INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX A APPENDIX B REFERENCES

OUR LABORATORY RECENTLY demonstrated that direct Fick principle calculated cardiac output using CO₂ (CO_CO2) agrees well with cardiac output using O₂ (CO_O2) in normal subjects at rest and during exercise (51), as predicted by Fick (16). Other investigators, to avoid blood sampling, have calculated CO_CO2 indirectly (7, 12, 15, 29, 36). This approach, although superficially attractive, pays insufficient attention to the effect of pH and change in buffer base on the accuracy and precision of the estimation of CO₂ concentration (CCO₂) from PCO₂, particularly during work rates at which lactate is increased. Blood CCO₂ is influenced by pH, PCO₂, Hb concentration, and oxyhemoglobin saturation (SO₂). During exercise, the CO₂ pressure-concentration relationship (PCO₂-CCO₂ relationship) may be more complex than the near-linear relationship depicted in textbooks and the original reports (8, 38, 56), because it is assumed that there is no change in buffer base as PCO₂ increases. A frequently referenced report, which presents formulas for refining the influences of PCO₂, Hb, and SO₂ on CCO₂ at rest and during exercise, concludes that "the relationship (PCO₂-CCO₂) is only slightly influenced by changes in pH" (34). Although some investigators recommend correction for acid-base changes (18, 27), most investigators have calculated the arteriovenous CCO₂ difference [mixed venous CCO₂ (C_CO2)-arterial CCO₂ (Ca_CO2)] for estimating cardiac output by the indirect Fick method from mixed venous PCO₂ (P_CO2) and arterial PCO₂ (Pa_CO2), correcting only for changes in Hb and SO₂, but ignoring pH changes (6, 9-11, 26, 31-33, 35, 37, 42).

It is well known that acidemia due to lactic acidosis occurs with symptom-limiting exercise, both in normal subjects and patients (3, 4, 21, 50-55), resulting in an almost stoichiometric decrease in bicarbonate concentration ([HCO]) as lactate increases (46, 48, 55). This intimate relationship challenges the concept that changes in pH only slightly influence CCO₂ during exercise. We measured pH, PCO₂, Hb, and SO₂ in both arterial and mixed venous blood during progressively increasing work rate exercise to maximum (Max) in normal subjects to determine the relative importance of the changes in pH, Hb, and SO₂ on the PCO₂-CCO₂ relationship and on each component in CO₂ transport. We hypothesized that, by ignoring changes in acid-base balance during exercise, major errors result in estimates of CCO₂ from PCO₂ at work rates above the lactic acidosis threshold (LAT). This could translate into major errors when these values are used to calculate cardiac output by the Fick principle.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX A APPENDIX B REFERENCES

Subjects, Protocol, and Measures

Subjects. The research protocol was approved by the Human Subjects Committee at Harbor-UCLA Medical Center. Informed consent was obtained from five healthy nonsmoking male subjects that participated in the study.

Catheter placement. A flow-directed pulmonary artery catheter (Arrow International, Reading, PA) was introduced via a femoral vein sheath (Cordis, Miami, FL), which had been inserted percutaneously into the right femoral vein and positioned in the main pulmonary artery under direct fluoroscopic guidance. An arterial catheter was placed percutaneously into the left brachial artery. Each catheter was attached to an infusion apparatus (Continu-Flo, Baxter Health Care, Deerfield, IL), which provided a slow, continuous flow (15 ml/h) of heparinized normal saline (1,000 U heparin/l) and allowed periodic bolus flushings.

Exercise protocols. An increasing work rate exercise test was performed on an electromagnetically braked cycle ergometer (type 18070, Gould-Godart, Bilthoven, the Netherlands). The rate of work rate increase (range 25-40 W/min) depended on a preliminary, noninvasive increasing work rate exercise test designed to achieve exhaust in ~10 min. Gas-exchange and heart rate measurements were averaged for each 30-s period during 3 min of rest and 3 min of unloaded pedaling and during the progressively increasing work rate test to maximum tolerance. Pedal frequency was maintained at 60 rpm.

