INTRODUCTION

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IN VIVO MEASUREMENTS of the arterial inflow to or venous outflow from a contracting muscle group can now be obtained quite precisely in humans (2, 16). Knee extensor exercise has commonly been used because the muscle activity has been verified to be confined to the quadriceps muscle, as found by applying T₂-weighted magnetic resonance imaging, electromyography, and intramuscular temperature measurements, as well as by comparing voluntary with electrically induced knee extensor contractions (1, 11, 15, 18). Moreover, during intense dynamic contractions the various portions of the quadriceps muscle appear to be close to or fully engaged (15, 18).

As the first thermodilution blood flow measurements were published (2), they were questioned as being too high. Subsequent studies by others have however confirmed these values (Table 1), and with even higher magnitudes during more extreme interventions (12, 13, 19-22, 24). The initial criticism was mainly based on traditional findings measuring blood flow with ¹³³Xe clearance and plethysmography. ¹³³Xe clearance has, however, been shown to be nonlinear and to underestimate blood flow (3, 5, 6, 23). In addition, plethysmography is subject to inherent limitations such as motion and occlusion artifacts confined to application at rest (4, 7, 28). Furthermore, the plethysmography measurements performed after or in between contractions only reflect an immediate postexercise value and not exercise per se. Moreover, as the magnitude of the thermodilution measurements has now been verified by other independent methods such as ultrasound Doppler and indocyanine green dye infusion (2, 10, 16, 26), and as these three techniques enable equivalent measurements during both the contraction and relaxation phases, they seem today to offer the best regional measurements of blood flow during exercise in humans (17).

View this table: [in this window] [in a new window]   Table 1.   Average values of peak work rate, , and O2

In the previous estimates of muscle perfusion, the size of the knee extensor muscle engaged has often been determined from anthropometric estimates (2). The mathematical formulas (2, 8) applied for the anthropometric estimates are based on the original model derived by Jones and Pearson (9). The quadriceps muscle mass is determined from a size relationship between the quadriceps and hamstring muscle in relation to the total limb volume, on the basis of a small number of autopsy studies (2). There are comparisons between such anthropometric estimates and imaging techniques, but the number of image scans have generally been too few (8). Recently, Ray and Dudley (15) also used MRI to determine the muscle volume engaged in contractions, but instead of measuring blood flow, they used data in the literature to estimate muscle perfusion.

Thus, to obtain accurate values of muscle perfusion, not only blood flow but also the muscle volume engaged in the exercise needs to be accurately determined in the same subjects. As the thermodilution measurements today have been verified as accurate by other methods, we aimed to address the impact of the muscle mass estimates. In the present study, muscle blood flow was determined with thermodilution in the femoral vein. The size of the knee extensor muscle was determined by multiple computed tomography (CT) image cross sections along the full length of the muscle. Moreover, to evaluate their validity, three different estimates of the quadriceps muscle length were applied to anthropometrically estimated knee extensor muscle volume. The present study, therefore, provides a guideline for the accuracy of muscle mass estimates and an improved knowledge of peak perfusion and oxygen uptake in human skeletal muscle.

	METHODS

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Subjects. Nine healthy human subjects, eight men and one woman, with a mean ± SE age of 26 ± 1 (range 22-35) yr, height of 178 ± 3 (range 158-186) cm, and weight of 78 ± 4 (range 49-98) kg, volunteered to participate in this study. The subjects' engagement in exercise varied from daily activities to regular endurance training. Before the experiments, the subjects were informed about the experimental procedures and potential risks and discomfort and were advised that they could withdraw at any time without consequences. They participated after first giving signed informed consent. The experiments were carried out with the approval of the Ethical Committees of Copenhagen and Fredriksberg (KF-01-403/95).

Experimental procedures. Before the experiments all subjects were familiarized with the one-legged, dynamic, knee extensor exercise model (1) by training at 60 rpm until they were comfortable and could fully relax the hamstring muscles, so that the work was done solely by the knee extensors. The isolation of the quadriceps muscle was supported by the kicking-force tracings collected during exercise. The peak power output that the subjects could sustain at 60 rpm was determined before the experiments by an incremental test starting at 10 W and increasing by 5-10 W every third minute until they could no longer sustain the rhythm.

Tissue volume measurements. A computer tomograph (model Prospeed VX, General Electric, Paris, France) was used to obtain precise estimates of muscle volume. It operated at a voltage of 120 kV, a current of 200 mA, and a scan-to-scan cycle time of 3.0 s. A standardized reconstruction algorithm and a 512 × 512 reconstruction matrix with a field of view of 35-45 cm were used, giving a spatial resolution in the range of 0.59-0.87 mm. In total, 21-25 serial segments, with a 10-mm thickness, were obtained every 20 mm along the thigh, starting distally from the patella and going in a proximal direction toward the spina iliaca anterior superior (SIAS). All segments taken of the first subject were analyzed by two experienced persons who were independent of the study. After comparison, verification, and agreement as to the definition of the different tissue structures, one of the analysts continued to analzye data from the remaining subjects.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Multiple cross sections (2 cm apart) of the thigh from proximal edge of patella toward spina iliaca anterior superior measured with computer tomography in 9 subjects. Cross sections of total thigh over the length of the quadriceps femoris muscle are shown. Values are means ± SE. CT, computed tomography.

