Body carbon dioxide NORMAN Department
storage capacity
L. JONES AND JANET E. JURKOWSKI of Medicine, ilkMaster University Medical
in exercise
Centre, Hamilton,
Ontario
L8S 4J9, Canada
were studied to provide a wide range of body size and fitness and to obtain a range of CO2 outputs during exercise (Table 1). The subjects exercised on a calibrated cycle ergometer (Elema model AM 360) at two power outputs, approximating 30 and 60% of the maximal oxygen uptake (Vozmax); th ese power outputs were chosen on the basis of a preliminary maximal exercise study, so that they could be maintained in a steady state for at least 30 min, and were at a sufficiently low level that ventilation could be voluntarily controlled. At each power output the subject breathed normally for 10 min to reach a steady state of 02 intake and CO2 output (Fig. 1); during the last 2 min of this “control” period measurements were made of ventilation (\j~), oxygen intake (voz), carbon dioxide output @co& endtidal PCO~ (PETco,), and rebreathing mixed venous PCO~ (PVco,). After a further 1 min to allow for the elimination of any CO2 retained during rebreathing, ventilation was voluntarily increased to lower PETCO, from about 40 to hyperventilation; mixed venous PCO~ about 25 Torr. Preliminary studies had indicated that if the increase in ventilation was achieved using the same or an increased tidal volume in combination with an THE BODY'S STORAGE CAPACITY for CO2 has been esti- increased frequency, this change of PET~Q, was associated mated in animals and humans by measuring the CO2 with a fall in PVco, of at least 10 Tar?. The subject excreted by hyperventilation or the CO2 retained by voluntarily maintained tidal volume and breathing frerebreathing. In hyperventilation experiments the storage quency at appropriately increased levels by watching capacity is calculated from the CO* excreted, and the PETCO, displayed on an oscilloscope. Although the ability change in mixed venous Pco~, and is expressed in terms to reduce PETCO, abruptly and maintain a low PETIT, in of body weight. Reviews of the topic (3, 9) have noted steady state varied from subject to subject, they were the wide variation in reported values, mainly accounted able to maintain PETROL within t 2.5 Torr of the target for by the duration of the studies. This factor was em- level. During the study, measurements of TE, v02, Tco~, phasized recently by Khambatta and Sullivan (20) in and PETE?, were available immediately, which enabled careful studies of the washout of CO2 induced by hyperhyperventilation to be continued until the respiratory ventilation of anesthetized dogs, in which they found that exchange ratio (R) had reached control (prehyperventi20-30 min were required to reach a new steady state of lation) values. A final measurement of PVco, was then Con output. A second factor not always allowed for in made. previous studies is the increase in metabolic production The subject breathed through a low-resistance lowof COa that accompanies alkalosis and if ignored will dead-space valve. Inspiratory ventilation was measured result in an overestimate of the CO2 washed out. by a dry gas meter (Parkinson Cowan CD-4). Expired gas During exercise, mixed venous PCO~ increases, and CO2 was led through a g-liter mixing chamber containing a is “stored.” Only two studies of body CO2 storage capacity fan and was analyzed both close to the lips and distal to in humans have examined exercise (5, 11); in both, the the mixing chamber, using 02 (Godart Rapox) and CO2 duration of the experiments was short. The present study (Godart Capnograph) analyzers. The gas analyzers were examined CO2 washout by hyperventilation at two levels calibrated with several gasesin the required range, which of exercise; frequent measurements of CO2 output were had been analyzed using the Lloyd-Haldane apparatus. made during each study to ensure that hyperventilation Analog data were displayed on an eight-channel recorder was continued until the steady state of CO2 output and (Mingograf 81, Siemens-Elema). In addition, the signals respiratory exchange ratio was reestablished. were processed by an analog-to-digital converter and presented to a PDP-8-1 computer, which made the necMATERIALS AND METHODS essary calculations and displayed variables on-line every Nine healthy subjects, six males and three females, 20 s (17). These measurements had been previously val-
JONES,NORMAN L., ANDJANETEJURKOWSKI. Bodycarbon dioxide storage capacity in exercise. J. Appl. Physiol.: Respirat. 1979.-Body COz Environ. Exercise Physiol. 46(4): 811415, storage capacity was measured in nine subjects at two levels of exercise, approximating 30 and 60% VOW max, by measuring the excess CO:! output associated with hyperventilation at constant end-tidal Pco~, and the change in mixed venous PCO~ (PVco,) measured by rebreathing. CO2 output was measured during 20s periods and monitored throughout the procedure; hyperventilation was continued until CO2 output had returned to control values. Washout of CO2 was more rapid than previously found at rest, 90% of the change following an increase in ventilation occurring within 4 min. CO2 storage capacity was 1.83 t 0.552 (SD) ml. kg-‘*Torr-* at the lower power output and 1.19 t 0.490 kg-’ Torr- ’ at the higher power output. The CO:! storage capacity was inversely related to Pvco, . It was concluded that the body’s capacity to store CO* decreases with increasing Pvco,; this may be one factor leading to the progressive increase in pulmonary CO2 output at high levels of exercise. ml