Respired-gas analysis. The subjects respired through a mouthpiece during the test. Expired air was directed to a Fleisch type 3 pneumotachograph via a breathing valve (100-ml dead space). The PO₂, PCO₂, and partial pressure of N₂ at the mouthpiece were continuously measured by mass spectrometry (MGA-1100, Perkin Elmer, Pomona, CA). Minute ventilation (BTPS) and O₂ uptake (O₂) and CO₂ production (CO₂) (both STPD) were calculated as whole breath averages for each 30-s exercise period, as previously reported (49). The LAT and maximal O₂, defined as the O₂ averaged over the last 30 s of exercise, were determined (4). Later, for tabular and graphic representations of the group response, the O₂ was normalized to LAT with O₂ at LAT set as 1.00 for each subject and all other values on either side of LAT set as a ratio of LAT. Values were calculated at each minute of exercise.

Blood samples. Blood was sampled simultaneously from the pulmonary artery and brachial artery during rest and unloaded cycling and at each minute of increasing work rate exercise. Blood-gas samples were drawn over a 15- to 20-s period. The samples were collected in glass syringes that contained a small amount (mean 0.14 ml) of liquid heparin (1,000 U/ml). The blood samples were agitated to prevent clotting and were immediately placed in an ice slurry.

Blood analyses. Blood-gas analyses were performed by using an Instrumentation Laboratory 1306 blood-gas analyzer (Lexington, MA) for pH, PCO₂, and PO₂ and an Instrumentation Laboratory 482 CO-oximeter for Hb and SO₂. The blood-gas analyzer was recalibrated every 20-30 min. Tonometered blood samples were used to verify accuracy. The measured values were corrected for heparin dilution and the known consistent underestimation of blood PCO₂ values >45 Torr (23, 51).

Calculation of CCO₂, C_CO2-Ca_CO2, CO_CO2, and Plasma [HCO]

Values of CCO₂, C_CO2-Ca_CO2, CO_CO2, and plasma [HCO]. The plasma CCO₂ (CCO_2 pl) (mM) and plasma [HCO] (mM) were calculated from the standard formula derived from the Henderson-Hasselbalch equation (13, 21, 34, 38)

(1)

(2)

where s is the plasma solubility coefficient (in mM/Torr) of CO₂ and pK' is the apparent dissociation constant of the CO₂-HCO system. The variable s is 0.0307 in plasma at 37°C and pH 7.4 in normal human subjects (1). The variable pK' was calculated from Kelman's equation (13, 28), assuming the temperature was stable at 37°C during short-period exercise (51)

(3)

Total blood CCO₂ (mM) was calculated from the equation of Douglas et al. (13), modified from Visser's equation (34) after taking into account the effects of changing pH during exercise on pK'

(4)

where [Hb] is Hb concentration.

(5)

Default values of CCO₂, C_CO2-Ca_CO2, and CO_CO2. To compare the magnitude of error caused by the failure to acknowledge changes in pH, Hb, and SO₂ from rest to exercise on the PCO₂-CCO₂ relationship, default (Def) values of CCO₂ were calculated by using the resting values of pH, Hb, and/or SO₂ for each individual. Def values are so named because they do not acknowledge the changes in one or more of the independent variables during exercise. For example, if the changing PCO₂, Hb, and SO₂ values were used to calculate CCO₂ during each minute of exercise but the pH remained at its resting (or Def) value, the CCO₂ would be identified as Def-pH CCO₂. In the same way, we calculated Def-Hb, Def-SO₂, and the combined Def-pH, Hb and SO₂, the latter when changes in all three values were not taken in to account. The percent errors of the Def values from the actual values of Ca_CO2, C_CO2, C_CO2-Ca_CO2, and CO_CO2 for each stage of exercise were calculated using the following formula: %error = 100 × (default  actual)  actual.

