Abstract-Developed in 1992, the flow-mediated dilation test is now the most commonly used noninvasive assessment of vascular endothelial function in humans. Since its inception, scientists have refined their understanding of the physiology, analysis, and interpretation of this measurement. Recently, a significant growth of knowledge has added to our understanding and implementation of this clinically relevant research methodology. Therefore, this tutorial provides timely insight into recent advances and practical information related to the ultrasonic assessment of vascular endothelial function in humans. (Hypertension. 2010;55:1075-1085.)

Muscle O2 uptake (VO2) kinetics in response to an augmented energetic requirement (on-transition) has never been directly determined in humans. We have developed a constant-infusion thermodilution technique that allowed rapid measurements of leg blood flow (Qleg) and, in conjunction with frequent serial measurement of arteriovenous O2 content difference across the leg [(Ca - Cv)O2leg], permitted the determination of the VO2 of the leg (VO2leg) at 3- to 4-s time intervals. VO2leg kinetics during the on-transition was taken as a close approximation of muscle VO2 (VO2mus) kinetics. Alveolar VO2 (VO2A), Qleg, leg O2 delivery [(Q.CaO2leg)], (Ca - Cv)O2leg, and VO2leg kinetics were determined in six trained subjects [age 22.8 +/- 4.4 (SD) yr; maximal O2 uptake 59.1 +/- 5.3 ml.kg-1.min-1] during the transition from unloaded pedaling to a workload (loaded pedaling; LP) (183 +/- 20 W) well below the previously determined ventilatory threshold. For all variables, two distinct phases were recognized. During the first 10-15 s of loaded pedaling (phase I), VO2A, Qleg, and (Q.CaO2)leg increased rapidly, whereas VO2leg increased only slightly and (Ca - Cv)O2leg actually decreased. After phase I, all variables showed a monoexponential increase (phase II), with similar time courses [slightly faster for (Ca - CV)O2leg]. In a consideration of both phases, the half times of the responses among variables were not significantly different: 25.5 +/- 2.6 s for VO2A, 26.6 +/- 7.6 s for Qleg, 26.9 +/- 8.3 s for (Q.CaO2leg, 23.5 +/- 1.3 s for (Ca - Cv)O2leg, and 27.9 +/- 5.7 s for VO2leg. We conclude that during the on-transition the kinetics of VO2A and VO2leg, as measured by these methods, are similar. The analysis of the early phase (first 10-15 s) of the on-transition indicates that bulk delivery of O2 to the working muscles is not limiting VO2leg kinetics. However, the present results cannot discriminate between maldistribution of blood flow/VO2 vs. inertia the intracellular oxidative machinery as the limiting factor.

The assumption that cellular oxygen pressure (Po2) is close to zero in maximally exercising muscle is essential for the hypothesis that 02 transport between blood and mitochondria has a finite conductance that determines maximum 02 consumption.

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During conventional cycle ergometry, as work rate (WR) is increased toward maximum, O2 extraction increases hyperbolically, typically achieving values of 80-90% at peak O2 uptake (VO2). In contrast, studies using isolated knee-extensor exercise report much higher mass-specific blood flows (Q) and lower maximal O2 extractions (approximately 70%), which have been interpreted as transit time limitation to O2 movement out of the muscle capillary. However, maximal achievable WR levels during conventional cycle ergometry are generally reached (over 10-15 min) after rapid increases in WR, whereas the reported knee-extensor studies have used only more lengthy protocols (45 min). The duration of these protocols may have prevented the attainment of high WR levels and thus high O2 extraction ratios. Accordingly, this investigation examined leg Q and O2 extraction responses during single-leg knee-extensor exercise incremented rapidly (steps of 15-25 W per 2- to 3-min interval), which produced fatigue in 13-15 min. Q and muscle VO2 increased linearly with WR to fatigue with Q-WR and VO2-WR slopes similar to those reported in previous knee-extensor studies. However, with the use of this protocol, very high maximal achievable WR [99 +/- 6 (SE) W] and muscle Q (385 +/- 26 ml.min-1 x 100 g-1) levels were attained, some 80% greater than previously reported. An O2 extraction of 84.6 +/- 2.1% was reached, giving a maximal VO2 of 60.2 +/- 5.8 ml.min-1 x 100 g-1. We conclude that, even under the high Q conditions of single-leg knee-extensor exercise, O2 extraction does not reach a plateau on the basis of short transit times and that previous conclusions to the contrary reflect failure to attain sufficiently high WR levels. Maximal VO2, Q, and O2 extraction in this model have yet to be defined.