INTRODUCTION

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HYPOXIA IS THE main stimulus for ventilatory and cardiovascular compensation for diminished arterial O₂ content (Ca_O2) (9, 20). However, there are reports suggesting a role for hemoglobin-induced variations in Ca_O2 to play a role as well (14). This relates primarily to systemic and limb blood flow being altered to maintain O₂ delivery. Thus, in chronic anemia, Sproule et al. (18) demonstrated cardiac output to be elevated both at rest and during exercise in severely anemic patients. In addition, a lower hemoglobin concentration ([Hb]) accounts for the higher cardiac output in women compared with men at a given submaximal work load (2). On the regional level, blood flow has been shown to vary in healthy people with varying [Hb] levels (14), a finding recently shown also to occur with acute anemia (10). The question then arises to what extent Ca_O2, independent of arterial O₂ tension (Pa_O2), can affect the compensatory regulation taking place when the human body is challenged by hypoxemia. To directly compare the effects of Pa_O2 and Ca_O2 on ventilatory and cardiovascular responses to exercise, a wide range of Ca_O2 was studied in subjects during hypoxia, acute isovolemic anemia, and combined hypoxia and acute anemia (anemia + hypoxia). The contributions of low Ca_O2 and low Pa_O2 were further elucidated by comparing responses to hypoxia with normal [Hb] (hypoxia) to responses seen in low [Hb] with normoxia (anemia), thus allowing contrast of two situations with identical Ca_O2. Another comparison is made between hypoxia and anemia + hypoxia, two conditions with near-identical levels of Pa_O2 but markedly different Ca_O2. Moreover, the anemia + hypoxia condition caused a very extreme arterial hypoxemia. We have previously reported some of these data, and they solely focused on comparing normoxia with hypoxia (9), or normal with low [Hb] (10).

	METHODS

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Subjects. Seven young men (age 24 ± 1 yr) participated in the study. Their mean height and weight were 183 ± 3 cm and 85.1 ± 4.6 kg, respectively. Their maximal pulmonary O₂ consumption (O₂), determined by cycle ergometry, was 55 ± 5 ml · kg¹ · min¹ (range 41-70), and maximal cardiac output (_TOT) was 26 ± 0.8 l/min (range 23-28). Additional description of anthropometric and muscle characteristics of these seven subjects as well as more details on methods and study design are available in a previous publication (10). All subjects were informed about the procedures and risks of the study before giving written informed consent to participate as approved by the Copenhagen Fredriksberg Ethical Committee.

Experimental protocol. Subjects were studied on two occasions: once with their normal [Hb] and at least 1 wk later after blood withdrawal with low [Hb]. The afternoon before the low [Hb] experiment, 1-1.5 l of whole blood (average 1.3 ± 0.05 l, ~20% of subject's blood volume) were withdrawn from each subject and replaced by an equal volume of human serum albumin (5% albumin). After normovolemic hemodilution, the blood volume was maintained (7.06 ± 0.46 to 6.93 ± 0.48 l, pre-post blood removal, P > 0.8), and [Hb] and hematocrit dropped ~20%, to 114.7 ± 1.9 g/l and 34.4 ± 0.4%, respectively (10). At the end of the low [Hb] experiment, the previously removed whole blood was reinfused to the subject. During the normal and low [Hb] experiments, the subjects inspired 0.21 and 0.11 FI_O2 in N₂ administered in random order. These tests were separated by at least 1 h of rest in the semirecumbent position while subjects breathed room air. In those subjects who breathed hypoxic gas first, in some cases the subsequent resting measurements for lactate were above baseline, although no effects of order of hypoxia were observed during exercise.

Measurements. LBF was measured in the femoral vein by constant-infusion thermodilution as described in detail elsewhere (1). Limb O₂ of the knee extensors was calculated by the Fick principle (LBF times femoral arteriovenous O₂ difference). Pulmonary O₂, CO₂, and E were measured with an on-line system (Medical Graphics CPX) while the subjects breathed through a low-resistance breathing valve. Gases with known O₂ and CO₂ concentration (micro-Scholander) were used for gas analyzer calibration.

