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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We have made in vivo 1H NMR measurements of the time course of pH and lactate in human skeletal muscle after exercise. Spectra were obtained in a 4.7-T 30-cm bore Bruker Biospec spectrometer with a 2.5-cm diameter single surface coil. pH was determined from the shift of the endogenous carnosine H-C2 peak while lactate concentrations were determined by comparison with endogenous total creatine, taken to be 28.5 mM/kg wet wt. Fitting the data shows that the exponential decay of lactate (-0.094 +/- 0.014 min-1. t1/2 = 10.6 min) is slower than that of pH (-0.147 +/- 0.015 min-1, t1/2 = 4.7 min), n = 7 with two different volunteers. These values are significantly different with P less than 0.0005. Relaxation times of lactate and creatine were also measured for lactate quantitation; creatine T1, 1.23 +/- 12 s, T2, 136.2 +/- 26.4 ms (both in resting human muscle); lactate T1 (in postmortem rabbit muscle), 1.0 +/- 11 s and T2, 80 ms (in postexercise human muscle). At the end of intense exercise, the lactate level reached was 25.3 +/- 4.0 mM and the average pH drop was 1.0 pH unit. We discuss the implications of these measurements in conjunction with existing data on other sources of H+ flux, phosphocreatine resynthesis, H+ transport, and contribution of inorganic phosphate to buffering.

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