Near-infrared spectroscopy estimation of combined skeletal muscle oxidative capacity and O2 diffusion capacity in humans

Abstract

The final steps of the O₂ cascade during exercise depend on the product of the microvascular-to-intramyocyte $P_{O_2}$ difference and muscle O₂ diffusing capacity ( $D{m}_{O_2}$ ). Non-invasive methods to determine $D{m}_{O_2}$ in humans are currently unavailable. Muscle oxygen uptake (m ${\dot{V}}_{O_2}$ ) recovery rate constant (k), measured by near-infrared spectroscopy (NIRS) using intermittent arterial occlusions, is associated with muscle oxidative capacity in vivo. We reasoned that k would be limited by $D{m}_{O_2}$ when muscle oxygenation is low (k_LOW ), and hypothesized that: (i) k in well oxygenated muscle (k_HIGH ) is associated with maximal O₂ flux in fibre bundles; and (ii) ∆k (k_HIGH - k_LOW ) is associated with capillary density (CD). Vastus lateralis k was measured in 12 participants using NIRS after moderate exercise. The timing and duration of arterial occlusions were manipulated to maintain tissue saturation index within a 10% range either below (LOW) or above (HIGH) half-maximal desaturation, assessed during sustained arterial occlusion. Maximal O₂ flux in phosphorylating state was 37.7 ± 10.6 pmol s^-1 mg^-1 (∼5.8 ml min^-1 100 g^-1 ). CD ranged 348 to 586 mm^-2 . k_HIGH was greater than k_LOW (3.15 ± 0.45 vs. 1.56 ± 0.79 min^-1 , P < 0.001). Maximal O₂ flux was correlated with k_HIGH (r = 0.80, P = 0.002) but not k_LOW (r = -0.10, P = 0.755). Δk ranged -0.26 to -2.55 min^-1 , and correlated with CD (r = -0.68, P = 0.015). m ${\dot{V}}_{O_2}$ k reflects muscle oxidative capacity only in well oxygenated muscle. ∆k, the difference in k between well and poorly oxygenated muscle, was associated with CD, a mediator of $D{m}_{O_2}$ . Assessment of muscle k and ∆k using NIRS provides a non-invasive window on muscle oxidative and O₂ diffusing capacity. KEY POINTS: We determined post-exercise recovery kinetics of quadriceps muscle oxygen uptake (m ${\dot{V}}_{O_2}$ ) measured by near-infrared spectroscopy (NIRS) in humans under conditions of both non-limiting (HIGH) and limiting (LOW) O₂ availability, for comparison with biopsy variables. The m ${\dot{V}}_{O_2}$ recovery rate constant in HIGH O₂ availability was hypothesized to reflect muscle oxidative capacity (k_HIGH ) and the difference in k between HIGH and LOW O₂ availability (∆k) was hypothesized to reflect muscle O₂ diffusing capacity. k_HIGH was correlated with phosphorylating oxidative capacity of permeabilized muscle fibre bundles (r = 0.80). ∆k was negatively correlated with capillary density (r = -0.68) of biopsy samples. NIRS provides non-invasive means of assessing both muscle oxidative and oxygen diffusing capacity in vivo.

Keywords: biopsy; capillary density; mitochondria; recovery kinetics; skeletal muscle.

Figure 1. Study design and muscle recovery rate constant (k) protocol by NIRS

Participants visited the laboratory on four occasions. Visit 1 was used to determine peak oxygen uptake (${\dot{V}}_{{\mathrm{O}}_2\mathrm{peak}}$) and gas exchange threshold (GET). Visits 2 and 3 were used to determine the physiological normalization (PN) of quadriceps TSI following sustained arterial occlusion, and the muscle ${\dot{V}}_{{\mathrm{O}}_2}$ recovery rate constant (k) in well oxygenated (HIGH) and poorly oxygenated (LOW) conditions. Two k measurements were performed at each visit. To measure k, participants initially cycled for 5 min at 80% GET, followed immediately by 10–20 intermittent arterial occlusions (300 mmHg). The duration and timing of repeated occlusions were modulated to maintain TSI in two different ranges: from 0% to 10% of PN (LOW) and from 50% to 60% of PN (HIGH). A muscle biopsy was obtained during visit 4.

Figure 2. Representative tissue saturation index (TSI) responses during repeated arterial occlusions of the quadriceps following moderate exercise in LOW and HIGH conditions

Representative muscle TSI profiles and m${\dot{V}}_{{\mathrm{O}}_2}$ recovery kinetics during intermittent arterial occlusions following 5 min moderate intensity cycling. A, TSI profile in poorly oxygenated (LOW; TSI = 0–10% of physiological normalization) condition. B, TSI profile in well oxygenated (TSI = 50–60% of physiological normalization) condition. C, m${\dot{V}}_{{\mathrm{O}}_2}$ recovery and exponential fit (black line) for calculation of k _LOW. D, m${\dot{V}}_{{\mathrm{O}}_2}$ recovery and exponential fit (black line) for calculation of k _HIGH. Grey point represents outlier, excluded from the analysis (see Methods). k is the recovery rate constant (n = 1).

Figure 3. Individual test–retest reliability of muscle oxidative capacity by NIRS

The individual test–retest reliability of recovery rate constant (k) assessed by NIRS in different days during both HIGH (black circles) and LOW (grey circles) for 12 participants. Straight line represents linear regression curve and dashed line represent 95% confidence intervals. Grey line represents identity. Circles represent k values obtained during first and second trial.

Figure 4. Muscle capillarization from two participants

Immunofluorescence identification of capillaries in cross‐sections of vastus lateralis from two participants. A, participant with the highest capillary density. B, participant with the lowest capillary density. After immunostaining using anti‐CD31, capillaries appear green (n = 2).

Figure 5. Association between in vivo and ex vivo estimation of skeletal muscle OXPHOS capacity and capillary density

Association between in vivo (NIRS) and ex vivo (biopsy) estimation of (A and B) skeletal muscle OXPHOS capacity and (C) skeletal muscle capillary density. Correlation between muscle oxidative phosphorylation capacity by high‐resolution respirometry and recovery rate constant by NIRS measured in HIGH (A) and LOW (B) conditions. C, correlation between Δk ( = k _HIGH – k _LOW) by NIRS and capillary density (CD) from biopsy. Δk is lower in participants with a greater muscle capillarization. Linear regression (solid) 95% confidence intervals (dash) (n = 12).