Blood analysis. Blood volume was determined after the subjects were supine for at least 45 min, at 10, 20, and 30 min after the injection of the tracer (¹³¹I-RISA, ~250 kBq). [Hb] and O₂ saturation (SO₂) were measured with a co-oximeter (AVL 912 Co-Oxylite). PO₂, PCO₂, and pH were determined by standard techniques (AVL Compact 2) and corrected for measured body temperature. Hematocrit was determined by microcentrifugation on triplicate samples and corrected for trapped plasma (1.5%). Blood O₂ content (Ca_O2 and Cv_O2) was computed from the saturation and [Hb] {i.e., (1.34[Hb] × SO₂)  (0.003 × PO₂)}. From blood gas and hemoglobin data at peak effort, O₂ conductance into the muscle cell (DO₂) was estimated by a numerical integration procedure (17, 22). Mean transit time (MTT) was estimated as previously described (14) from data in a companion paper on these subjects (see Ref. 9, Table 1). Plasma K⁺ was measured with an ion-sensitive electrode (AVL 983-S). Whole blood lactate concentrations were measured with Triton X-100 as an erythrocyte-lysing agent (YSI 2300 Stat Plus). Leg lactate release was calculated as the product of LBF and the venous-arterial lactate difference. Plasma norepinephrine (NE) and epinephrine concentrations were measured by HPLC with electrochemical detection (6). NE spillover into plasma was calculated using the following equation (16): NE spillover = [(C_v  C_a) + C_a(A_e)]LPF, where C_v and C_a are plasma concentrations in the femoral vein and artery, respectively; A_e is the fractional extraction of epinephrine; and LPF is the leg plasma flow calculated from LBF and hematocrit. NE extraction, determined from the fractional extraction of [³H]NE, has been shown to be ~68% of A_e under steady-state conditions in three subjects (r = 0.88; Ref. 16).

Statistical analysis. Differences in the measured variables among conditions and exercise levels were analyzed with two-way ANOVA for repeated measures, with condition and work load as within-subjects factors. The Newman-Keuls post hoc test was used to assign specific differences in the ANOVA when F was significant. Simple linear regression analyses were performed to determine the relation between variables. Significance was accepted at P < 0.05. Data are reported as means ± SE.

	RESULTS

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Interventions. The Ca_O2 in anemia and hypoxia (FI_O2 = 0.11) ranged from 150.8 ± 7.2 to 163.9 ± 6.8 ml/l (P < 0.01; see matched content in Fig. 1A). Combining anemia + hypoxia resulted in a further drop in Ca_O2 from control of 58 to 76 ml/l (P < 0.01). The Pa_O2 in these conditions was on average 41.9 ± 1.5 Torr (see matched Pa_O2 in Fig. 1B). The lowest Pa_O2 values were reached in anemia + hypoxia, with a mean value of 40.0 ± 1.2 Torr at peak effort, and individual values as low as 32.5 Torr.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Indexes of arterial and femoral venous oxygenation in control, anemia, hypoxia, and anemia + hypoxia at rest (open bars), 30 W (gray bars), and peak (solid bars) dynamic knee extensor exercise. CaO2, arterial O2 content; PaO2, arterial O2 tension; CfvO2, femoral venous O2 content; PfvO2, femoral venous O2 tension.  P < 0.01 vs. control;  P < 0.01 vs. all conditions; * P < 0.01 vs. control and anemia; § P < 0.01 vs. control and hypoxia; §§ P < 0.01 vs. hypoxia alone.

Whole body responses. Pulmonary O₂ rose linearly with increasing power output from rest to peak effort (slope = 0.02 l O₂ · min¹ · W¹), and this rise was independent of hypoxia or anemia. At rest and 30 W, pulmonary O₂, CO₂, and E/O₂ were nearly the same among conditions (Table 1). The matching of Ca_O2 between hypoxia and anemia resulted in similar peak power outputs and pulmonary O₂, values ~19% lower than control (P < 0.01) but ~25% higher than in anemia + hypoxia (Table 1). Thus, although Pa_O2 was matched between hypoxia and anemia + hypoxia, peak power output was ~25% lower in anemia + hypoxia (P < 0.01). Taking power output into account revealed similar pulmonary O₂ per watt at peak effort in all conditions.

View this table: [in this window] [in a new window]   Table 1.   Pulmonary O2, E/O2 , and two-leg O2 and femoral venous lactate concentration, lactate release, and pH during two-legged knee extensor exercise in conditions with varied oxygen and hemoglobin levels

View larger version (18K): [in this window] [in a new window]   Fig. 2.   A: at 30 W (open bars), cardiac output was slightly higher in anemia and anemia + hypoxia than control values ( P < 0.05). At peak effort (solid bars), cardiac output was slightly lower in hypoxia compared with all conditions ( P < 0.05). B: leg blood flow (l/min) is shown in all 4 conditions at 30 W and peak effort. P < 0.01 vs. control; P < 0.01 vs. all conditions. See text and Fig. 3 regarding relationship of rise in leg blood flow and CaO2.