Relative importance of multiple factors on the PCO₂-CCO₂ relationship during exercise. Besides physically dissolved CO₂ ([CO₂]), which depends only on PCO₂ under isothermic conditions, the two major factors influencing the PCO₂-CCO₂ relationship in the Douglas equation are the HCO factor (F_bic, i.e., the effect of pH on the quantity of [HCO] in both plasma and red blood cell) and the Hb factor (F_Hb, i.e., the effect of Hb binding to CO₂). The F_Hb consists of three subfactors, the Hb concentration (F_HbHb), the SO₂ (F_HbSO2), and the pH (F_HbpH), on Hb binding of CO₂. The formulas for the two major factors and the three subfactors are given in APPENDIX A. After calculation of actual values of each factor and subfactor using these equations, the relative importance of the changes in each factor and subfactor at each stage of exercise was calculated. Thus the influence of a specific factor or subfactor at any stage of exercise depends on how far its ratio differs from 1.00.

Estimation of Each Component of Blood CO₂ During Exercise

Data Analysis and Statistics

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX A APPENDIX B REFERENCES

The subject's physical characteristics and aerobic parameters were as follows: age, 25 ± 5 (20-34) yr; height, 179 ± 3 (173) cm; body weight, 73 ± 5 (68-80) kg; work rate at LAT, 126 ± 25 (98-154) W; maximum work rate, 302 ± 47 (225-360) W; O₂ at LAT, 2.00 ± 0.31 (1.50-2.35) l/min; and maximal O₂, 3.91 ± 0.61 (2.74-4.31) l/min. From rest to Max, O₂ increased over 10-fold, from 0.37 ± 0.04 to 3.91 ± 0.61 l/min; CO₂ increased over 16-fold, from 0.29 ± 0.04 to 4.84 ± 0.71 l/min; heart rate increased nearly threefold, from 63 ± 6 to 178 ± 12 beats/min; whereas cardiac output increased over threefold, from 7.06 ± 2.37 to 25.38 ± 3.90 l/min. The rate of increase in O₂ as related to work rate increase was 10.03 ± 0.34 (9.50-10.68) ml · min¹ · W¹, similar to that previously reported (24, 25, 52).

Actual Changes in Blood CO₂ Content

View larger version (27K): [in this window] [in a new window]   Fig. 1.   PCO2 (A), CO2 concentration (B), pH (C), and O2 concentration (D) in both mixed venous (solid symbols) and arterial (open symbols) blood as related to O2 uptake (O2) normalized to the lactic acidosis threshold (@LAT) for 5 normal subjects. The first symbol on the left of each plot identifies the resting value, whereas the second symbol of each plot identifies the value at unloaded pedaling. Subsequent symbols from left to right are at approximately minute intervals during incre mental work to maximum (Max). The x-axis is normalized so that O2@LAT = 1.0. The actual average O2 at LAT is 2.0 with a range of 1.50-2.35 l/min. Note the widening differences between mixed venous and arterial values for all variables as work intensity increases. The differences become more marked above the LAT. The concentration of O2 was calculated from the following equation: concentration of O2 (mM) = (1.34 × Hb × SO2 + 0.00326 × PO2)  2.226, where SO2 is oxyhemoglobin saturation (51). The decrease in CCO2 during heavy exercise, despite the concurrent rise in PCO2, is noteworthy. Values are means ± SE.