Statistics and data analysis. Parametric statistics were used for data analysis. Analysis of variance for repeated measures was used when more than two data groups were compared, and Tukey's honestly significant difference post hoc tests were used to distinguish the differences. Linear regression and Pearson's correlation were employed. P < 0.05 was considered as statistically significant. A nonstatistically significant comparison is indicated by P = NS. The values given in the text are means ± SE.

	RESULTS

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Estimates of muscle volume and mass. The CT quadriceps femoris muscle volume averaged 2.36 ± 0.17 (range 1.31-3.27) liters, which corresponded to a mass (M_qf.CT) of 2.48 ± 0.18 (range 1.37-3.43) kg. The anthropometric muscle mass estimates overestimated (P < 0.02) the CT measurement by ~21% (M_qf.PB, 3.00 ± 0.18 kg, range 2.60-3.99), 29% (M_qf.IL, 3.21 ± 0.19 kg, range 2.15-4.20), and 46% (M_qf.SIAS, 3.61 ± 0.22 kg, range 2.26-4.64), respectively (Fig. 2A). However, they all correlated with and were linearly related to each other (0.93  r  0.98, P < 0.001)

View larger version (17K): [in this window] [in a new window]   Fig. 2.   A:quadriceps femoris muscle mass. B and C: peak muscle perfusion (peak mus) and peak muscle oxygen uptake (O2 peak mus), respectively, during 1-legged, dynamic, knee extensor exercise. Values are means ± SE. Quadriceps femoris muscle volume was measured with CT as well as anthropometrically with different muscle lengths estimated from patella to pubic bone (PB), inguinal ligament (IL), and spina iliaca anterior superior (SIAS). Quadriceps femoris muscle mass values measured with CT and anthropometry were all significantly different from each other (P < 0.02). As a consequence of different muscle mass estimates using anthropometric procedure, peak mus and O2 peak mus were underestimated compared with CT-scan measurements by ~18-33%. Significant differences (P < 0.05): * from CT, § from CT and PB, and  from CT, PB, and IL estimates.

Knee extensor exercise. Leg blood flow (_leg) and leg oxygen uptake (O_{2 leg}) increased (P < 0.05) during submaximal exercise at 30 W from their resting baseline values of 0.24 ± 0.02 (range 0.17-0.35) l/min and 18.7 ± 2.6 (range 9.4-34.2) ml/min, respectively, to 3.57 ± 0.25 (range 2.69-5.01) l/min and 434 ± 24 (range 361-528) ml/min, respectively. A further increase (P < 0.05) was observed at peak effort (67 ± 7, range 55-100 W) to 5.99 ± 0.66 (range 4.15-9.52) l/min and 856 ± 109 (range 590-1,521) ml/min for _leg and O_{2 leg}, respectively.

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	DISCUSSION

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This study demonstrates that anthropometric muscle volume estimates markedly overestimate the knee extensor muscle volume, which in turn means that previous anthropometrically based estimates of peak muscle perfusion and oxygen uptake in humans, using various types of blood flow measurements, are underestimated by up to ~30% (2, 21, 24). In light of the fact that the initial thermodilution blood flow measurements were criticized for being too high, as peak muscle perfusion and oxygen uptake were studied in a single active muscle group (2), these findings are of major importance because they emphasize that the peak values are even higher than previously found. As the magnitude of the thermodilution measurements also have been verified by ultrasound Doppler and indocyanine green dye infusion (2, 10, 16, 26), the low peak values found by ¹³³Xe clearance and plethysmography seem to be even less believable.

In this study multiple CT image scans were taken along the full length of the quadriceps muscle (Fig. 1) of subjects with a large variation in the length (L) of the quadriceps muscle (L_CT 42.4 ± 0.73 cm, range 38-46) and its points of insertion. Furthermore, the large variation in muscle length with the three different anthropometric approaches (L_PB 38.5 ± 0.62 cm, range 36-41; L_IL 41.0 ± 0.54 cm, range 39-43.5; L_SIAS 45.3 ± 0.65 cm, range 40.5-47) induces a great source of error in the muscle volume and mass calculations. Moreover, the configuration of the muscle varies markedly along its length (Fig. 1). Thus only three circumference measurements or CT image scans along the thigh may not give an accurate estimate of muscle volume (2, 8, 27). In addition, the mathematical formulas used to estimate the muscle volume on the basis of anthropometry do not account for the variation in muscle configuration, because they are based on the fact that the muscle is constructed from two symmetrical "cones" that narrow toward the ends of the muscle. More importantly, the anthropometric formulas do not account for either the relative area that the quadriceps muscle group occupies in the thigh region or the fact that the quadriceps muscle volume may vary. In the present study, this variation in quadriceps muscle volume ranged from ~1.31 to 3.27 liters. The present study thus provides a set of formulas that relate the anthropometric estimates to the CT measurements, which potentially can be used to correct for the anthropometric overestimations. However, one should be careful with such a generalization because the formulas are based on results from nine subjects only and the relationship is likely to be affected by different types and levels of activity, inactivity, gender, and age. If anthropometry is used to estimate muscle mass, muscle length estimated from the patella to the PB induces the smallest error in the muscle volume and mass estimate.