Leg responses. LBF (l/min) rose in all subjects in all conditions above control values during 30-W exercise (P < 0.001; Fig. 2B). The rise was related to the fall in Ca_O2 across conditions (r = 0.99, P < 0.01), not Pa_O2 (Fig. 3, B and D). At peak effort, LBF expressed as a function of work load was also higher than control (P < 0.01) in all subjects in anemia, in six of seven in hypoxia, and in all subjects in anemia + hypoxia. The trend of changes in LBF at peak effort also followed Ca_O2, not Pa_O2 (r = 0.78, P < 0.001; Fig. 3, A and C). The increase in LBF above control in all experimental conditions was sufficient to maintain leg O₂ delivery at rest and 30 W. At peak effort, leg O₂ delivery both in hypoxia and anemia fell 23% from control values and a further 15% in anemia + hypoxia (P < 0.001 compared with all other conditions). O₂ delivery to the muscle in relation to power output (or O₂ consumed) appears nearly constant (1, 13), a relationship that was unchanged by hypoxia or anemia. The 38% lower leg O₂ delivery observed at peak effort in anemia + hypoxia compared with control accounted for 92% of the 0.68 l/min decrement in leg O₂ from control to anemia + hypoxia. Leg O₂ was similar in all conditions at rest and 30 W, and at peak effort when Ca_O2 was matched between hypoxia and anemia (Table 1). The relationship of leg O₂ to power output from rest through peak exercise in anemia + hypoxia was 13.2 ml O₂ · min¹ · W¹, a value similar to previous reports from subjects breathing normoxic air and having normal [Hb] (12) (see Fig. 3D in Ref. 9 and Fig. 3A in Ref. 10). Leg O₂ extraction was at rest 52% and similar among conditions. At 30 W, leg O₂ extraction rose in anemia + hypoxia to 73 ± 2%, a value slightly higher than observed in any other condition (P < 0.001; Fig. 4A). At peak effort, leg O₂ extraction in hypoxia reached 79 ± 2% (Fig. 4A). The femoral arteriovenous O₂ difference was, as expected, similar when Ca_O2 was matched between hypoxia and anemia and lower than control (and higher than anemia + hypoxia) from rest to peak effort (P < 0.01; Fig. 4B). In anemia + hypoxia at rest, femoral arteriovenous O₂ difference was 71 ± 4 ml/l, increasing at 30 W to 84 ± 2 ml/l and reaching 98 ± 3 ml/l at peak effort (P < 0.001 vs. all conditions from rest to peak effort; Fig. 4B). Estimated leg DO₂ at peak effort reached 23.5 ± 2.2 and 25 ± 2.6 ml · min¹ · Torr¹ in control and hypoxia, respectively, and dropped as [Hb] fell in anemia and anemia + hypoxia to 19.8 ± 2.1 and 21 ± 2.5 ml · min¹ · Torr¹ (P < 0.05 for both conditions compared with control and hypoxia), respectively. At peak effort, estimated MTT was 529 ± 31 ms in control, a value that rose to 565 ± 29 ms (P < 0.05) in hypoxia, and was similar to values seen in anemia (525 ± 28 ms) and anemia + hypoxia (533 ± 31 ms).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Individual data for limb blood flow (l · min1 · W1) responses to varied CaO2 for 30 W and peak effort (A). Each condition has a large symbol for 30 W and a small symbol for peak effort. In B, rise in limb blood flow (l/min) with falling CaO2 is shown at 1 work load (30 W). In C, leg blood flow (l/min)-to-PO2 relationship is shown for 30 W and peak effort. In D, lack of relationship of limb blood flow to PaO2 at 30 W is shown.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Leg O2 extraction (A) was higher at 30 W in anemia + hypoxia compared with all other conditions ( P < 0.01). At peak effort (solid bars), leg O2 extraction in hypoxia rose above control and anemia (** P < 0.05). In B drop from control in femoral arteriovenous O2 difference (a-vfem O2 diff; in ml/l) with exercise in all experimental conditions ( P < 0.01 vs. control;  P < 0.01 vs. all conditions) is shown. At peak effort (solid bars), femoral arteriovenous O2 difference (ml/l) in anemia + hypoxia reaches values near those observed when exercising at only 30 W (open bars) in anemia or hypoxia.

Catecholamine responses. At peak effort, arterial NE reached 8.4 ± 1.1 nM in anemia + hypoxia, a value lower than in control (10.3 ± 1.0 nM; P < 0.05), but similar to hypoxia (9.3 ± 0.9 nM) or anemia (9.2 ± 1.7 nM). NE spillover was elevated above baseline only at peak effort, reaching 4.6 ± 1.0 nM/min in all conditions (P < 0.01 vs. baseline), with no differences due to anemia or hypoxia. Epinephrine was only higher than baseline at peak effort in all conditions (P < 0.05 vs. rest, average values of 0.8 ± 0.2 nM at 30 W and 2.5 ± 0.2 nM at peak effort), also with no notable effects of anemia or hypoxia.