l
l
0161-7567/79/oooO-0000$01.25
Copyright
0 1979 the American
Physiological
Society
811
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812
N. L.JONES
idated by comparisons with expired gas collections using a Tissot spirometer. Oxygenated Pvco, was measured by a rebreathing equilibration technique (17, 19). The “washout” of CO2 was followed every 20 s using the computed data and plotted in the form of a graph; a line of best fit was drawn and the CO2 washed out could be calculated from the area under the curve (Fig. 1). In many but not ail studies, an increase in oxygen intake accompanied hyperventilation. To allow for the associated increase in metabolic CO2 production, a metabolic respiratory quotient (RQ) was assumed equal to the R obtained in the steady-state control conditions. Thus, for 1. Anthropometric details experimental results ____-~
TABLE
and -~~_~---- _ ___-~-
Low Power Output Subj
AR 0433 yr, 86 kg, 185 cm) NJ 0% 43 yr, 97 kg, 188 cm) RT (M, yr, 84 kg, 185 cm) JH W, 23 yr, 53 kg, 157 cm) MM W, 26 yr, 77 kg, 165 cm) JS M 32 VP84 kg, 185 cm) EH E 31 yr, 34 kg, 163 cm) JM M 32 p, 94 kg, 174 cm) MS 0% 32 v, 78 kg, 173 cm)
26
co*
i Initial
Final
storagy, dT;$ 1-
1,040
64.4
50.1
2.48
1,290
76.9
56.1
1,440
69.7
820
con
Ph‘p Initial
Final
storag:, y-k.& I *
1,590
64.9
49.8
1.70
1.09
1,910
79.1
55.8
0.74
50.8
2.76
1,930
74.4
58.6
2.39
60.7
42.7
1.62
1,180
71.3
57.3
1.07
940
70.0
48.5
2.01
1,340
75.2
56.2
0.73
1,910
72.3
61.4
2.27
2,190
80.0
62.0
1.69
47.4
2.41
1,260
84.6
71.0
0.82
1,110
52.1
1.44
1,820
74.3
60.0
1.12
1,300
57.4
1.05
2,~
78.9
58.9
0.98
VO2, I
I
ml-min-’
Ton-
VOZ,
ml-min-’
AND J. RJURKOWSKI
all subjects, “net” CO2 washed out was calculated by subtracting measured voz x steady-state R from the measured CO2 output. The CO2 washed out was also plotted as a function of time on semilogarithmic graph paper (Fig. 2); two exponential functions were derived graphically to obtain half times for each function. The total CO2 washed out was divided by the change in Pvco, and by body weight to obtain a CO2 storage capacity expressed as ml CO2 kg- ’ Torr- ‘. l
l
RESULTS
The increase in VE and step change in PETUI, led to an increase in %02 that in most subjects, ran-a time course similar to that shown in Fig. 1. An increase in v02 did not accompany hyperventilation invariably: when it did, the increase was small in amount, on average 42 ml. min-’ (range o-120). There was variation in this effect between subjects but not between loads; a subject showing an increase at one work rate would also have an increase at the second. This was not related quantitatively to the reduction in PVcc, or PETco,. In most studies two exponential functions of CO2 washout with time were identified (Fig. 2). The half time (t& of the initial rapid component was 1 min or less in all studies in which an abrupt step change in Pc02 was achieved (Table 2); no consistent difference was found between the two exercise intensities. The t1,2 of the slower component varied between 2.3 and 7 min and was usually shorter at the higher exercise level (Table 2). Hyperventilation led to an average fall in Pvco, amounting to 17.8 Torr (range 10.9-23.8 Torr) (Table l), which was similar at the two power outputs. Carbon dioxide storage capacity was greater at the lower power output (1.83 t 0.552 (SD) ml. kg-’ l Torr-‘) than the higher (1.19 t 0.409 (SD) ml. kg-loTorr-l; P < 0.001). The storage capacity was related to the average PVco, in each study (Fig. 3), the relationship being expressed by the following equation y = 4.48 -0.052x
FIG. 1. Oxygen intake and CO2 output during one experiment in a subject exercising at 400 kpm/min. Measurements began after 5 min exercise. At 1st arrow PTco, was measured. Hyperventilation began at 9 min (2nd arrow) and was continued until 24 min when a second measurement of PVc:o, was made (3rd arrow). Note that scales for 02 intake and CO2 output are displaced to avoid overlap.
I //5
I 15
10 TIME
1 20
25
(MINUTES)
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CO:! STORES
IN
813
EXERCISE
‘?io 1
OXYGENATED
1 50
1 60
MIXED
VENOUS
I
I
70
80 PcO
2
mm
Hg
FIG. 3. Relationship between CO:! storage capacity and the average of initial and final measurements of Pvco, for individual studies: two points are shown for each subject.
2
4
6
8
10
12
MINUTES 2. Volum: of CO2 washed out (Akoz) plotted as a function of time following onset of hyperventilation in a typical subject (JM) during exercise. A%02 is expressed as ml. min-’ and corrected for subject’s weight (kg) and change in PE~I,, allowing comparison with data obtained in a resting subject by Brandi and Clode (1). Washout curve has been analyzed in terms of two exponential functions (dashed Lines). Corresponding functions obtained by Brandi and Clode are shown dotted. FIG.
