31P magnetization transfer measurements of Pi→ATP flux in exercising human muscle

Abstract

Fundamental criticisms have been made over the use of (31)P magnetic resonance spectroscopy (MRS) magnetization transfer estimates of inorganic phosphate (Pi)→ATP flux (VPi-ATP) in human resting skeletal muscle for assessing mitochondrial function. Although the discrepancy in the magnitude of VPi-ATP is now acknowledged, little is known about its metabolic determinants. Here we use a novel protocol to measure VPi-ATP in human exercising muscle for the first time. Steady-state VPi-ATP was measured at rest and over a range of exercise intensities and compared with suprabasal oxidative ATP synthesis rates estimated from the initial rates of postexercise phosphocreatine resynthesis (VATP). We define a surplus Pi→ATP flux as the difference between VPi-ATP and VATP. The coupled reactions catalyzed by the glycolytic enzymes GAPDH and phosphoglycerate kinase (PGK) have been shown to catalyze measurable exchange between ATP and Pi in some systems and have been suggested to be responsible for this surplus flux. Surplus VPi-ATP did not change between rest and exercise, even though the concentrations of Pi and ADP, which are substrates for GAPDH and PGK, respectively, increased as expected. However, involvement of these enzymes is suggested by correlations between absolute and surplus Pi→ATP flux, both at rest and during exercise, and the intensity of the phosphomonoester peak in the (31)P NMR spectrum. This peak includes contributions from sugar phosphates in the glycolytic pathway, and changes in its intensity may indicate changes in downstream glycolytic intermediates, including 3-phosphoglycerate, which has been shown to influence the exchange between ATP and Pi catalyzed by GAPDH and PGK.

Keywords: 31P magnetization transfer; Pi↔ATP exchange; exercising muscle; saturation transfer.

Fig. 1.

Schematic representation of the ³¹P magnetic resonance spectroscopy (MRS) exercise protocol. Solid lines symbolize sequence blocks, gray shaded regions correspond to when exercise occurred. Time from onset of exercise is illustrated by the timeline. In the first exercise section, once exercising steady-state conditions were met, spectra were obtained with saturation of the γ-ATP resonance and then control saturation placed equidistant to the inorganic phosphate (P_i) resonance (SAT-CONT expts). Spectra were also obtained with a long repetition time (TR) of 44 s for calculation of metabolite concentrations (METAB). Following exercise cessation a phosphocreatine (PCr) recovery measurement (REC) was used to assess the immediate end-of-exercise oxidative ATP synthesis rate. Within the second exercise, once in steady state, the inversion recovery data were acquired with varying times between the inversion and subsequent excitation pulse (TIs) (IR expts) with an effective TR of 6 s, and a measure of M_o was also obtained. The four stars represent comparison sites for steady-state conditions.

Fig. 2.

Individual time course of metabolite concentrations obtained during steady-state exercise with alternating γ-ATP and control irradiation. Representative (group B volunteer) metabolite concentration time course of PCr (squares), P_i (circles), and γ-ATP (triangles), obtained during steady-state exercise conditions with alternating frequency of saturation (SAT-CONT section in Fig. 1). Each x-axis point corresponds to a single spectrum. Even scan numbers correspond to spectra obtained with saturation of γ-ATP (SAT) and odd scan numbers to the equivalent control saturation frequency equidistant to P_i (CONT). Consecutive points are joined by gray dashed (PCr), solid black (P_i), and dotted black (γ-ATP) lines to aid visualization.

Fig. 3.

³¹P MRS measurements of P_i→ATP flux at rest and during steady-state exercise. Representative saturation transfer (ST) spectra at rest (A) and during steady-state exercise (C), with saturation of the γ-ATP resonance (SAT) (A and C, lower right) and corresponding control spectrum (CONT) (A and C, upper right). The CONT spectra show the phosphomonoester (PME), P_i, and phosphodiester (PDE) resonances (A and C, left), superimposed with the SAT P_i resonance to show the difference (Δ) in P_i resonance. Corresponding inversion recovery plot for measurement of the P_i T₁ in the presence of γ-ATP saturation both at rest (B) and during steady-state exercise (D).

Fig. 4.

Steady-state rates of exercising P_i→ATP flux and its increment above basal levels, compared with measures of oxidative ATP synthesis rates. Exercising steady-state rates of P_i→ATP flux (V_Pi-ATP) (A) and its increment above basal levels (B), plotted against oxidative ATP synthesis rates (V_ATP) as measured from the immediate end-of-exercise PCr resynthesis rate. Black stars represent individuals in group A, and multiple scans of the three volunteers in group B are denoted by circles of black, gray, and white, respectively. The solid line represents unity equivalence of the two rates.

Fig. 5.

Paired-samples difference (Δ) in surplus P_i→ATP flux and substrate concentrations of the enzymes GAPDH and phosphoglycerate kinase (PGK) between steady-state exercise and resting conditions. Paired-samples (n = 9) mean difference ± SE (exercising-resting values) for surplus V_Pi-ATP and substrate concentrations of the enzymes GAPDH and PGK; P_i, ADP, and H⁺. Surplus V_Pi-ATP was calculated by subtracting the net rate of oxidative ATP synthesis, V_ATP (estimated as the immediate postexercise PCr resynthesis rate), from the rate of P_i→ATP flux during exercise (V_Pi-ATP) to provide an estimate of the component of the ST measurement not explained by suprabasal mitochondrial ATP synthesis. Data from volunteers in group B have been averaged to provide one value per person to avoid inappropriate weighting (hence n = 9). A paired-samples t-test was used to test for significant differences between resting and exercising conditions (P values shown).

Fig. 6.

Relationship of the P_i→ATP flux with the concentration of phosphomonoester (PME), at rest and during steady-state exercise. A: correlation of resting P_i→ATP flux (V_Pi-ATP) with resting [PME] (r = 0.740, P < 0.001, n = 18). B: relationship of exercising V_Pi-ATP with exercising [PME] (r = 0.730, P = 0.001, n = 18). As in Fig. 4, black stars represent the individuals in group A, and the multiple scans of the three volunteers in group B are denoted by circles of black, gray, and white, respectively. C: surplus V_Pi-ATP relative to [PME] at rest (white diamonds, n = 18) and during exercise (gray and black diamonds, n = 18). Surplus V_Pi-ATP was calculated by subtracting the rate of suprabasal oxidative ATP synthesis, V_ATP (estimated as the immediate postexercise PCr resynthesis rate), from the exercising V_Pi-ATP. Resting V_Pi-ATP alone was used for the equivalent measure in resting muscle, where suprabasal ATP synthesis is by definition zero. Linear regression using both resting and exercising data (n = 36) found that in addition to [PME], V_ATP was also a significant negative predictor of surplus V_Pi-ATP (both [PME] and V_ATP P < 0.001). This is illustrated schematically here by dividing the exercising data into low (0.0–14.9 mM/min) and high (15.0–30.5 mM/min) exercising V_ATP groups denoted by gray and black diamonds, respectively. To aid visualization the dashed and solid black lines represent the trend lines for resting and high-exercising V_ATP groups, respectively, and highlight the association of V_ATP with reductions in surplus V_Pi-ATP for a given [PME].