Metabolic responses. With hypoxia (both hypoxia alone and anemia + hypoxia), venous pH was higher at 30 W and peak effort compared with values in anemia or control (P < 0.01; Table 1). Also at peak effort, similar femoral venous lactate values were reached in hypoxia and anemia, largely because of the nearly identical peak power outputs in these conditions (Table 1). In contrast, as peak power output in anemia + hypoxia only reached 59% of the control values, lactate at peak effort was also lower, reaching only 5.0 ± 0.5 mM (P < 0.001 vs. all conditions, Table 1). Expression of lactate at peak effort relative to peak power output reveals that in anemia + hypoxia, lactate per watt was matched to all other conditions. Leg lactate release was higher at peak effort in hypoxia compared with all conditions (P < 0.05; Table 1). Venous K⁺ rose with increasing work in all conditions (r = 0.7, P < 0.001), but the increase was not greater with hypoxia or anemia.

	DISCUSSION

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The major new finding of this study is the key role that Ca_O2 plays in the regulation of muscle blood flow during exercise, which is likely due to vasodilatation as it occurs in the face of an unchanging MAP, and hence invariant perfusion pressure. On the other hand, the effects of Pa_O2 seem limited during exercise to carotid body-linked stimulation of pulmonary ventilation. Evidence that a low Pa_O2 alone does not cause vasodilation comes from the observation of similar limb blood flows in the two conditions with nearly identical Ca_O2 but widely different Pa_O2. Moreover, despite using the knee extensor exercise model and thus not taxing fully the capacity of the cardiovascular system at the highest work loads, we found that blood flow (both cardiac output and limb blood flow) rose to an upper level beyond which no further elevation was achieved regardless of intervention. The similarity of responses between hypoxia and anemia illustrates the dependence of power output on O₂ delivery (blood flow × Ca_O2).

The apparent difference between Ca_O2 and Pa_O2 in the regulation of the LBF response to hypoxemia suggests that factors related to [Hb] play a role in the vasodilatation. Recently, such an important role has been proposed for the hemoglobin molecule in the regulation of peripheral vasodilatation in the face of altered O₂ concentrations. Stamler et al. (19) have shown that hemoglobin acts as a nitric oxide scavenger in vitro and in vivo in an O₂-dependent manner, resulting in more nitric oxide available for local vasodilatation when fewer O₂ binding sites are occupied, as happens with a lowering of Ca_O2, but less so when only Pa_O2 was lowered. Another, although also speculative, possible similar mechanism proposed by Ellsworth et al. (4) is O₂-dependent ATP release. Arguing against a role for an anemia- or hypoxia-mediated [K⁺] release playing a dominant role in the regulation of vasodilatation was the similarity in all conditions in [K⁺] response both in arterial and femoral venous blood as well as release from active muscles.

Low [Hb] or hypoxia or a combination does not further elevate the highest attained blood flow in two-legged kicking. This is surprising because the LBF only amounts to a fraction of the attainable maximal cardiac output. Blood pressure is not further enhanced either. There is also some indication of a maintained perfusion of the splanchnic region despite the exhaustive effort and HR in the range of 150-160 beats/min (9, 10). Moreover, at peak effort, _TOT reached 18-20 l/min as compared with _TOT in ordinary cycle exercise of ~26 l/min. Thus, despite substantial reserve capacity, _TOT rose no further. In this connection, the comparison with the classic study of Sproule et al. (18) is worth mentioning. _TOT was up to 23 l/min during maximal treadmill exercise. This, however, resulted in only 1.8 l/min O₂ or an O₂ uptake in the same range as in the present subjects exercising only with the knee extensors of the two legs. In whole body exercise, it is easy to understand the lack of a further increase in _TOT at peak effort when Ca_O2 is reduced, since the upper limit in the pump capacity of the heart is reached already in normoxia. The reason for this lack of compensation in the present study where an additional 3-4 l/min in _TOT would have been sufficient is unknown. The explanation most likely lies in the size of the muscle mass involved in the exercise. Dynamic knee-extensor exercise inhibits the parasympathetic activity to the heart but has limited effect on the sympathetic drive to the heart and vascular beds of noncontracting tissue (15).