TABLE
.-
2. Half times of CO2 washout .--. -.----- _~_-_-~Low Power Output
Subj
t11’2r (initial) min
0.6 0.6
AR NJ RT JH MM JS EH JM MS
1.0 0.7 0.9 0.8 * Abrupt
step decreases
~--
High Power Output t1 , L’9 (slow) min
7.0 4.5 * 5.4 3.0 3.9 * 4.8 * in PET~O, were not
twr (initial) min
0.6 0.6 1.0 0.5 0.8 0.9 0.8 0.7 achieved
tm, (slow) min
4.2 2.8 3.2 2.3 * 4.4 3.6 3.5 3.7 in these studies.
where y is storage capacity in ml kg-‘. Torr-’ and x is the average oxygenated PVco,, i.e., control PVco, - [(control PVco, - final PVco,)/2] Torr. The correlation coefficient of the relationship was 0.600. DISCUSSION
The primary purpose of the present study was to obtain estimates of CO2 storage during exercise. PVco,
increases progressively with increasing work rates, indicating that storage of CO2 during exercise may be substantial, but measurements of CO* storage capacity in exercising subjects have not been extensive (5, 11). Two methods have been used in previous studies of body CO2 storage capacity (3). In the first and more widely used method, the volume of CO2 excreted following an increase in ventilation is measured and related to the fall in arterial, alveolar, or mixed venous Pco~. The second method employs rebreathing; body CO2 production is assumed to remain constant during the time of rebreathing and the increase in alveolar PCO~ is measured. In hyperventilation and CO2 breathing experiments at rest, a steady state of CO2 output equal to that preceding hyperventilation is not achieved for 0.5 h (1,3,20, 23). Brandi and Clode (1) emphasized the importance of using a step change in alveolar Pco~, as distinct from a step change in ventilation, in measuring the rate of CO2 washout. They found that CO2 washout in one subject was described by two exponential functions. The first, having a half time of 1.12 min, accounted for only 25% of the storage capacity and was consistent with CO2 stores in the lungs, blood, and well-perfused organs. The second had a half time of 13.4 min and accounted for 75% of the CO2 washed out, representing a large volume of CO2 in less well-perfused tissue, probably muscle. Finally, there is also a pool of CO2 that turns over very slowly (3) and consists of CO2 in bone; the turnover rate is so slow as to be irrelevant to the present discussion. In the present studies it was possible to identify two rates of CO2 clearance, both substantially more rapid than those found by Brandi and Clode (1) in man at rest (Fig. 2). The initial clearance, in addition to having a shorter half time, accounted for over 75% of the CO2 washed out. Thus, although it takes 15 min for 90% of the CO2 to be washed out at rest (l), during exercise the equivalent time is reduced to 4 min. This increase in the rate of CO2 washout is consistent with a shift of the large muscle CO2 pool into a rapidly clearing well-perfused compartment. The increase in initial clearance rate is probably explained by the increase in cardiac output in exercise, which will increase the volume of blood presented to the lungs in a given time. Also, as the metabolic CO2 production is increased in exercise; a given reduction in alveolar PcoB requires an alveolar ventilation up to 10 times greater than at rest, resulting in a more rapid excretion of CO2 from stores.
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814
N. L. JONES
Our findings are consistent with experiments in which the rate of equilibration of CO2 was measured in isolated muscle. Cherniack and colleagues (4) found that the apparent slope of the CO2 dissociation curve for resting muscle did not reach a steady value (3.5 mlkg-’ 9 TOW’) until 20 min after a step change in Pco~. They explained this finding by the inhomogeneity of muscle perfusion and supported this theory by finding an increase in the slope of the dissociation curve and the rate of CO2 equilibration after the intravenous administration of dinitrophenol, which increases muscle metabolism and perfusion. Studies of “j3Xe clearance from muscle have shown that muscle perfusion increases from resting levels of l-3 ml0 min-’ 100 g-’ to over 40 in exercise (14). Thus exercise may lead to an apparent increase in the body storage capacity for CO2 by more rapid equilibration of CO2 due to both the increase in turnover of CO2 and an increase in muscle perfusion. Two previous studies have examined CO2 storage capacity during exercise. Fowle and Campbell (11) measured the rate of rise in PcoB during rebreathing. The immediate CO2 storage capacity calculated by this *Tori? at rest, increasing in method was 0.56 mlkg-’ two subjects to 0.6-0.8 ml. kg-’ l Torr-’ during exercise. Clode, Clark, and Campbell (5) found values close to 1 ml kg-’ •T~rr-’ during exercise using rebreathing and 6 min of hyperventilation. Because rebreathing experiments in exercise can seldom be continued for longer than 1.5 min (II), it is likely that rebreathing measurements represent changes in the most accessible CO2 stores and may not represent fully the capacity for CO2 storage that is available during exercise maintained for several minutes. Following the practice established previously in studies of CO2 storage capacity in humans (5,11,12), the change in tissue PCO~ was assumed equal to the change in oxygenated Pv co, measured by rebreathing. The relationship of this change to changes in “true” (pulmonary artery) PCO~ is influenced by at least two factors. First, several studies in exercising humans have shown that the Pcoz at which equilibration occurs during the rebreathing of CO2 in 02 may be appreciably higher than the Pcoz of blood drawn from a systemic artery during rebreathing (18, 19) or from the pulmonary artery (6). Controversy still exists regarding the cause of this difference. The equilibrium PCO~ may represent the instantaneous PCO~ (lo), which because of the finite reaction time of CO2 in whole blood (16) may be higher than the PCO~ measured by electrodes some minutes later. However, recent studies by Effros et al. (8) suggest that equilibration of CO2 in blood traversing the lungs is enhanced by pulmonary tissue carbonic anhydrase. These findings tend to support the existence of a true alveolar-to-capillary PCO~ difference (15). The presence of such a difference in rebreathing theoretically will lead to a small overestimate of “true” PVco, and thus to a small underestimate of “true” CO2 storage capacity. Second, the relationship between oxygenated PVco, , as measured by the equilibrium rebreathing technique, and true PVco, is influenced by the effect of 02 saturation on the position of the CO2 dissociation curve (the Christiansen-Douglas-Haldane effect) (23). As “oxygenated” and l
