Dai Okushima

Validation of a high-power time resolved near-infrared spectroscopy system for measurement of superficial and deep muscle deoxygenation during exercise

Near-infrared assessment of skeletal muscle is restricted to superficial tissues due to power limitations of spectroscopic systems. We reasoned that understanding of muscle deoxygenation may be improved by simultaneously interrogating deeper tissues. To achieve this, we modified a high-power (∼8 mW), time-resolved, near-infrared spectroscopy system to increase depth penetration. Precision was first validated using a homogenous optical phantom over a range of inter-optode spacings (OS). Coefficients of variation from 10 measurements were minimal (0.5-1.9%) for absorption (μa), reduced scattering, simulated total hemoglobin, and simulated O2 saturation. Second, a dual-layer phantom was constructed to assess depth sensitivity, and the thickness of the superficial layer was varied. With a superficial layer thickness of 1, 2, 3, and 4 cm (μa = 0.149 cm(-1)), the proportional contribution of the deep layer (μa = 0.250 cm(-1)) to total μa was 80.1, 26.9, 3.7, and 0.0%, respectively (at 6-cm OS), validating penetration to ∼3 cm. Implementation of an additional superficial phantom to simulate adipose tissue further reduced depth sensitivity. Finally, superficial and deep muscle spectroscopy was performed in six participants during heavy-intensity cycle exercise. Compared with the superficial rectus femoris, peak deoxygenation of the deep rectus femoris (including the superficial intermedius in some) was not significantly different (deoxyhemoglobin and deoxymyoglobin concentration: 81.3 ± 20.8 vs. 78.3 ± 13.6 μM, P > 0.05), but deoxygenation kinetics were significantly slower (mean response time: 37 ± 10 vs. 65 ± 9 s, P ≤ 0.05). These data validate a high-power, time-resolved, near-infrared spectroscopy system with large OS for measuring the deoxygenation of deep tissues and reveal temporal and spatial disparities in muscle deoxygenation responses to exercise.

Muscle deoxygenation in the quadriceps during ramp incremental cycling: Deep vs. superficial heterogeneity

Muscle deoxygenation (i.e., deoxy[Hb + Mb]) during exercise assesses the matching of oxygen delivery (Q̇O2) to oxygen utilization (V̇O2). Until now limitations in near-infrared spectroscopy (NIRS) technology did not permit discrimination of deoxy[Hb + Mb] between superficial and deep muscles. In humans, the deep quadriceps is more highly vascularized and oxidative than the superficial quadriceps. Using high-power time-resolved NIRS, we tested the hypothesis that deoxygenation of the deep quadriceps would be less than in superficial muscle during incremental cycling exercise in eight males. Pulmonary V̇O2 was measured and muscle deoxy[Hb + Mb] was determined in the superficial vastus lateralis (VL), vastus medialis (VM), and rectus femoris (RF-s) and the deep rectus femoris (RF-d). deoxy[Hb + Mb] in RF-d was significantly less than VL at 70% (67.2 ± 7.0 vs. 75.5 ± 10.7 μM) and 80% (71.4 ± 11.0 vs. 79.0 ± 15.4 μM) of peak work rate (WR(peak)), but greater than VL and VM at WR(peak) (87.7 ± 32.5 vs. 76.6 ± 17.5 and 75.1 ± 19.9 μM). RF-s was intermediate at WR(peak) (82.6 ± 18.7 μM). Total hemoglobin and myoglobin concentration and tissue oxygen saturation were significantly greater in RF-d than RF-s throughout exercise. The slope of deoxy[Hb + Mb] increase (proportional to Q̇O2/V̇O2) in VL and VM slowed markedly above 70% WR(peak), whereas it became greater in RF-d. This divergent deoxygenation pattern may be due to a greater population of slow-twitch muscle fibers in the RF-d muscle and the differential recruitment profiles and vascular and metabolic control properties of specific fiber populations within superficial and deeper muscle regions.