It is impressive how little change took place in C_CO2 from rest (23 mM) to Max (24 mM), despite large changes in P_CO2 (47-78 Torr) (see Figs. 1 and 2 and Table 2). Initially, as exercise became more intense, C_CO2 increased more than Ca_CO2. C_CO2 then stabilized, whereas Ca_CO2 began to decrease (P < 0.05 or P < 0.01) (Fig. 1). As exercise increased to maximum, the C_CO2 decreased from its peak level (Table 2, at 5 min of incremental exercise) to slightly above its rest value in the case of C_CO2 (P < 0.01) and below the rest value in the case of mixed venous [HCO] (Fig. 2B). In contrast, Ca_CO2 decreased to a much greater degree with little change in Pa_CO2. Thus the progressive increase in C_CO2-Ca_CO2 below LAT was due to an increasing C_CO2, whereas above LAT it was primarily due to a decreasing Ca_CO2 (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   CO2 (CCO2; A) and plasma bicarbonate concentrations ([HCO]; B) as related to PCO2 at minute intervals in both mixed venous (solid symbols) and arterial (open symbols) blood in response to increasing work rate to Max. Arrows connect the corresponding arterial and mixed venous values at rest, LAT, and Max. Dotted lines, iso-pH values, assuming a Hb of 15 g/dl and a SO2 of 100%. A: from rest to LAT, CCO2 increased as PCO2 increased. Above LAT, CCO2 temporarily stabilized and then decreased, despite the increasing PCO2. CaCO2 increased slightly as PaCO2 and H+ increased below the LAT. Above the LAT, CaCO2 sharply decreased because of a decrease in [HCO], pH, and PaCO2. Mixed venous-arterial differences [CCO2-CaCO2] progressively widened as exercise intensity increased (Table 2). Below the LAT, increases in CCO2-CaCO2 were mainly due to the increases in CCO2; above LAT increases in CCO2-CaCO2 were primarily due to decreases in CaCO2. B: the similarity of the patterns in the changes in plasma [HCO] indicates the dominance of HCO as the primary form of CO2 in the PCO2-CCO2 relationship during exercise.

Influence of pH on the PCO₂-CCO₂ and PCO₂-[HCO] relationships during exercise. As shown in Fig. 2A and Table 2, the PCO₂-CCO₂ relationship was markedly influenced by pH changes during exercise. From rest to LAT, C_CO2 increased relatively less than P_CO2 because of a moderate decline in pH (P < 0.001). Transiently, above LAT, C_CO2 stabilized, despite further increases in P_CO2 due to the start of a fall in [HCO] reflected by the decrease in pH. Eventually, despite the continuously increasing P_CO2 (P < 0.001), C_CO2 decreased at Max (P < 0.05, Max vs. LAT) due to the marked decrease in pH (P < 0.001, Max vs. LAT). Simultaneously, on the arterial blood side, Ca_CO2 increased insignificantly at work rates below LAT (P > 0.05), whereas Pa_CO2 increased (P < 0.05) and pH_a decreased slightly (P < 0.05). Above LAT, Ca_CO2 and [HCO] decreased when lactic acid production increased, causing a significant decrease in pH_a (P < 0.001) with only slight decreases in Pa_CO2 (Fig. 2).

Errors in Def CCO₂ and CO_CO2 During Exercise

Figure 3 shows the percent errors of specific Def values of C_CO2-Ca_CO2, despite the use of correct measurements of PCO₂, when no change during exercise is assumed in Hb, SO₂, and/or pH. Defaulting changes in Hb during exercise results in trivial error (only 0.2 to 0.6%). Defaulting the change in SO₂ results in slight underestimation in C_CO2-Ca_CO2 over the entire exercise period (6 to 3%; P < 0.05). In contrast, defaulting changes in either pH alone or pH, Hb, and SO₂ together cause large errors, i.e., a progressive overestimation in C_CO2-Ca_CO2 as exercise intensity increases, reaching ~50% at LAT and 100% at Max.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Errors in CCO2-CaCO2 during exercise caused by ignoring changes in pH, Hb, and/or SO2 from resting values. Horizontal dotted line, actual value at each point calculated from measured mixed venous and arterial PCO2, pH, Hb, and SO2 at rest and during exercise, normalized to 0. Values are means ± SE. Each symbol indicates percent errors of CCO2-CaCO2 from the actual values caused by ignoring exercise-induced changes from rest in pH, Hb, and/or SO2 in mixed venous and arterial blood, despite correct values for PCO2 and PaCO2. Ignoring changes in pH or all 3 variables from rest causes overestimation of CCO2-CaCO2 exceeding 100% at Max. Ignoring changes in SO2 from rest causes underestimation of CCO2-CaCO2 by 3-6%. Ignoring changes in values of Hb causes trivial underestimation of CCO2-CaCO2. O2/O2@LAT, ratio of O2 to O2@LAT.

