Intracellular pH is calculated from the median chemical shift of the inorganic phosphate (Pi) peak relative to that of PCr in the MRS spectra. Human skeletal muscle has a mixed-fiber type composition with large differences in the contractile and metabolic characteristics between fibers. Single-fiber analysis demonstrates that a considerable heterogeneity exists between and within fiber types in postexercise contents of phosphocreatine (PCr) and lactate (6, 10). The concentration of Pi increases nearly stoichiometrically to the fall in PCr. Fibers with the greatest change in PCr and Pi will have the greatest change in pH. The chemical shift of the Pi peak in the MRS spectra will predominately reflect fibers with a high Pi concentration. Estimated pH by the MRS technique will therefore primarily reflect pH in fibers with a high concentration of Pi (i.e., fibers with the greatest change in pH). The overall change in muscle pH will consequently be overestimated. The degree of heterogeneity (and hence the degree of error in pH estimates) will be dependent on the type and intensity of the exercise. During some conditions (e.g., onset of exercise) the error is likely to be considerable, whereas under steady-state conditions, when lactate concentration will be similar between fibers due to diffusion, the error may be negligible. This reasoning is supported by measurements from human muscle where the change in muscle pH estimated by MRS was greater than with conventional biochemical methods in some (1, 4) but not all studies (12).
With the MRS technique, lactate accumulation (and rate of glycolysis) is calculated from muscle buffer capacity and the change in muscle pH (2). Buffer capacity is derived from the initial increase in muscle pH and the observed decrease in PCr. An underlying assumption is that lactate accumulation is negligible during the initial period of contraction until pH reaches its peak (2050 s) (2, 5). However, this assumption is not valid for human muscle. It is well documented that there is no lag period of glycolysis in human muscle during ischemic contraction. Increases in muscle lactate have been observed already after 15 s of contraction (7, 8). PCr degradation and lactate accumulation occur virtually concurrently throughout the exercise period although the relative rates of these processes will change over time.
The authors validate their estimates of glycolytic flux with a separate method based on the assumptions that ATP turnover per twitch is maintained constant throughout the contraction period and that lactate accumulation is negligible during the initial period of contraction (2). However, the first of these assumptions is questionable (see p. C311 of Ref. 2) and the second is incorrect (see above). In a separate article, Conley and colleagues (11) argue that the estimates of glycolytic flux with the MRS method are confirmed by parallel measurements of muscle lactate efflux during rattling in the rattle snake. However, there are several confounding factors in this study that prevent a comparison between the two techniques. First, glycolytic flux was estimated by MRS during 30-s ischemic rattling but from lactate efflux after 25 min of aerobic rattling. Second, muscle lactate accumulation was ignored during aerobic rattling. Furthermore, species differences in the metabolic response between human and snake muscle cannot be ruled out.
Considering the potential errors in estimates of pH in a muscle with mixed fiber types and the unjustified assumption of a lag in lactate accumulation there is a considerable uncertainty in glycolytic flux estimated by MRS. Until the reliability of the MRS technique to estimate glycolytic flux in human contracting muscle is confirmed, one has to remain skeptical to previous conclusions of metabolic control based on this technique (2, 3, 5, 9).
REFERENCES
1. Bangsbo J, Johansen L, Quistorff B, and Saltin B. NMR and analytic biochemical evaluation of CrP and nucleotides in the human calf during muscle contraction. J Appl Physiol 74: 20342039, 1993.[Abstract]
2. Conley KE, Blei ML, Richards TL, Kushmerick MJ, and Jubrias SA. Activation of glycolysis in human muscle in vivo. Am J Physiol Cell Physiol 273: C306C315, 1997.
3. Conley KE, Kushmerick MJ, and Jubrias SA. Glycolysis is independent of oxygenation state in stimulated human skeletal muscle in vivo. J Physiol 511: 935945, 1998.
4. Constantin-Teodosiu D, Greenhaff PL, McIntyre DB, Round JM, and Jones DA. Anaerobic energy production in human skeletal muscle in intense contraction: a comparison of 31P magnetic resonance spectroscopy and biochemical techniques. Exp Physiol 82: 593601, 1997.[Abstract]
5. Crowther GJ, Carey MF, Kemper WF, and Conley KE. Control of glycolysis in contracting skeletal muscle. I. Turning it on. Am J Physiol Endocrinol Metab 282: E67E73, 2002.
6. Essen B and Haggmark T. Lactate concentration in type I and II muscle fibres during muscular contraction in man. Acta Physiol Scand 95: 344346, 1975.[ISI][Medline]
7. Henriksson J, Katz A, and Sahlin K. Redox state changes in human skeletal muscle after isometric contraction. J Physiol 380: 441451, 1986.[Abstract]
8. Hultman E and Sjoholm H. Substrate availability. In: Biochemistry of Exercise, edited by Knuttgen HG, Vogel JA, and Poortmans J. Champaign, IL: Human Kinetics, 1983, p. 6375.
9. Jubrias SA, Crowther GJ, Shankland EG, Gronka RK, and Conley KE. Acidosis inhibits oxidative phosphorylation in contracting human skeletal muscle in vivo. J Physiol 553: 589599, 2003.
10. Karatzaferi C, de Haan A, van Mechelen W, and Sargeant AJ. Metabolism changes in single human fibres during brief maximal exercise. Exp Physiol 86: 411415, 2001.
11. Kemper WF, Lindstedt SL, Hartzler LK, Hicks JW, and Conley KE. Shaking up glycolysis: sustained, high lactate flux during aerobic rattling. Proc Natl Acad Sci USA 98: 723728, 2001.
12. Sullivan MJ, Saltin B, Negro-Vilar R, Duscha BD, and Charles HC. Skeletal muscle pH assessed by biochemical and 31P-MRS methods during exercise and recovery in men. J Appl Physiol 77: 21942200, 1994.