Greater V̇O2peak is correlated with greater skeletal muscle deoxygenation amplitude and hemoglobin concentration within individual muscles during ramp‐incremental cycle exercise

It is axiomatic that greater aerobic fitness (V̇O2peak) derives from enhanced perfusive and diffusive O2 conductances across active muscles. However, it remains unknown how these conductances might be reflected by regional differences in fractional O2 extraction (i.e., deoxy [Hb+Mb] and tissue O2 saturation [StO2]) and diffusive O2 potential (i.e., total[Hb+Mb]) among muscles spatially heterogeneous in blood flow, fiber type, and recruitment (vastus lateralis, VL; rectus femoris, RF). Using quantitative time‐resolved near‐infrared spectroscopy during ramp cycling in 24 young participants (V̇2peak range: ~37.4–66.4 mL kg−1 min−1), we tested the hypotheses that (1) deoxy[Hb+Mb] and total[Hb+Mb] at V̇O2peak would be positively correlated with V̇O2peak in both VL and RF muscles; (2) the pattern of deoxygenation (the deoxy[Hb+Mb] slopes) during submaximal exercise would not differ among subjects differing in V̇O2peak. Peak deoxy [Hb+Mb] and StO2 correlated with V̇O2peak for both VL (r = 0.44 and −0.51) and RF (r = 0.49 and −0.49), whereas for total[Hb+Mb] this was true only for RF (r = 0.45). Baseline deoxy[Hb+Mb] and StO2 correlated with V̇O2peak only for RF (r = −0.50 and 0.54). In addition, the deoxy[Hb+Mb] slopes were not affected by aerobic fitness. In conclusion, while the pattern of deoxygenation (the deoxy[Hb+Mb] slopes) did not differ between fitness groups the capacity to deoxygenate [Hb+Mb] (index of maximal fractional O2 extraction) correlated significantly with V̇O2peak in both RF and VL muscles. However, only in the RF did total[Hb+Mb] (index of diffusive O2 potential) relate to fitness.

Reduction of V̇O2 slow component by priming exercise: novel mechanistic insights from time‐resolved near‐infrared spectroscopy

Novel time‐resolved near‐infrared spectroscopy (TR‐NIRS), with adipose tissue thickness correction, was used to test the hypotheses that heavy priming exercise reduces the V̇O2 slow component (V̇O2SC) (1) by elevating microvascular [Hb] volume at multiple sites within the quadriceps femoris (2) rather than reducing the heterogeneity of muscle deoxygenation kinetics. Twelve subjects completed two 6‐min bouts of heavy work rate exercise, separated by 6 min of unloaded cycling. Priming exercise induced faster overall V̇O2 kinetics consequent to a substantial reduction in the V̇O2SC (0.27 ± 0.12 vs. 0.11 ± 0.09 L·min−1, P < 0.05) with an unchanged primary V̇O2 time constant. An increased baseline for the primed bout [total (Hb + Mb)] (197.5 ± 21.6 vs. 210.7 ± 22.5 μmol L−1, P < 0.01), reflecting increased microvascular [Hb] volume, correlated significantly with the V̇O2SC reduction. At multiple sites within the quadriceps femoris, priming exercise reduced the baseline and slowed the increase in [deoxy (Hb + Mb)]. Changes in the intersite coefficient of variation in the time delay and time constant of [deoxy (Hb + Mb)] during the second bout were not correlated with the V̇O2SC reduction. These results support a mechanistic link between priming exercise‐induced increase in muscle [Hb] volume and the reduced V̇O2SC that serves to speed overall V̇O2 kinetics. However, reduction in the heterogeneity of muscle deoxygenation kinetics does not appear to be an obligatory feature of the priming response.

Blood flow occlusion-related O2 extraction "reserve" is present in different muscles of the quadriceps but greater in deeper regions after ramp-incremental test

