Measurement of human skeletal muscle oxidative capacity by 31P-MR spectroscopy: a cross-validation with in vitro measurements

Abstract

Purpose: To cross-validate skeletal muscle oxidative capacity measured by (31)P-MR spectroscopy with in vitro measurements of oxidative capacity in mitochondria isolated from muscle biopsies of the same muscle group in 18 healthy adults.

Materials and methods: Oxidative capacity in vivo was determined from PCr recovery kinetics following a 30-s maximal isometric knee extension. State 3 respiration was measured in isolated mitochondria using high-resolution respirometry. A second cohort of 10 individuals underwent two (31)P-MRS testing sessions to assess the test-retest reproducibility of the method.

Results: Overall, the in vivo and in vitro methods were well-correlated (r = 0.66-0.72) and showed good agreement by Bland Altman plots. Excellent reproducibility was observed for the PCr recovery rate constant (CV = 4.6%; ICC = 0.85) and calculated oxidative capacity (CV = 3.4%; ICC = 0.83).

Conclusion: These results indicate that (31)P-MRS corresponds well with gold-standard in vitro measurements and is highly reproducible.

Figure 1. ³¹P MRS spectra and PCr recovery in a single subject

Representative stackplot of phosphorous spectra at rest (first spectrum, 60s average) and throughout the 30s muscle contraction (2s resolution) and 10 minute recovery (top panel). Monoexponential fit of PCr during the recovery period in the same individual is shown by the solid black line (bottom panel).

Figure 2. Average PCr, Pi, and pH during rest, exercise and recovery

Phosphocreatine (black circles) transiently decreased during 30 seconds of exercise, while inorganic phosphate (grey circles) increased. Both metabolites recovered rapidly upon cessation of the muscle contraction. pH (white circles) exhibited the expected alkalosis during exercise and minimal acidosis during recovery. Data are mean ± SEM.

Figure 3. Comparison of in vitro to in vivo indices of muscle oxidative capacity

Panels A, B, and C show correlations between oxidative capacity measured by ³¹P-MRS (Qmax) and maximal respiration in isolated mitochondria in the presence of substrates for respiratory chain complex I (state 3, CI, Panel A), complex I+II (state 3, CI+II, Panel B), and complexII (state 3, CII, Panel C). Pearson correlation coefficients are given for each relationship. Solid black lines represent linear regression between each variable with the 95% confidence intervals as dashed lines. Panels D, E, and F show the level of agreement between in vitro and in vivo measurements as Bland-Altman plots of z-scores calculated for Qmax vs. complex I (panel D), Qmax vs. complex I+II (panel E), and Qmax vs. complex II (panel F).

Figure 4. Reproducibility of 31P-MRS for measuring oxidative capacity

Individual values for the PCr recovery rate constants (K_PCr, panel A) and computed oxidative capacity (Qmax, panel B) measured on 2 different days. Panels C and D show correlations between K_PCr (C) and Qmax (D) for each study day with the solid black line representing the line of identity. Intraclass correlation coefficients (ICC) are given for each variable.