l
AND
J. E. JURKOWSKI
“reduced” curves are almost parallel, a given change in oxygenated Pvco, is accompanied by a similar change in true Pvco,. However, at high work loads when P&O., is high and venous 02 low the absolute true PVco, will- be lower in relation to measured PVco, than at lower levels of exercise when venous 02 is higher. This means that the flattening of the body in vivo CO2 dissociation curve at higher levels of exercise may actually be more apparent when expressed in terms of changes in true P&O, than oxygenated Pvco,. A source of variation in previous studies of CO2 storage capacity is the assumption of unchanged metabolic CO2 production during hyperventilation. Cain (2) and Khambatta and Sullivan (20) studied dogs at rest, passively hyperventilated to arterial PCO~ levels of lo-15 Torr and arterial pH of over 7.7, and observed an increase in oxygen uptake that was linearly related to the fall in H+ concentration (2). Thus, if the associated increase in CO2 output was not allowed for, the calculated CO2 storage capacity would have been in error. In view of the fact that the increase in oxygen uptake amounted to some 20% of the control oxygen intake, this effect was appreciable in their studies, which were performed at a low level of tissue metabolism. During exercise and in studies where hyperventilation is associated with smaller changes in arterial PCO~ and arterial H+ concentration the relative increase in J?o~ is less (7). In our studies the largest increase in 02 intake amounted to 6% of the control (prehyperventilation) value. The increases in To2 associated with hyperventilation are accompanied by increases in plasma lactate, which may also increase CO2 output. The mechanism underlying both changes is thought to be an increase in glycolysis due to a pH-linked activation of the rate-limiting enzyme phosphofructokinase (PFK) in liver (13) and red blood cells (25). The possible effects of these changes on the assumptions made in calculating CO:! washed out of stores by hyperventilation are impossible to calculate with present information. However, it seems likely that in situations where the changes in PCO~and pH are less than those used in the previous in vitro and animal studies, the effects on metabolism will be insignificant, especially when related to the high body metabolism in exercise. Previous studies of CO2 storage capacity have not examined the linearity of the whole-body CO* dissociation curve. As the dissociation curve in blood is nonlinear, it would be surprising if the whole-body curve were linear. Farhi and Rahn (9) made an extensive theoretical analysis based on an electrical analog model. They assumed that the slope of the CO2 dissociation curve in blood and tissues was constant in the physiological range while realizing that departures from linearity outside this range would affect calculation of CO2 storage capacities. In support of this they pointed out that one of the highest reported values for the whole-body CO2 dissociation curve, 3.8 ml. kg-’ *TOW’ (21), might be accounted for by the low final PCO~ of 15 Torr following the hyperventilation used in the washout of COZ. An important difference wiIl exist between rest and exercise with respect to the venous blood pool and tissues. At rest, hyperventilation may lead to both venous and arterial PCO~ reaching
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CO2 STORES
IN
815
EXERCISE
levels at which the CO2 dissociation curve is steep, whereas in exercise the increased venoarterial PCO~ difference will tend to maintain venous and tissue Pcoz within the flatter portion of the curve. Extrapolation of our data (Fig. 3) to the left would indicate a CO2 storage capacity calculated during hyperventilation experiments at rest ( Pvco, falling from 45 to 30 Torr) of about 3.2 ml. kg-’ Torr-‘, which is consistent with results of experiments at rest lasting longer than 20 min (1, 3). Our finding of a lower CO2 storage capacity at higher levels of exercise suggest that the relationship between PCO~ and body CO2 stores is not linear and tends to flatten at high Pcoz. Thus the ability to store CO2 may fall with increasing PVco, during heavy exercise due to a progressive flattening of the in vivo CO2 dissociation curve. Another factor that presumably will be important at higher levels of exercise than we studied is the fall in
plasma and muscle HCOs concentrations accompanying lactic acid accumulation. Although a fall in HCOs and pH will have little effect on the slope of the CO2 dissociation curve (22), the absolute CO2 stores for a given Pvco, will fall. Thus in exercise of progressively increasing power, a diminishing CO2 storage capacity may lead to a progressive increase in CO2 excretion if large increases in Pvco,, and thus of tissue PCO~ and H+ ion concentration, are to be avoided. This mechanism may play an important part in the relative increase in ventilation that is observed above the so-called “anaerobic threshold” (24) associated with a progressive increase in CO2 output and plasma lactate concentration. This study was supported Research Council of Canada. Received
16 May
1978; accepted
by Grant in final
MA-4234 form
from
28 November
the
Medical
1978.