It was recently demonstrated that an O2 extraction reserve, as assessed by the near-infrared spectroscopy (NIRS)-derived deoxygenation signal ([HHb]), exists in the superficial region of vastus lateralis (VL) muscle during an occlusion performed at the end of a ramp-incremental test. However, it is unknown whether this reserve is present and/or different in magnitude in other portions and depths of the quadriceps muscles. We tested the hypothesis that an O2 extraction reserve would exist in other regions of this muscle but is greater in deep compared with more superficial portions. Superficial (VL-s) and deep VL (VL-d) as well as superficial rectus femoris (RF-s) were monitored by a combination of low- and high-power time-resolved (TRS) NIRS. During the occlusion immediately post-ramp-incremental test there was a significant overshoot in the [HHb] signal ( P < 0.05). However, the magnitude of this increase was greater in VL-d (93.2 ± 42.9%) compared with VL-s (55.0 ± 19.6%) and RF-s (47.8 ± 14.0%) ( P < 0.05). The present study demonstrated that an O2 extraction reserve exists in different pools of active muscle fibers of the quadriceps at the end of a ramp exercise to exhaustion. The greater magnitude in the reserve observed in the deeper portion of VL, however, suggests that this portion of muscle may present a greater surplus of oxygenated blood, which is likely due to a greater population of slow-twitch fibers. These findings add to the notion that the plateau in the [HHb] signal toward the end of a ramp-incremental exercise does not indicate the upper limit of O2 extraction. NEW & NOTEWORTHY Different portions of the quadriceps muscles exhibited an untapped O2 extraction reserve during a blood flow occlusion performed at the end of a ramp-incremental exercise. In the deeper portion of the vastus lateralis muscle, this reserve was greater compared with superficial vastus lateralis and rectus femoris. These data suggest that the O2 extraction reserve may be dependent on the vascular and/or oxidative capacities of the muscles.

Near infrared spectroscopy of superficial and deep rectus femoris reveals markedly different exercise response to superficial vastus lateralis

To date our knowledge of skeletal muscle deoxygenation as measured by near‐infrared spectroscopy (NIRS) is predicated almost exclusively on sampling of superficial muscle(s), most commonly the vastus lateralis (VL‐s). Recently developed high power NIRS facilitates simultaneous sampling of deep (i.e., rectus femoris, RF‐d) and superficial muscles of RF (RF‐s) and VL‐s. Because deeper muscle is more oxidative with greater capillarity and sustains higher blood flows than superficial muscle, we used time‐resolved NIRS to test the hypotheses that, following exercise onset, the RF‐d has slower deoxy[Hb+Mb] kinetics with reduced amplitude than superficial muscles. Thirteen participants performed cycle exercise transitions from unloaded to heavy work rates. Within the same muscle (RF‐s vs. RF‐d) deoxy[Hb+Mb] kinetics (mean response time, MRT) and amplitudes were not different. However, compared with the kinetics of VL‐s, deoxy[Hb+Mb] of RF‐s and RF‐d were slower (MRT: RF‐s, 51 ± 23; RF‐d, 55 ± 29; VL‐s, 18 ± 6 s; P < 0.05). Moreover, the amplitude of total[Hb+Mb] was greater for VL‐s than both RF‐s and RF‐d (P < 0.05). Whereas pulmonary V˙O2 kinetics (i.e., on vs. off) were symmetrical in heavy exercise, there was a marked on‐off asymmetry of deoxy[Hb+Mb] for all three sites i.e., MRT‐off > MRT‐on (P < 0.05). Collectively these data reveal profoundly different O2 transport strategies, with the RF‐s and RF‐d relying proportionately more on elevated perfusive and the VL‐s on diffusive O2 transport. These disparate O2 transport strategies and their temporal profiles across muscles have previously been concealed within the “global” pulmonary V˙O2 response.

The effect of dietary nitrate supplementation on the spatial heterogeneity of quadriceps deoxygenation during heavy‐intensity cycling

This study investigated the influence of dietary inorganic nitrate (NO3−) supplementation on pulmonary O2 uptake ( V˙ O2) and muscle deoxyhemoglobin/myoglobin (i.e. deoxy [Hb + Mb]) kinetics during submaximal cycling exercise. In a randomized, placebo‐controlled, cross‐over study, eight healthy and physically active male subjects completed two step cycle tests at a work rate equivalent to 50% of the difference between the gas exchange threshold and peak V˙ O2 over separate 4‐day supplementation periods with NO3−‐rich (BR; providing 8.4 mmol NO3−∙day−1) and NO3−‐depleted (placebo; PLA) beetroot juice. Pulmonary V˙ O2 was measured breath‐by‐breath and time‐resolved near‐infrared spectroscopy was utilized to quantify absolute deoxy [Hb + Mb] and total [Hb + Mb] within the rectus femoris, vastus lateralis, and vastus medialis. There were no significant differences (P > 0.05) in the primary deoxy [Hb + Mb] mean response time or amplitude between the PLA and BR trials at each muscle site. BR significantly increased the mean (three‐site) end‐exercise deoxy [Hb + Mb] (PLA: 91 ± 9 vs. BR: 95 ± 12 μmol/L, P < 0.05), with a tendency to increase the mean (three‐site) area under the curve for total [Hb + Mb] responses (PLA: 3650 ± 1188 vs. BR: 4467 ± 1315 μmol/L sec−1, P = 0.08). The V˙ O2 slow component reduction after BR supplementation (PLA: 0.27 ± 0.07 vs. BR: 0.23 ± 0.08 L min−1, P = 0.07) correlated inversely with the mean increases in deoxy [Hb + Mb] and total [Hb + Mb] across the three muscle regions (r2 = 0.62 and 0.66, P < 0.05). Dietary NO3− supplementation increased O2 diffusive conductance across locomotor muscles in association with improved V˙ O2 dynamics during heavy‐intensity cycling transitions.