As seen in Fig. 4, the percent errors in CO_CO2 that result from defaulting the influence of Hb, SO₂, and/or pH on the PCO₂-CCO₂ relationship are always opposite to those on C_CO2-Ca_CO2. As with its effect on C_CO2-Ca_CO2, the change in Hb causes a trivial effect on CO_CO2 (only 0.6 to 0.2%), whereas the changes in SO₂ cause the estimate of CO_CO2 to be 3-6% higher than actual (P < 0.05). In contrast, defaulting on either pH alone or pH, Hb, and SO₂ together causes errors in CO_CO2 by >30% at LAT and >50% at Max.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Errors in cardiac output using CO2 (COCO2) during exercise caused by ignoring changes in pH, Hb, and/or SO2 from resting values. Horizontal dotted line, actual value calculated from measured mixed venous and arterial PCO2, pH, Hb, and SO2 at rest and during exercise and CO2 production, normalized to 0. Values are means ± SE. Each symbol indicates percent errors of COCO2 from the actual values caused by ignoring exercise-induced changes in pH, Hb, and/or SO2 in mixed venous and arterial blood, despite correct values for PCO2 and PaCO2. Ignoring changes in all 3 variables or pH alone from rest causes underestimation of COCO2 exceeding 50% at Max. Ignoring changes in SO2 from rest causes overestimation of COCO2 by 3-6%. Ignoring changes in Hb from rest causes trivial overestimation of COCO2.

Relative importance of multiple factors on the PCO₂-CCO₂ relationship during exercise. The influence of each of several factors on the PCO₂-CCO₂ relationship is shown in Table 3, with factor influence increasing as its value deviates from 1.0. Thus the deviations during exercise of F_bic (which are due to pH changes) far exceed those of F_Hb, both in mixed venous and arterial blood, during exercise (Table 3). Considering the subfactors of F_Hb, the deviations during exercise of F_HbpH (due to the effect of pH changes on [NH-CO₂]) exceed those of F_HbSO2 and F_HbHb, both in mixed venous and arterial blood. Thus the changes and influence of pH (F_bic and F_HbpH) are quantitatively much more important in blood CO₂ transport during exercise, both in mixed venous and arterial blood, than are the changes and influences of change in Hb and SO₂. These findings contrast importantly with the conclusions of McHardy (34).

Changes in Blood Components of CCO₂ During Exercise

View larger version (56K): [in this window] [in a new window]   Fig. 5.   Changes in CCO2 at 5 levels of exercise (rest, mild, moderate, heavy, and very heavy). In each group of 3 bars, left bar shows the total mixed venous () CCO2, middle bar is arterial (a) CCO2, and right bar is mixed venous-arterial (-a) CCO2 difference. Each bar is divided into the [HCO], carbamino concentration ([NH-CO2]), and physically dissolved CO2 concentration ([CO2]) components for whole blood. During exercise, changes in [CO2] (PCO2 dependent) and [NH-CO2] (PCO2, pH, Hb, and SO2 dependent) fractions are dwarfed by the changes in [HCO] (both PCO2 and pH dependent). Ex-2 and Ex-7, 2 and 7 min of incremental exercise, respectively.