REFERENCES 1. BRANDI, G., AND M. CLODE. CO2 washout during hyperventilation in man. Respir. Physiol. 7: 163-172, 1969. 2. CAIN, S. M. Increased oxygen uptake with passive hyperventilation of dogs. J. AppZ. Physiol. 28: 4-7, 1970. 3. CHERNIACK, N. S., AND G. S. LONGOBARDO. Oxygen and carbon dioxide gas stores of the body. PhysioZ. Reu. 50: 196-243, 1970. 4. CHERNIACK, N. S., G. S. LONGOBARDO, AND A. P. FISHMAN. Transients in carbon dioxide stores. In: Carbon Dioxide and Metabolic Regulations, edited by G. Nahas and K. E. Shaefer. New York: Springer-Verlag, 1974, p. 324-338. 5. CLODE, M., T. J. H. CLARK, AND E. J. M. CAMPBELL. The immediate CO2 storage capacity of the body during exercise. CZin. Sci. 32: 161-165, 1967. 6. DENISON, D., R. H. T. EDWARDS, G. JONES, AND H. POPE. Direct and rebreathing estimates of the 02 and CO2 pressures in mixed venous blood. Respir. Physiol. 7: 326-334, 1969. 7. EDWARDS, R. H. T., AND M. CLODE. The effect of hyperventilation on the lactacidaemia of muscular exercise. CZin. Sci. 38: 269-276, 1970. 8. EFFROS, R. M., R. S. Y. CHANG, AND P. SILVERMAN. Acceleration of plasma bicarbonate conversion to carbon dioxide by pulmonary carbonic anhydrase. Science 199: 427-429, 1978. 9. FARHI, L. E., AND H. RAHN. Dynamics of changes in carbon dioxide stores. Anesthesiology 2 1: 604-614, 1960. 10. FORSTER, R. E. Can alveolar PCO~ exceed pulmonary end-capillary COZ? No. J. AppZ. Physiol.: Respirat. Environ. Exercise Physiol. 42: 323-328, 1977. 11. FOWLE, A. S. E., AND E. J. M. CAMPBELL. The immediate carbon dioxide storage capacity of man. CZin. Sci. 27: 41-49, 1964. 12. FOWLE, A. S. E., C. M. E. MATTHEWS, AND E. J. M. CAMPBELL. The rapid distribution of ‘Hz0 and “COe in the body in relation to the immediate carbon dioxide storage capacity. CZin. Sci. 27: 51-65, 1964. 13. GEVERS, W., AND E. DOWDLE. The effect of pH on glycolysis in vitro. CZin. Sci. 25: 343-349, 1963.
14. GRIMBY, G., E. HAGGENDAL, AND B. SALTIN. Local xenon 133 clearance from the quadriceps muscle during exercise in man. J. AppZ. Physiol. 22: 305-310, 1967. 15. GURTNER, G. H. Can alveolar PCO~exceed pulmonary end-capiIlary COZ? Yes. J. AppZ. PhysioZ.: Respirat. Environ. Exercise Physiol. 42: 323-326, 1977. 16. HILL, E. P., G. G. POWER, AND R. D. GILBERT. Rate of pH changes in blood plasma in vitro and in vivo. J. AppZ. Physiol.: Respirat. Environ. Exercise Physiol. 42: 928-934, 1977. 17. JONES, N. L., E. J. M. CAMPBELL, R. H. T. EDWARDS, AND D. G. ROBERTSON. CZinicaZ Exercise Testing. Philadelphia, PA: Saunders, 1975. 18. JONES, N. L., E. J. M. CAMPBELL, R. H. T. EDWARDS, AND G. WILKOFF. Alveolar to blood PCO~ difference during rebreathing in exercise. J. AppZ. Physiol. 27: 356-360, 1969. 19. JONES, N. L., E. J. M. CAMPBELL, G. J. R. MCHARDY, B. E. HIGGS, AND M. CLODE. The estimation of mixed venous blood during exercise. CZin. Sci. 32: 311-327, 1967. 20. KHAMBATTA, H. J., AND S. F. SULLIVAN. Carbon dioxide production and washout during passive hyperventilation alkalosis. J. AppZ. PhysioZ. 37: 665-669, 1974. 21. LILLEHEI, J. P. AND B. BALKE. Studies OfHyperuentiZation. USAF School of Med. Rept. 55-62, 1955. 22. MCHARDY, G. J. R. The relationship between the differences in pressure and content of carbon dioxide in arterial and venous blood. CZin. Sci. 32: 299-309, 1967. 23. SHEPHARD, R. J. The carbon dioxide balance-sheets of the body: Their determination in normal subjects and in cases of congenital heart disease. J. Physiol. London 129: 142-158, 1955. 24. WASSERMAN, K., B. J. WHIPP, S. M. KOYAL, AND W. L. BEAVER. Anaerobic threshold and respiratory gas exchange during exercise. J. AppZ. Physiol. 35: 236-243, 1973. 25. ZBOROWSKA-SLUIS, D. T., AND G. A. KLASSEN. Carbon dioxide mediated glycolysis. II. Respir. Physiol. 19: 162-175, 1973.