Reply to Dr. Grassi

TO THE EDITOR: We appreciate Dr. Grassi’s insights (1) on our study demonstrating an O2 extraction reserve in different portions of the quadriceps muscles (2). Dr. Grassi commented that the existence of a “reserve” in O2 extraction is a wellestablished concept demonstrated by previous studies in which, differently from our approach, blood flow occlusion occurred a “few minutes” (rather than immediately) after exercise cessation. Dr. Grassi contended that differences in procedures are “irrelevant” to the interpretation of the results. We disagree with this position. Our study determined whether a potential to further increase microvascular O2 extraction in the vastus lateralis muscle existed in the presence of a plateau in the near-infrared (NIRS)-derived deoxy-hemoglobin ([HHb]) signal, with pulmonary V̇O2 still increasing. In this context, occluding blood flow immediately at the end of the ramp exercise (i.e., V̇O2max) allowed us to evaluate the [HHb] response at a metabolic rate and O2 tension associated with maximal exercise intensity for the proposed exercise. Had we occluded blood flow “after a few minutes of recovery,” this procedure would have not allowed us to answer our experimental question, because during this time muscle V̇O2 and vascular dynamics (e.g., hematocrit, capillary recruitment, O2 diffusive capacity) would have been substantially different, thus affecting the [HHb] response. This is clear in a previous study showing that occlusions of blood flow before the ramp exercise resulted in lower amplitudes in the [HHb] signal compared with those performed immediately after the ramp exercise (3). Thus, although we are aware of Dr. Grassi’s studies, it is difficult to understand how they apply to our experimental conditions. Regarding the plateau in the [HHb] signal, we reasoned that, based on the Fick equation, the absence of a further increase in O2 extraction in the presence of increasing V̇O2 implies that a greater O2 availability is supporting the increased metabolic rate. Dr. Grassi highlighted that data from different groups indicated that there are “. . . substantial amounts of O2 in the venous blood draining from maximally working muscles” and that “muscle O2 extraction does not reach 100% during incremental exercise,” thus the plateau reflects a limitation of the muscle to extract O2. It should be noted that substantial amounts of O2 are to be expected in the venous circulation when samples are collected from a vessel that is draining blood from both highly active and less active muscles. For this reason, the arterial-venous O2 difference is never 20 ml/dl of blood. However, by evaluating the [HHb] signal of the active tissues, this limitation can be somewhat circumvented. Although critical O2 tensions will eventually affect the O2 diffusion gradient and 100% of the O2 will never be extracted, it is highly probable that the NIRS-derived estimation of O2 extraction is more likely to approximate the highest level of O2 extraction in the active fibers compared with other more “systemic” measures. To conclude, this exchange with Dr. Grassi is certainly a positive stimulus for science to progress. For this reason, and in line with the title of this letter, we believe that wellestablished concepts can only benefit from new ideas.

Greater Vo2peak Is Associated With Deoxygenation Amplitude, But Not Deoxygenation Kinetics, Across The Active Muscles: 117 Board #6 June 1, 9: 30 AM - 11: 30 AM.

Greater Vo2peak Is Associated With Deoxygenation Amplitude, But Not Deoxygenation Kinetics, Across The Active Muscles Dai Okushima, David C. Poole, FACSM, Thomas J. Barstow, FACSM, Harry B. Rossiter, FACSM, T. Scott Bowen, Tatsuro Amano, Narihiko Kondo, Shunsaku Koga. Kobe Design University, Kobe, Japan. Kansas State University, Manhattan, KS. Los Angeles Biomedical Research Institute at Harbor–UCLA Medical Center, Torrance, CA. Leipzig University, Leipzig, Germany. Kobe University, Kobe, Japan.