Kent Sahlin
College of P.E. and Sports
Stockholm University College
PO Box 5626
SE 114 86 Stockholm
Sweden
E-mail: kent.sahlin{at}ihs.se
Heterogeneity of muscle properties. Muscle fiber types differ in glycolytic, oxidative, and contractile properties and these differences become apparent at high rates of exercise where high flux rates in fast-twitch fibers greatly exceed slow-twitch fibers. This heterogeneity problem applies to all of the metabolic and contractile properties and whether muscle is studied noninvasively or by biopsy, and is therefore an important cautionary note for any study of muscle metabolism in vivo. We have dealt with this well-established problem in two ways. First, we take care to use experimental protocols that activate all motor units, so that the results are not contaminated by recruitment of different motor units over time or exercise level (see Ref. 3 and references therein). Second, we focus on moderate rates of activity where the impact of the heterogeneity of contractile and metabolic properties is small.
Errors in estimating the muscle buffer capacity. The rise in pH at the onset of exercise in muscle indicates that H+ uptake by the creatine kinase reaction exceeds H+ generation by glycolysis. This alkalinization is used to estimate the intrinsic cell buffer capacity, which together with the buffering by ions (e.g., inorganic phosphate), determines the buffer capacity of the cell. Adams et al. (1) have shown agreement between this method and direct measurements of buffer capacity in tissue homogenates. We have shown agreement of separate measures of glycolysis: one uses the buffer capacity (stoichiometric method or "pH method") and an independent method that does not use the buffer capacity ("PCr method") (4). Thus controls indicate that any systematic error in determining this component of the buffer capacity has little effect on our calculation of glycolytic flux. However, it may not be possible to use this method to estimate buffer capacity at extreme rates of exercise that elicit a rapid onset of glycolysis, as Dr. Sahlin has noted.
Validation of the stoichiometric approach in vivo. We verified the stoichiometric approach in vivo using a muscle of uniform properties that recruits all fibers during contraction: the rattlesnake tail shaker muscle (5). We made our measurements at different times during ischemic and aerobic contractions to permit measuring glycolysis during ischemic rattling before significant acidosis for comparison to lactate efflux into blood during aerobic rattling. We performed two controls to check the validity of this approach. First, we tested that the separate measurements of ATP supply and demand yielded an ATP balance, as found in steady-state rattling. Figure 3 of Kemper et al. (5) shows that we accounted for all the ATP supply and it equaled the ATP demand. Second, we tested that the ATP fluxes measured at different times during ischemia and during aerobic rattling were the same. Figure 4 of Kemper et al. (5) shows that glycolytic ATP supply during ischemia agreed with the ATP supply calculated for lactate efflux into the blood during aerobic ratting. Similarly, the recovery ATP synthesis after ischemia agreed with the ATP synthesis expected from O2 uptake from the blood. No net accumulation of lactate in the tailshaker muscle is expected during sustained rattling given that other energetic metabolites reach a steady state by 50 s of rattling [see Fig. 2 of Kemper et al. (5)] and we took our samples at 25 min of sustained rattling. Our control experiments support a lack of lactate accumulation by the agreement of the sources and sinks for ATP separated in the ischemic experiment (and separated in time from the aerobic rattling measurements) with those determined during aerobic rattling. Thus our magnetic resonance methods for quantifying ATP fluxes are validated by direct measurements of lactate and O2 flux in muscle.
Finally, Dr. Sahlin questioned whether potential errors due to estimates of pH in muscle with mixed fiber types could nullify our conclusions concerning the control of glycolysis during exercise. However, our basic conclusion from these pH measurements is the same conclusion that Dr. Sahlin and colleagues published (7): " parameters other than PCr, ATP, Pi, calculated pH, free ADP and free AMP regulate glycolysis and glycogenolysis of human skeletal muscle". Thus our stoichiometric method and simple measures of muscle pH have both shown the inadequacy of the current models of glycolytic control. The advantage of the stoichiometric method is that it permits taking the next step: testing new mechanisms of regulation of glycolysis in vivo by quantifying the glycolytic flux responsible for changes in muscle pH.
REFERENCES
1. Adams GR, Foley JM, and Meyer RA. Muscle buffer capacity estimated from pH changes during rest-to-work transition. J Appl Physiol 69: 968972, 1990.
2. Conley KE, Blei ML, Richards TL, Kushmerick MJ, and Jubrias SA. Activation of glycolysis in human muscle in vivo. Am J Physiol Cell Physiol 273: C306C315, 1997.
3. Crowther GJ and Gronka RK. Fiber recruitment affects oxidative recovery measurements of human muscle in vivo. Med Sci Sports Exerc 34: 17331737, 2002.[CrossRef][ISI][Medline]
4. Crowther GJ, Kemper WF, Carey MF, and Conley KE. Control of glycolysis in contracting skeletal muscle. II. Turning it off. Am J Physiol Endocrinol Metab 282: E74E79, 2002.
5. Kemper WF, Lindstedt SL, Hartzler LK, Hicks JW, and Conley KE. From the Cover: shaking up glycolysis: sustained, high lactate flux during aerobic rattling. Proc Natl Acad Sci USA 98: 723728, 2001.
6. Kushmerick MJ. Multiple equilibria of cations with metabolites in muscle bioenergetics. Am J Physiol Cell Physiol 272: C1739C1747, 1997.
7. Quistorff B, Johansen K, and Sahlin K. Absence of phosphocreatine resynthesis in human calf muscle during ischaemic recovery. Biochem J 291: 681686, 1992.[ISI]
Kevin E. Conley
Department of Radiology
School of Medicine
University of Washington
Seattle, WA 98195
E-mail: kconley{at}u.washington.edu
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