In Fig. 5, both mixed venous [CO₂] and [NH-CO₂] increased progressively from rest to Max (P < 0.05). Smaller percent but larger absolute changes in [HCO] dwarfed the impact of the larger percent but smaller absolute changes in mixed venous [CO₂] and [NH-CO₂]. Below LAT, trends in change in C_CO2 were similar to those in [CO₂] or [NH-CO₂], but they differed markedly above LAT. The arterial [CO₂] and [NH-CO₂] increased only slightly from rest at LAT (P < 0.05) and then returned to near resting values at the highest work rate. In contrast to the relatively stable arterial [CO₂] and [NH-CO₂], large declines in arterial [HCO] above the LAT caused marked decreases in Ca_CO2. Thus changes in C_CO2 and Ca_CO2 conformed mainly to changes in [HCO] with changes in [CO₂] and [NH-CO₂] having a relatively small effect.

Referring to Table 4 (selected exercise intensities), ratios of CCO₂ to [CO₂] (CCO₂/[CO₂]) were calculated by dividing the CCO₂ by the sum of plasma and red blood cell components of [CO₂]. Note that the CCO₂/[CO₂] for mixed venous blood progressively decreased as the exercise intensity increased (P < 0.05), especially above LAT (P < 0.01). The CCO₂/[CO₂] in arterial blood, which did not change significantly below LAT (P > 0.05), progressively decreased at and above LAT (P < 0.05), because of the reduction in [HCO]. Thus CCO₂/[CO₂] is not constant but depends on the source of blood and the intensity of exercise.

It is also evident from Table 4 and Fig. 5 that plasma and red blood cell [HCO] together comprise ~85% (mixed venous) and 90% (arterial) of the CCO₂ at rest and during exercise. Even though the absolute values of mixed venous [HCO] are always greater than those of the arterial [HCO], the mixed venous [HCO]/CCO₂ are always lower than the arterial [HCO]/CCO₂ (P < 0.001) because of the greater amount of [CO₂].

Relative contributions of the three forms of CO₂-to-CO₂ exchange are shown for each level of exercise in Table 5. From rest to Max, [HCO] exchange remained large and quite constant at ~76-77% of total CO₂ excreted, i.e., C_CO2-Ca_CO2. In contrast, [CO₂] and [NH-CO₂] accounted for ~9 and 14%, respectively, of total CO₂ excreted at rest. Above LAT, the relative contribution to CO₂ output of [CO₂] progressively increased to 13% (P < 0.05) and that of [NH-CO₂] decreased to ~10% (P < 0.01).

Finally, to support the validity of our calculated values for Ca_CO2, C_CO2, and C_CO2-Ca_CO2 at all levels of exercise, we compared the CO_CO2 measured by the Fick principle with CO_O2 at rest and each level of exercise for the five subjects in this study (Table 6). At each level but Max, the mean values are in good agreement. As noted in Table 6, in other quite similar exercise studies (51), the CO_CO2 was insignificantly different from CO_O2 at peak and all other levels of exercise.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX A APPENDIX B REFERENCES

Major Findings

The Dominant Role of pH in the PCO₂-CCO₂ Relationship During Exercise

As commonly graphed, the PCO₂-CCO₂ relationship is depicted as nearly linear between PCO₂ values of ~30-80 Torr and CCO₂ values of ~12-28 mM but without reference to or depiction of the effect of a pH change (8, 38, 56). Changes in CCO₂ are sometimes fractionated into plasma and red blood cell components, and the differences between mixed venous and arterial blood due to Hb and SO₂ are also considered, again with exclusion of the effect of pH (8, 38, 56). However, as seen in Figs. 1 and 2A and Tables 1 and 2, widely divergent P_CO2 values (47-78 Torr) may occur with reasonably similar C_CO2 values (22.8-23.9 mM). This is due to relatively large decreases in the buffer base reflected in the change in pH (7.362-7.130) (Table 1 and Fig. 1). Furthermore, reasonably similar Pa_CO2 values (41-38 Torr) may pair with widely different Ca_CO2 values (20.9-15.2 mM) because of relatively large differences in pH. Thus when the C_CO2 is calculated from P_CO2, the change in blood pH during exercise must not be ignored. During heavy exercise, CCO₂ is not linearly related to the PCO₂ in either arterial or mixed venous blood (see Fig. 2A). In fact, PCO₂ and CCO₂ change in opposite directions in mixed venous blood as metabolic acid is added to the blood by the muscles. In contrast to original reports and textbooks (8, 38, 56), Fig. 2 shows that C_CO2 does not increase as a function of P_CO2 during exercise above the LAT.

