivo and ex vivo (8 -13), and of muscle extracts (8,14). Specific applications have been made to muscle in resting (8,10,11,(13)(14)(15)(16), diseased (16), caffeinetreated (14), and exercised (9,11,12) states. Pan et al (11) and Hitzig et al (13) found high correlations between pH I measurements by the C-2 and P i methods.…”

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confidence: 99%

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The carnosine C‐2 proton's chemical shift reports intracellular pH in oxidative and glycolytic muscle fibers

Damon

Hsu

Stark

et al. 2003

Magnetic Resonance in Med

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The appearance of new peaks in the 7.7-8.6 and 6.8 -7.4 ppm regions of the postexercise 1 H spectrum of frog muscle is reported. These new peaks result from the splitting of single pre-exercise carnosine C-2 and C-4 peaks into two peaks, representing the intracellular pH (pH I ) of oxidative and glycolytic fibers. The following data support this conclusion: 1) comparison of means and regression analysis indicates equivalence of the pH I measurements by 1 H and 31 P NMR; 2) the pre-and poststimulation concentrations of carnosine are equal; 3) in ischemic rat hindlimb muscles, the presence of a single, more acidic peak in the plantaris; a single, less acidic peak in the soleus; and two peaks (more and less acidic) in the gastrocnemius correspond to published values for the fiber-type composition of these muscles; and 4) in muscles treated with iodoacetate prior to and during stimulation, a second peak never appears. These data indicate that it is feasible to measure separately the pH I of oxidative and glycolytic fibers using 1 H NMR spectroscopy. Measuring intracellular pH (pH I ) is important in studies of many cellular processes. In studies of muscle metabolism and exercise, it is desirable to measure separately the pH I of oxidative and glycolytic fibers. pH I measurements are commonly made using 31 P NMR spectroscopy, using the inorganic phosphate (P i ) peak's chemical shift. Following intense muscle exercise, the single pre-exercise P i peak frequently splits into two or more poststimulation P i peaks (P i,A and P i,B ), which are believed to correspond to P i in glycolytic and oxidative fibers, respectively (1-7). However, because of the low NMR sensitivity of the phosphorus nucleus, it is sometimes not possible to distinguish a P i peak from noise when the concentration of P i is low. Extreme broadening of the P i peak during exercise has also been observed (5), interfering with the ability to resolve individual peaks.The C-2 and C-4 protons on the imidazole ring of carnosine (␤-alanyl histidine) have pK A 's of approximately 7.0 and chemical shifts that are sensitive to pH in the physiological range, providing an alternative means of measuring pH noninvasively (8). Several authors have taken advantage of this pH sensitivity to measure the pH I of muscle in vivo and ex vivo (8 -13), and of muscle extracts (8,14). Specific applications have been made to muscle in resting (8,10,11,(13)(14)(15)(16), diseased (16), caffeinetreated (14), and exercised (9,11,12) states. Pan et al. (11) and Hitzig et al. (13) found high correlations between pH I measurements by the C-2 and P i methods.While studying exercising ex vivo frog gastrocnemii, we also observed the C-2 peak of carnosine at ϳ8.0 ppm prior to stimulation. However, we noted two peaks (one at ϳ8.53 and one at ϳ8.37 ppm) following exercise. In this study, we tested the hypothesis that they both originate from C-2 protons of carnosine. We also tested the hypothesis that the downfield peak (which we call carnosine C-2 A ) originates from a more acidic compartm...

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Section: Both Peaks Are Carnosine C-2mentioning

confidence: 69%

Section: Compartmentation Results From Pools Of Oxidative and Glycolymentioning

confidence: 97%

Section: Both Peaks Are Carnosine C-2mentioning

confidence: 99%

mentioning

confidence: 99%

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The carnosine C‐2 proton's chemical shift reports intracellular pH in oxidative and glycolytic muscle fibers

Damon

Hsu

Stark

et al. 2003

Magnetic Resonance in Med

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“…This method was extended by Yoshizaki et al (3), who initially used the histidine-containing dipeptide carnosine (j3-Ala-His) to determine pH in excised frog muscle. Subsequent analogous studies have been made of extracted human muscle (4) and in vivo rat muscle using anserine (J3-Ala, methyl-His) (5,6). We recently reported the in vivo observation of lactic acid production by 'H NMR and its clearance in the human forearm muscle after exercise.…”

mentioning

confidence: 99%

Intracellular pH in human skeletal muscle by 1H NMR.