Besides providing a relationship between anthropmetric and CT estimates of muscle volume, knowing the number of CT or magnetic resonance image (MRI) sections that need to be performed is also of interest to allow a reasonable measure of accuracy. In the present data, the maximum width of the quadriceps muscle was located approximately one-half the distance between the greater trochanter and the top of patella. Thus, if muscle volume is estimated by using an area from the section of maximum width and from two other sections, imaged at one-half the distance between the maximum width and the greater trochanter as well as at one-half the distance between the maximum width and the patella, respectively, the three sections would give a muscle volume of ~2.20 liters, i.e., an underestimation of ~7%. If an estimate of the missing muscle volume proximally and distally, respectively, to the two outer sections is added, assuming a decrease in muscle size toward its end points, as in the case of two narrowing cones, then the muscle volume would be ~2.64 liters, i.e., an overestimation of ~12%. If the estimate is based on five sections, the first section halfway between the greater trochanter and patella and then four additional sections measured every 8 cm, i.e., two on each side of the first section, then the muscle volume would be ~2.37 liters, i.e., an overestimation of ~0.5%. By adding the missing outer cone-shaped muscle volumes, the volume would be ~2.46 liters, i.e., an overestimation of ~4.2%. Similarly, 13 sections taken every 4 cm, starting from the same midpoint between the greater trochanter and patella, would render an overestimation of only ~0.4%. Thus, to carefully describe muscle volume, as many imaged sections as are in the range of every every 2-4 cm are needed, because the shape of the muscle varies individually along its full length. However, five sections may give a reasonable estimate, but one should be aware that our sample of subjects is fairly small.

There is ample evidence for the whole quadriceps muscle to be engaged in very intense knee extensor exercise (11, 15, 18). However, at submaximal efforts the recruitment pattern has been less clear. Electromyography tracings from the various portions of the quadriceps muscle suggest a gradual and equal involvement. Ray and Dudley's (15) recent findings using T₂-weighted MRI scans support this notion but more importantly add a value for the muscle mass being recruited at different intensities. On the basis of their findings, with a muscle volume "engagement" of ~16% at rest, ~54% at 20 W, and ~94% at 40 W, and assuming ~100% at peak effort, a linear relationship can be expressed as the recruited muscle volume (%) = 25.187 + 1.285 · load (W). Interpolation gives the notion that in the present study ~64% of the muscle volume could have been engaged at 30 W, rendering values for muscle perfusion and oxygen uptake of ~235 ± 24 (range 154-383) and 28 ± 2.3 (range 21-41) ml · min^l · 100 g^l, respectively, on the basis of blood flow and oxygen uptake in the present study (Fig. 3). This can be compared with ~151 ± 15 (range 99-245) and 18 ± 1.4 (range 14-26) ml · min^l · 100 g^l, respectively, values that are found if, as in the past (2), blood flow and oxygen uptake in the present study are related to the whole quadriceps muscle mass. Thus, as pointed out by Ray and Dudley, peak muscle perfusion may be achieved in certain regions at already submaximal intensities. However, muscle oxygen uptake appears to be further elevated at peak effort, possibly due to a larger oxygen extraction (Fig. 3). A tentative explanation for higher oxygen uptake per unit of muscle at increasing work intensities despite recruitment of more muscle to perform the work could be that first the frequency of activation is increased and then energy turnover.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Muscle perfusion (mus; A) and muscle oxygen uptake (O2 mus; B) during 1-legged, dynamic, knee extensor exercise, at 30 W and at peak effort (~ 67 ± 7 W), on the basis of and O2 of the present study. Values are means ± SE. Values are estimated, as if the whole quadriceps femoris (qf) muscle volume was recruited (qf 100%), or only a portion of the muscle (qf 64%) on the basis of findings of Ray and Dudley (15). Thus peak mus may be achieved in portions of muscle already at intensities lower than peak effort. Significant differences (P < 0.05): * from exercise at 30 W related to recruitment of 100% of qf muscle mass, and ** from exercise at 30 W related to recruitment of 100 and 64% of qf muscle mass.

In conclusion, anthropometric overestimates of muscle volume in the past have markedly underestimated peak muscle perfusion and oxygen uptake. Thus peak perfusion and oxygen uptake in human skeletal muscle may be up to ~30% higher than previous estimates on the basis of blood flow measurements with techniques equivalent to themodilution (2, 16, 19-22, 26), making values obtained by ¹³³Xe clearance and plethysmography even less believable. Moreover, to obtain an estimate of muscle mass recruited when muscle perfusion and muscle oxygen uptake are estimated at intensities lower than at peak effort, T₂-weighted MRI is a necessity, in addition to either CT or MRI muscle volume determinations.