We suggest that the addition of pH (or [H⁺]) isopleths to diagrams depicting the plasma [HCO]-PCO₂ and CCO₂-PCO₂ relationships, such as shown in Fig. 2, conveys necessary and important information that is lacking in the present standard depictions of these relationships (8, 38, 56). Such isopleths clarify and reinforce the importance of the acid-base changes that can occur during exercise.

Total blood [HCO] comprises ~85% (mixed venous) or 90% (arterial) of the CCO₂ (Table 4). Although the percent changes in mixed venous [CO₂] and [NH-CO₂] are appreciable (Tables 4 and 5 and Fig. 5), their impact is dwarfed by the magnitude of the large absolute changes in [HCO]. Additionally, Tables 2 and 3 and Fig. 3, which give Def values and factor ratios, analyze the relative importance of the components of CCO₂ in the Douglas equation and confirm the dominance of pH factors. In Table 3 and Fig. 3, it is shown that the pH change factor (F_bic from 1.00 at rest to 0.59 at Max) dominates over other factors. Within the red blood cell, the pH factor even dominates over the change in Hb concentration and SO₂ factors (Table 3). Thus making adjustments for the influences of Hb and SO₂ while ignoring pH to calculate C_CO2 or Ca_CO2 during exercise, as was done earlier (34), results in major errors.

Relevance of the CCO₂-PCO₂ Relationship to the Accuracy and Precision of Estimation of C_CO2-Ca_CO2 and CO_CO2

It would be convenient if errors in measurement of C_CO2 and Ca_CO2 were to be offset in the calculation of C_CO2-Ca_CO2, but this is not the case. It is evident from Table 2 that omitting the change in pH in calculating C_CO2-Ca_CO2 causes a much larger error than when C_CO2 and Ca_CO2 are calculated individually. Figure 4 shows that, when these erroneous measurements are applied to calculate cardiac output, errors of large magnitude result. In contrast, ignoring the changes in [Hb] and SO₂ during exercise results in relatively unimportant errors compared with ignoring changes in pH.

Possible Measurement and Calculation Errors in Our Data

We did not measure blood or body temperatures during exercise. From other studies using a similar exercise protocol, we estimated that the temperature increase from rest to peak exercise would be 0.2-0.8°C (51). We calculated that a temperature rise of 0.5°C during exercise would affect the values of Ca_CO2, C_CO2, and C_CO2-Ca_CO2 by <1% at peak exercise (51). Therefore, this small temperature change would not significantly alter our findings.

Considering the diversity of sources and complexity of equations in APPENDIX B that were used to calculate the six components of CCO₂, the sum values for each blood sample from the six sources agree remarkably well with those calculated from the simpler Douglas equation. The CCO₂ values calculated from the Douglas equation have excellent linear correlation with the total sum values at all levels of CCO₂ for both arterial and mixed venous blood (r = 0.9998, Table 4).

Possible Errors Due to a Disequilibria of pH and CO₂

1) End-tidal PCO₂ (PET_CO2) exceeds Pa_CO2 during exercise with a maintained negative Pa_CO2-PET_CO2 differnce of ~4 Torr, despite the increase in CO₂ to very high levels (54). If there were a disequilibrium, Pa_CO2-PET_CO2 difference and Pa_CO2 should increase relative to PET_CO2 as CO₂ increases. However, the change in PET_CO2 parallels the change in Pa_CO2 as CO₂ increases to maximum and remains above it (54).