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storage capacity
L. JONES AND JANET E. JURKOWSKI of Medicine, ilkMaster University Medical
in exercise
Centre, Hamilton,
Ontario
L8S 4J9, Canada
were studied to provide a wide range of body size and fitness and to obtain a range of CO2 outputs during exercise (Table 1). The subjects exercised on a calibrated cycle ergometer (Elema model AM 360) at two power outputs, approximating 30 and 60% of the maximal oxygen uptake (Vozmax); th ese power outputs were chosen on the basis of a preliminary maximal exercise study, so that they could be maintained in a steady state for at least 30 min, and were at a sufficiently low level that ventilation could be voluntarily controlled. At each power output the subject breathed normally for 10 min to reach a steady state of 02 intake and CO2 output (Fig. 1); during the last 2 min of this “control” period measurements were made of ventilation (\j~), oxygen intake (voz), carbon dioxide output @co& endtidal PCO~ (PETco,), and rebreathing mixed venous PCO~ (PVco,). After a further 1 min to allow for the elimination of any CO2 retained during rebreathing, ventilation was voluntarily increased to lower PETCO, from about 40 to hyperventilation; mixed venous PCO~ about 25 Torr. Preliminary studies had indicated that if the increase in ventilation was achieved using the same or an increased tidal volume in combination with an THE BODY'S STORAGE CAPACITY for CO2 has been esti- increased frequency, this change of PET~Q, was associated mated in animals and humans by measuring the CO2 with a fall in PVco, of at least 10 Tar?. The subject excreted by hyperventilation or the CO2 retained by voluntarily maintained tidal volume and breathing frerebreathing. In hyperventilation experiments the storage quency at appropriately increased levels by watching capacity is calculated from the CO* excreted, and the PETCO, displayed on an oscilloscope. Although the ability change in mixed venous Pco~, and is expressed in terms to reduce PETCO, abruptly and maintain a low PETIT, in of body weight. Reviews of the topic (3, 9) have noted steady state varied from subject to subject, they were the wide variation in reported values, mainly accounted able to maintain PETROL within t 2.5 Torr of the target for by the duration of the studies. This factor was em- level. During the study, measurements of TE, v02, Tco~, phasized recently by Khambatta and Sullivan (20) in and PETE?, were available immediately, which enabled careful studies of the washout of CO2 induced by hyperhyperventilation to be continued until the respiratory ventilation of anesthetized dogs, in which they found that exchange ratio (R) had reached control (prehyperventi20-30 min were required to reach a new steady state of lation) values. A final measurement of PVco, was then Con output. A second factor not always allowed for in made. previous studies is the increase in metabolic production The subject breathed through a low-resistance lowof COa that accompanies alkalosis and if ignored will dead-space valve. Inspiratory ventilation was measured result in an overestimate of the CO2 washed out. by a dry gas meter (Parkinson Cowan CD-4). Expired gas During exercise, mixed venous PCO~ increases, and CO2 was led through a g-liter mixing chamber containing a is “stored.” Only two studies of body CO2 storage capacity fan and was analyzed both close to the lips and distal to in humans have examined exercise (5, 11); in both, the the mixing chamber, using 02 (Godart Rapox) and CO2 duration of the experiments was short. The present study (Godart Capnograph) analyzers. The gas analyzers were examined CO2 washout by hyperventilation at two levels calibrated with several gasesin the required range, which of exercise; frequent measurements of CO2 output were had been analyzed using the Lloyd-Haldane apparatus. made during each study to ensure that hyperventilation Analog data were displayed on an eight-channel recorder was continued until the steady state of CO2 output and (Mingograf 81, Siemens-Elema). In addition, the signals respiratory exchange ratio was reestablished. were processed by an analog-to-digital converter and presented to a PDP-8-1 computer, which made the necMATERIALS AND METHODS essary calculations and displayed variables on-line every Nine healthy subjects, six males and three females, 20 s (17). These measurements had been previously val-
JONES,NORMAN L., ANDJANETEJURKOWSKI. Bodycarbon dioxide storage capacity in exercise. J. Appl. Physiol.: Respirat. 1979.-Body COz Environ. Exercise Physiol. 46(4): 811415, storage capacity was measured in nine subjects at two levels of exercise, approximating 30 and 60% VOW max, by measuring the excess CO:! output associated with hyperventilation at constant end-tidal Pco~, and the change in mixed venous PCO~ (PVco,) measured by rebreathing. CO2 output was measured during 20s periods and monitored throughout the procedure; hyperventilation was continued until CO2 output had returned to control values. Washout of CO2 was more rapid than previously found at rest, 90% of the change following an increase in ventilation occurring within 4 min. CO2 storage capacity was 1.83 t 0.552 (SD) ml. kg-‘*Torr-* at the lower power output and 1.19 t 0.490 kg-’ Torr- ’ at the higher power output. The CO:! storage capacity was inversely related to Pvco, . It was concluded that the body’s capacity to store CO* decreases with increasing Pvco,; this may be one factor leading to the progressive increase in pulmonary CO2 output at high levels of exercise. ml
l
l
0161-7567/79/oooO-0000$01.25
Copyright
0 1979 the American
Physiological
Society
811
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812
N. L.JONES
idated by comparisons with expired gas collections using a Tissot spirometer. Oxygenated Pvco, was measured by a rebreathing equilibration technique (17, 19). The “washout” of CO2 was followed every 20 s using the computed data and plotted in the form of a graph; a line of best fit was drawn and the CO2 washed out could be calculated from the area under the curve (Fig. 1). In many but not ail studies, an increase in oxygen intake accompanied hyperventilation. To allow for the associated increase in metabolic CO2 production, a metabolic respiratory quotient (RQ) was assumed equal to the R obtained in the steady-state control conditions. Thus, for 1. Anthropometric details experimental results ____-~
TABLE
and -~~_~---- _ ___-~-
Low Power Output Subj
AR 0433 yr, 86 kg, 185 cm) NJ 0% 43 yr, 97 kg, 188 cm) RT (M, yr, 84 kg, 185 cm) JH W, 23 yr, 53 kg, 157 cm) MM W, 26 yr, 77 kg, 165 cm) JS M 32 VP84 kg, 185 cm) EH E 31 yr, 34 kg, 163 cm) JM M 32 p, 94 kg, 174 cm) MS 0% 32 v, 78 kg, 173 cm)
26
co*
i Initial
Final
storagy, dT;$ 1-
1,040
64.4
50.1
2.48
1,290
76.9
56.1
1,440
69.7
820
