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...

As the end product of anaerobic metabolism and a source of H+, lactic acid is important in metabolism and pH regulation. Several methods have been introduced to calculate changes in the lactate anion (Lac–) concentration in exercising skeletal muscle from information derived from the 31P spectrum. Alternatively, Lac–may be observed directly with 1H MRS. Both 1H and 31P spectroscopy have potential problems, which could prevent accurate determination of [Lac–]. It is demonstrated that quantitatively accurate 1H MRS measurements of changes in [Lac–] due to exercise are possible in isolated muscle. In general, calculation by 31P MRS overestimates Lac–production. An analysis is presented of possible sources of errors in the 1H and 31P MRS methods. Magn Reson Med 44:418–426, 2000. © 2000 Wiley‐Liss, Inc.

Skeletal muscle contraction and glycogenolysis are closely coupled. The standard explanation for this coupling, as taught in modern biochemistry textbooks, is that the metabolic products of contraction (ADP, AMP, P i ) feed back to activate glycogenolytic enzymes, thus providing for resynthesis of ATP. However, both in vivo 31 P MRS analyses and chemical analyses of muscle extracts have provided results that are contrary to this theory, at least in its simplest form. The MRS studies suffer from ambiguous assumptions. More importantly, in 31 P MRS studies the dependent and independent variables are often confounded because the glycogenolytic rate is calculated from the same data which are used to calculate the other metabolic variables. The analysis of biopsies has been necessarily quite limited, and suffers from a different set of experimental artifacts. Thus, the problem of contraction-glycogenolysis-coupling was reassessed using a quantitatively accurate 1 H MRS method. It is confirmed that glycogenolysis and contractions are closely coupled during repetitive exercise, while glycogenolysis and P-metabolite concentrations are not. A simple metabolic feedback system cannot explain contraction-glycogenolysis-coupling.Magn Despite its fundamental importance in all prokaryotic and eukaryotic life, the in vivo regulation of glycolysis is still not fully understood. In standard textbook descriptions, glycolysis is activated by a feedback mechanism at phosphofructokinase (PFK) by metabolites such as ADP and P i , direct products of the actomyosin-ATPase, and AMP, which is in equilibrium with ADP through adenylate kinase (1). By this simple feedback mechanism, glycolysis is activated whenever the direct and indirect products of ATP hydrolysis are increased. However, during the past 20 years it has been shown that, as calculated from changes in the 31 P MRS spectrum of intact ex vivo and in vivo animal and human muscle, glycolysis is closely coupled to contractions and not to metabolite levels (2-6).Similar results have been obtained using muscle biopsies and extracts (7,8).Close coupling of glycolysis or glycogenolysis to contraction is referred to as contraction-glycolysis-coupling or contraction-glycogenolysis-coupling (CGC). In this article, glycogenolysis refers to the degradation of glycogen to lactate. In the absence of oxygen, CGC is characterized by a partial metabolic recovery from contraction, followed by metabolic stasis during anaerobic rest, i.e., very low glycogenolytic rate with little to no net change in metabolite concentrations. During and closely following contraction the glycogenolytic rate is increased by orders of magnitude over the resting rate and then it falls to near zero without restoring P-metabolites to their precontractile values. In other words, glycogenolysis and contractions are coupled temporally. Glycogenolysis and contractions are also coupled quantitatively, since the amount of lactate (Lac -) produced is proportional to the magnitude of mechanical activity.The methods previously us...

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