2) If there were a disequilibrium, calculated dead space volume/tidal volume should increase as work rate increases, because Pa_CO2 would be increased relative to PET_CO2. This happens when a right-to-left shunt opens during exercise but does not happen normally. Dead space volume/tidal volume decreases with exercise and remains decreased to similar levels or decreases further as work rate increases to the maximum in normal subjects.

3) If there were a disequilibrium, CO₂ would not increase appropriately as high-metabolic rates are achieved and Pa_CO2 would increase. Increasing CO₂ keeps pace with increasing O₂ even for very fit men and exceeds O₂ once lactic acidosis occurs. If metabolic CO₂ plus CO₂ released from buffer were retained because of an alveolar-capillary disequilibrium as blood passed from pulmonary artery to pulmonary vein, we might expect to see a higher Pa_CO2 and arterial [HCO]. In fact, Pa_CO2 and arterial [HCO] decrease, without any evidence of CO₂ retention.

4) The decrease in arterial [HCO] is approximately equal to the increase in arterial lactate during high levels of exercise. If there were a disequilibrium, [HCO] would be relatively high, because it would not dissociate adequately in its passage from pulmonary artery to systemic artery. In fact, despite the very high P_CO2 for work above the LAT (Fig. 1), the Pa_CO2 and [HCO] decrease more than that of the mixed venous blood. Our major finding is that the decrease in Ca_CO2 is the primary explanation for the increase in C_CO2-Ca_CO2 during exercise at high work rates above the LAT, not the increase in C_CO2. This contradicts the changes we would see if there were a disequilibrium of significance.

Relative Contributions of Components of Blood CO₂-to-CO₂ Exchange Across the Lung

The traditional dissociation curve for CO₂ over the range of 30 to 70-80 Torr depicted in textbooks (8, 38, 56) suggests that a large part of the C_CO2-Ca_CO2 is dependent on the SO₂ difference between mixed venous and arterial blood. Considering the relative contributions of all components to CO₂ exchange that account for the increase in C_CO2-Ca_CO2 during exercise, our study shows that [HCO] accounts for over three-fourths of the total difference at rest and at all levels of exercise, whereas [CO₂] and [NH-CO₂], in combination, account for less than one-fourth of the total difference. Of this, approximately three-fifths come from [NH-CO₂] and two-fifths come from [CO₂] below LAT. This relationship reverses as work rate increases above LAT.

The difference in CCO₂ caused by changing the state of oxygenation of the blood at the same PCO₂ is fully attributable to the change in red blood cell [NH-CO₂]. Our calculations of [NH-CO₂] in C_CO2 and in Ca_CO2 at rest are not in agreement with earlier reports (38, 44), which estimated that one-third of CO₂ exchange was attributable to oxygen-induced changes in [NH-CO₂]. These earlier studies used blood devoid of 2,3-diphospho-D-glycerate (DPG). Later studies in blood that considered the effect of DPG (2, 30) found that the [NH-CO₂] contribution to CO₂ exchange at rest is only between 10 and 15%, in close agreement with our finding of 14%. If we had ignored the DPG effect, our CCO₂ values calculated from APPENDIX B would have deviated further from the Douglas equation CCO₂ values.

It is clear that the dissociation of [HCO] plays the dominant role in CO₂ exchange at the lung, whereas [CO₂] and [NH-CO₂] play smaller roles in total CO₂ exchange. Although PCO₂ differences account for the transfer of CO₂ out of blood, >75% of the quantity transferred comes from the dissociation of mixed venous [HCO]. We have shown that C_CO2 in blood actually decreases during exercise above the LAT, despite increasing PCO₂. The major reason for this is that the CO₂ dissociation curve is shifted downward when lactic acid is generated during exercise. Simultaneously, the P_CO2 increases as additional CO₂ over that produced from metabolism is released from the HCO buffering of lactic acid. Because of changing [HCO] and pH during exercise, it is inappropriate to determine CCO₂ from extrapolations that assume a near-linear CCO₂-PCO₂ relationship.