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

Abstract

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...

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

“…4B to be 7.348 -0.0154 T, where T is temperature in degrees Celsius. The values of pK Im and ⌬pK Im /⌬T are comparable to those in previous reports (1,9,13,26).…”

“…where ␦ o equals the chemical shift of the imidazole (C-2 proton) signal relative to creatine (ϪCH 3 signal), and ␦ acid and In agreement with the previous reports (1,9,26), the limiting chemical shifts were unaffected by temperature. The ␦ acid and ␦ base were estimated to be 5.533 and 4.625 relative to the creatine signal, respectively.…”

Detoxification of aldehydes by histidine-containing dipeptides: From chemistry to clinical implications

“…MRS method has recently been used to show that carnosine is depleted in the gastrocnemius muscle of type 2 diabetic patients [78]. The NMR signal of carnosine has been used to investigate the dynamics of intracellular pH in working muscles [79–81]. Like the MS based methods, NMR is also a method of choice for metabolomics studies, and levels of carnosine measured by this method have been reported [82, 83].…”

Effects of Beta-Alanine on Muscle Carnosine and Exercise Performance: A Review of the Current Literature

“…Subsequent studies examining this relationship in human muscle tissue followed thereafter [13,14,29,30,31,32]. When skeletal muscles are involved in moderate to intense exercise, there is typically a generation of lactic acid and subsequent dissociation into lactate and H+, which can alter the pH levels.…”