Pan

Hamm²,

Rothman³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

126

101

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We report here the in vivo observation of the imidazole protons (C-2 and C-4) ofcarnosine (fl-Ala-His) by 1HNMR at 4.7 T in human skeletal muscle. The relationship between the pH and chemical shift of the C-2 and C-4 resonances was determined analytically. These titration curves were used to measure the resting pH of human muscle in vivo, 7.01 x 0.04 (C-2 proton) and 6.97 + 0.10 (C-4 proton). An in vivo titration curve of the C-2 proton resonance was determined by interleaving 1H and 31P NMR spectra after exhaustive exercise, during which muscle pH recovers from an acidic value of 6.1. We observed excellent agreement between the pH values as determined by the C-2 resonance of carnosine and that of inorganic phosphate. Carnosine provides an excellent pH indicator since (i) its concentration is relatively stable and (ii) it allows measurement of proton metabolites and pH to be achieved through the same coil, thus enabling a better signal/noise ratio and better localization.The imidazole protons of histidine have long been useful as pH indicators in NMR. In studies of proteins and intracellular pH of erythrocytes (1, 2), the chemical shift ofthe C-2 and C-4 ring protons with pH has yielded much information about the environment of the histidine residue. This method was extended by Yoshizaki et al. (3), who initially used the histidine-containing dipeptide carnosine (j3-Ala-His) to determine pH in excised frog muscle. Subsequent analogous studies have been made of extracted human muscle (4) and in vivo rat muscle using anserine (J3-Ala, methyl-His) (5, 6). We recently reported the in vivo observation of lactic acid production by 'H NMR and its clearance in the human forearm muscle after exercise. Simultaneous tissue acidification was followed by 3'P NMR (7). Lactate was measured to have a clearance time of 10 min; however, parallel 31p spectra showing recovery of muscle pH by using the pHsensitive chemical shift of the Pi resonance was not always possible because of the acute drop in the concentration of Pi after exercise (8). Thus, an alternative method of in vivo pH determination would be very useful. We report here the in vivo observation of the C-2 and C-4 proton resonances of camosine in human forearm muscle and show how they can be used to measure intracellular pH at rest and during recovery from exhaustive exercise. METHODSThe relationship between pH and the chemical shift ofthe C-2 and C-4 protons was established by titrating a solution containing 150 mM KCl, 20 mM phosphocreatine (as a chemical shift reference), 30 mM carnosine, and, where specified, 1 mM MgCI2. These analytic titration curves were determined on a Bruker AM-360 MHz wide-bore system using a 5-mm Helmholtz probe. Fully relaxed spectra were acquired with a spin-echo sequence with a 40-ms echo delay (all echo delay values in this paper are given as 1/2 of the entire spin-echo duration) with a total repetition time of 8 s.Water suppression was achieved by applying a single frequency presaturation pulse at 40 mW for 1 s and using carrier-...

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Regeneration and myogenic cell proliferation correlate with taurine levels in dystrophin- and MyoD-Deficient muscles

et al. 1998

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This study coupled proton magnetic resonance spectroscopy (1H-NMR) and in situ hybridization plus autoradiography in a novel examination of different phenotypes of adult myogenesis that arise from genetic disruptions in mice. Study of muscle extracts from normal and dystrophin-deficient mdx limb and diaphragm muscles confirmed our previous findings linking taurine and muscle regeneration at the peak of damage and repair. 1H-NMR distinguished biochemical differences in regenerating muscles that were consistent with the extent of repair in three strains: mdx dystrophic mice; MyoD(-/-) mice that lack expression of the early myogenic regulatory gene MyoD; and a double-mutant mdx:MyoD(-/-) strain lacking expression of both MyoD and dystrophin. We tested the hypothesis that differences in spectra according to genotype and the regeneration phenotype are related specifically to proliferation by committed myogenic precursor cells. 1H-NMR distinguished the three mutant strains: Taurine was highest in mdx muscles, with the phenotype of most effective regeneration; lowest in MyoD(-/-) muscles, with the least effective formation of new muscle in repair, as reported previously; and intermediate in double-mutant muscles, now reported to show an intermediate repair phenotype. The early and late muscle precursors (mpcs) expressing myf5 and myogenin were examined for proliferation. Eighteen percent of mdx myf5-positive mpcs were proliferative, whereas myf5-positive mpcs did not proliferate in regenerating muscles that lacked MyoD expression. By contrast, whereas 30% of myogenin-positive mpcs were proliferative in mdx muscles, almost none were proliferative in MyoD(-/-) muscles, and 12% were proliferative in double-mutant muscles. Therefore, the extent of accumulated structural regeneration, taurine levels, and proliferation of late mpc (expressing myogenin) were congruent across genotypes. Proliferation by early mpc (expressing myf5) was inhibited by the lack of MyoD expression during muscle regeneration. These studies indicate the potential for 1H-NMR monitoring of muscle status in disease, regeneration, and treatment.

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Application of high-field 1H-NMR spectroscopy for the study of perifused amphibian and excised mammalian muscles

Cited by 42 publications

References 35 publications

The carnosine C‐2 proton's chemical shift reports intracellular pH in oxidative and glycolytic muscle fibers

The carnosine C‐2 proton's chemical shift reports intracellular pH in oxidative and glycolytic muscle fibers

Intracellular pH in human skeletal muscle by 1H NMR.

Regeneration and myogenic cell proliferation correlate with taurine levels in dystrophin- and MyoD-Deficient muscles

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