con
Ph‘p Initial
Final
storag:, y-k.& I *
1,590
64.9
49.8
1.70
1.09
1,910
79.1
55.8
0.74
50.8
2.76
1,930
74.4
58.6
2.39
60.7
42.7
1.62
1,180
71.3
57.3
1.07
940
70.0
48.5
2.01
1,340
75.2
56.2
0.73
1,910
72.3
61.4
2.27
2,190
80.0
62.0
1.69
47.4
2.41
1,260
84.6
71.0
0.82
1,110
52.1
1.44
1,820
74.3
60.0
1.12
1,300
57.4
1.05
2,~
78.9
58.9
0.98
VO2, I
I
ml-min-’
Ton-
VOZ,
ml-min-’
AND J. RJURKOWSKI
all subjects, “net” CO2 washed out was calculated by subtracting measured voz x steady-state R from the measured CO2 output. The CO2 washed out was also plotted as a function of time on semilogarithmic graph paper (Fig. 2); two exponential functions were derived graphically to obtain half times for each function. The total CO2 washed out was divided by the change in Pvco, and by body weight to obtain a CO2 storage capacity expressed as ml CO2 kg- ’ Torr- ‘. l
l
RESULTS
The increase in VE and step change in PETUI, led to an increase in %02 that in most subjects, ran-a time course similar to that shown in Fig. 1. An increase in v02 did not accompany hyperventilation invariably: when it did, the increase was small in amount, on average 42 ml. min-’ (range o-120). There was variation in this effect between subjects but not between loads; a subject showing an increase at one work rate would also have an increase at the second. This was not related quantitatively to the reduction in PVcc, or PETco,. In most studies two exponential functions of CO2 washout with time were identified (Fig. 2). The half time (t& of the initial rapid component was 1 min or less in all studies in which an abrupt step change in Pc02 was achieved (Table 2); no consistent difference was found between the two exercise intensities. The t1,2 of the slower component varied between 2.3 and 7 min and was usually shorter at the higher exercise level (Table 2). Hyperventilation led to an average fall in Pvco, amounting to 17.8 Torr (range 10.9-23.8 Torr) (Table l), which was similar at the two power outputs. Carbon dioxide storage capacity was greater at the lower power output (1.83 t 0.552 (SD) ml. kg-’ l Torr-‘) than the higher (1.19 t 0.409 (SD) ml. kg-loTorr-l; P < 0.001). The storage capacity was related to the average PVco, in each study (Fig. 3), the relationship being expressed by the following equation y = 4.48 -0.052x
FIG. 1. Oxygen intake and CO2 output during one experiment in a subject exercising at 400 kpm/min. Measurements began after 5 min exercise. At 1st arrow PTco, was measured. Hyperventilation began at 9 min (2nd arrow) and was continued until 24 min when a second measurement of PVc:o, was made (3rd arrow). Note that scales for 02 intake and CO2 output are displaced to avoid overlap.
I //5
I 15
10 TIME
1 20
25
(MINUTES)
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CO:! STORES
IN
813
EXERCISE
‘?io 1
OXYGENATED
1 50
1 60
MIXED
VENOUS
I
I
70
80 PcO
2
mm
Hg
FIG. 3. Relationship between CO:! storage capacity and the average of initial and final measurements of Pvco, for individual studies: two points are shown for each subject.
2
4
6
8
10
12
MINUTES 2. Volum: of CO2 washed out (Akoz) plotted as a function of time following onset of hyperventilation in a typical subject (JM) during exercise. A%02 is expressed as ml. min-’ and corrected for subject’s weight (kg) and change in PE~I,, allowing comparison with data obtained in a resting subject by Brandi and Clode (1). Washout curve has been analyzed in terms of two exponential functions (dashed Lines). Corresponding functions obtained by Brandi and Clode are shown dotted. FIG.
TABLE
.-
2. Half times of CO2 washout .--. -.----- _~_-_-~Low Power Output
Subj
t11’2r (initial) min
0.6 0.6
AR NJ RT JH MM JS EH JM MS
1.0 0.7 0.9 0.8 * Abrupt
step decreases
~--
High Power Output t1 , L’9 (slow) min
7.0 4.5 * 5.4 3.0 3.9 * 4.8 * in PET~O, were not
twr (initial) min
0.6 0.6 1.0 0.5 0.8 0.9 0.8 0.7 achieved
tm, (slow) min
4.2 2.8 3.2 2.3 * 4.4 3.6 3.5 3.7 in these studies.
where y is storage capacity in ml kg-‘. Torr-’ and x is the average oxygenated PVco,, i.e., control PVco, - [(control PVco, - final PVco,)/2] Torr. The correlation coefficient of the relationship was 0.600. DISCUSSION
The primary purpose of the present study was to obtain estimates of CO2 storage during exercise. PVco,
increases progressively with increasing work rates, indicating that storage of CO2 during exercise may be substantial, but measurements of CO* storage capacity in exercising subjects have not been extensive (5, 11). Two methods have been used in previous studies of body CO2 storage capacity (3). In the first and more widely used method, the volume of CO2 excreted following an increase in ventilation is measured and related to the fall in arterial, alveolar, or mixed venous Pco~. The second method employs rebreathing; body CO2 production is assumed to remain constant during the time of rebreathing and the increase in alveolar PCO~ is measured. In hyperventilation and CO2 breathing experiments at rest, a steady state of CO2 output equal to that preceding hyperventilation is not achieved for 0.5 h (1,3,20, 23). Brandi and Clode (1) emphasized the importance of using a step change in alveolar Pco~, as distinct from a step change in ventilation, in measuring the rate of CO2 washout. They found that CO2 washout in one subject was described by two exponential functions. The first, having a half time of 1.12 min, accounted for only 25% of the storage capacity and was consistent with CO2 stores in the lungs, blood, and well-perfused organs. The second had a half time of 13.4 min and accounted for 75% of the CO2 washed out, representing a large volume of CO2 in less well-perfused tissue, probably muscle. Finally, there is also a pool of CO2 that turns over very slowly (3) and consists of CO2 in bone; the turnover rate is so slow as to be irrelevant to the present discussion. In the present studies it was possible to identify two rates of CO2 clearance, both substantially more rapid than those found by Brandi and Clode (1) in man at rest (Fig. 2). The initial clearance, in addition to having a shorter half time, accounted for over 75% of the CO2 washed out. Thus, although it takes 15 min for 90% of the CO2 to be washed out at rest (l), during exercise the equivalent time is reduced to 4 min. This increase in the rate of CO2 washout is consistent with a shift of the large muscle CO2 pool into a rapidly clearing well-perfused compartment. The increase in initial clearance rate is probably explained by the increase in cardiac output in exercise, which will increase the volume of blood presented to the lungs in a given time. Also, as the metabolic CO2 production is increased in exercise; a given reduction in alveolar PcoB requires an alveolar ventilation up to 10 times greater than at rest, resulting in a more rapid excretion of CO2 from stores.
