Intracellular pH in human skeletal muscle by 1H NMR.

Abstract

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-...

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

“…3 in Ref. 11, there is broadening of the C-2 peak and the development of a shoulder upfield to the main peak, similar to the results reported here. The existence of a distinct second peak may have been obscured by a low reported signal-to-noise ratio (SNR) (5.5-8.25:1) as compared to that in the present study (ϳ28:1).…”

“…Some of these, such as the higher sensitivity of the 1 H nucleus; the shorter T 1 of the carnosine C-2 proton; more stable concentrations of carnosine throughout rest, exercise, and recovery; the relatively higher frequency range of the pH-induced chemical shift; and narrower linewidth of the C-2 resonance combine to improve temporal resolution and chemical sensitivity. Another advantage of using the C-2 peak to measure pH I is the relative insensitivity of its chemical shift to the concentrations of divalent cations (9,11), particularly in light of the fact that intracellular [Mg 2ϩ ] increases during exercise (e.g., Ref. 35).…”

“…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.…”

ASIC3 in muscle mediates mechanical, but not heat, hyperalgesia associated with muscle inflammation

“…A decrease in pH is observed following inflammation, hematomas and isometric exercise (Hood et al, 1988;Issberner et al, 1996;Pan et al, 1988;Revici et al, 1949), and decreases in pH in muscle produce pain in humans (Issberner et al, 1996). Peripheral initiators of muscle pain are virtually unknown, but are likely key to development of chronic pain after muscle insult.…”

Significant differences in proton trimethyl ammonium signals between human gastrocnemius and soleus muscle

“…These studies significantly enhance our understanding of the underlying biophysics of the proton spectra of muscles, and have demonstrated potential applications of 1 H MRS in muscles. In vivo proton spectroscopy of muscles can directly monitor creatine (4,5), carnitine (6,7), extramyocelluar lipid (EMCL), intramyocellular lipid (IMCL) (8 -11), pH-sensitive imidazole protons of histidine (12), and deoxymyoglobin (13). Because of the discovery of the effect of fiber orientation on 1 H metabolites (14,15), in vivo 1 H MRS of skeletal muscle offers the potential to investigate both physical changes and biochemical changes induced by a diseased process or physiological activity.…”