A lowering of blood viscosity has previously been reported to be a cause for an increase in _TOT with low Ca_O2 (11), although _TOT was not studied when Ca_O2 was held constant and viscosity altered. Matching Ca_O2 in the present study between hypoxia and anemia allows comparison of the effects of viscosity and Ca_O2 and reveals a close coupling of blood flow to Ca_O2, independent of viscosity within the range studied. In support of this are observations made by Jones et al. (8) on cerebral blood flow over a wide range of Ca_O2, Pa_O2, and hematocrit in sheep. As Ca_O2 fell, there was a reciprocal rise in cerebral blood flow, independent of Pa_O2 or hematocrit. Thus, although viscosity may play a role in vasodilatation in extreme hypovolemia, or in the polycythemia of chronic altitude exposure, it is unlikely that viscosity contributes to the regulation of blood flow in acute, isovolemic anemia. Furthermore, the findings of Jones et al. (8) suggest that the close coupling of Ca_O2 to regulation of blood flow to exercising skeletal muscle as observed in the present study also may be a widespread mechanism of O₂ sensing in other vascular beds.

A remarkable finding in the present study is that the subjects could tolerate the very marked acute drop in Ca_O2 to 126.6 ± 2.9 ml/l and Pa_O2 to 40.0 ± 1.2 Torr that occurred in the anemia + hypoxia intervention. Other situations exist with such low Ca_O2 and Pa_O2 values as seen in the present intervention. In chronic anemia (3, 18) but not in chronic hypoxia (21) an elevated blood flow contributes in maintaining O₂ delivery. O₂ extraction, expressed as a percentage of arterially transported O₂, is enlarged in both conditions as a result of a right shift in the O₂ dissociation curve. In acute anemia or hypoxia (or the combination of the two, see Fig. 4A), blood flow is up both on the systemic and regional level, but the systemic O₂ extraction is some 10% lower than in chronic exposure (~65 vs. 70-80%). Of note is, however, that these high O₂ extractions occur at much lower power outputs.

Recently, Ferretti et al. (5) have further developed the idea that Ca_O2 and blood flow are regulated to maintain a fixed femoral Cv_O2 or mixed venous Cv_O2 at a given exercise level. In the present study, with cardiac output measured by dye dilution and direct measurement of femoral Cv_O2, we were not able to verify a constancy of _TOT × Cv_O2. The explanation for this discrepancy from the report of Ferretti et al. (5) is likely that the slope of O₂ × _TOT(Ca_O2) is <1, which violates a key assumption in their theoretical analysis. Furthermore, the lower slope of the O₂ × _TOT(Ca_O2) relationship (values ranged from 0.6 to 0.8) is explained to a large degree by the much wider range in [Hb] and FI_O2 in the present study compared with the conditions studied by Ferretti et al. (5).

A large amount of O₂ was left in the femoral vein in all interventions also at peak effort. In the present study, hypoxia resulted at peak effort in femoral Cv_O2 of 28.8 ± 1.0 and 33.4 ± 2.1 ml/l in anemia + hypoxia and hypoxia, respectively. A too short transit time for full O₂ off-loading could be one explanation for the higher femoral Pv_O2 or Cv_O2. This was not the case, since MTT shows no relationship to femoral Pv_O2 or Cv_O2. Thus factors affecting off-loading of O₂, or its further transport to and utilization by the mitochondria, must explain the observed high residual femoral venous tension and content. A low pH is one factor that would alter the O₂ dissociation curve to favor an increase in end-capillary off-loading of O₂. This may have been the case in the present study because a linear drop was noted in femoral Pv_O2 as pH rose at peak effort in anemia + hypoxia. The higher pH values with hypoxia were due largely to the respiratory stimulation that resulted in a marked drop in Pa_CO2. Furthermore, O₂ conductance into the muscle cell estimated by numerical integration (17, 22) shows a close coupling of the predicted DO₂ values and the resulting femoral venous PO₂, but not the femoral Cv_O2. Combining pH and DO₂ into a regression equation to predict femoral venous PO₂ reveals that ~60% of the variance in femoral Pv_O2 was accounted for by pH and DO₂ (R² = 0.59, F = 20.3, P < 0.0001).

In conclusion, cardiac output and LBF rise as Ca_O2 falls, suggesting that O₂ delivery, rather than the regulation of capillary PO₂, is the main regulatory goal of the vasodilatation. The mechanisms necessary to adjust blood flow according to local demands for O₂ delivery are likely situated in the tissue. Several compounds await further investigation as likely regulators of blood flow according to changes in Ca_O2 or [Hb]. These include red blood cell ATP release (4), arachidonic acid metabolites released in response to changes in O₂ levels (7), and a [Hb] specific effect on scavenging of nitric oxide to effect vasodilatation in the face of lowered [Hb] (19).