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814
N. L. JONES
Our findings are consistent with experiments in which the rate of equilibration of CO2 was measured in isolated muscle. Cherniack and colleagues (4) found that the apparent slope of the CO2 dissociation curve for resting muscle did not reach a steady value (3.5 mlkg-’ 9 TOW’) until 20 min after a step change in Pco~. They explained this finding by the inhomogeneity of muscle perfusion and supported this theory by finding an increase in the slope of the dissociation curve and the rate of CO2 equilibration after the intravenous administration of dinitrophenol, which increases muscle metabolism and perfusion. Studies of “j3Xe clearance from muscle have shown that muscle perfusion increases from resting levels of l-3 ml0 min-’ 100 g-’ to over 40 in exercise (14). Thus exercise may lead to an apparent increase in the body storage capacity for CO2 by more rapid equilibration of CO2 due to both the increase in turnover of CO2 and an increase in muscle perfusion. Two previous studies have examined CO2 storage capacity during exercise. Fowle and Campbell (11) measured the rate of rise in PcoB during rebreathing. The immediate CO2 storage capacity calculated by this *Tori? at rest, increasing in method was 0.56 mlkg-’ two subjects to 0.6-0.8 ml. kg-’ l Torr-’ during exercise. Clode, Clark, and Campbell (5) found values close to 1 ml kg-’ •T~rr-’ during exercise using rebreathing and 6 min of hyperventilation. Because rebreathing experiments in exercise can seldom be continued for longer than 1.5 min (II), it is likely that rebreathing measurements represent changes in the most accessible CO2 stores and may not represent fully the capacity for CO2 storage that is available during exercise maintained for several minutes. Following the practice established previously in studies of CO2 storage capacity in humans (5,11,12), the change in tissue PCO~ was assumed equal to the change in oxygenated Pv co, measured by rebreathing. The relationship of this change to changes in “true” (pulmonary artery) PCO~ is influenced by at least two factors. First, several studies in exercising humans have shown that the Pcoz at which equilibration occurs during the rebreathing of CO2 in 02 may be appreciably higher than the Pcoz of blood drawn from a systemic artery during rebreathing (18, 19) or from the pulmonary artery (6). Controversy still exists regarding the cause of this difference. The equilibrium PCO~ may represent the instantaneous PCO~ (lo), which because of the finite reaction time of CO2 in whole blood (16) may be higher than the PCO~ measured by electrodes some minutes later. However, recent studies by Effros et al. (8) suggest that equilibration of CO2 in blood traversing the lungs is enhanced by pulmonary tissue carbonic anhydrase. These findings tend to support the existence of a true alveolar-to-capillary PCO~ difference (15). The presence of such a difference in rebreathing theoretically will lead to a small overestimate of “true” PVco, and thus to a small underestimate of “true” CO2 storage capacity. Second, the relationship between oxygenated PVco, , as measured by the equilibrium rebreathing technique, and true PVco, is influenced by the effect of 02 saturation on the position of the CO2 dissociation curve (the Christiansen-Douglas-Haldane effect) (23). As “oxygenated” and l
l
AND
J. E. JURKOWSKI
“reduced” curves are almost parallel, a given change in oxygenated Pvco, is accompanied by a similar change in true Pvco,. However, at high work loads when P&O., is high and venous 02 low the absolute true PVco, will- be lower in relation to measured PVco, than at lower levels of exercise when venous 02 is higher. This means that the flattening of the body in vivo CO2 dissociation curve at higher levels of exercise may actually be more apparent when expressed in terms of changes in true P&O, than oxygenated Pvco,. A source of variation in previous studies of CO2 storage capacity is the assumption of unchanged metabolic CO2 production during hyperventilation. Cain (2) and Khambatta and Sullivan (20) studied dogs at rest, passively hyperventilated to arterial PCO~ levels of lo-15 Torr and arterial pH of over 7.7, and observed an increase in oxygen uptake that was linearly related to the fall in H+ concentration (2). Thus, if the associated increase in CO2 output was not allowed for, the calculated CO2 storage capacity would have been in error. In view of the fact that the increase in oxygen uptake amounted to some 20% of the control oxygen intake, this effect was appreciable in their studies, which were performed at a low level of tissue metabolism. During exercise and in studies where hyperventilation is associated with smaller changes in arterial PCO~ and arterial H+ concentration the relative increase in J?o~ is less (7). In our studies the largest increase in 02 intake amounted to 6% of the control (prehyperventilation) value. The increases in To2 associated with hyperventilation are accompanied by increases in plasma lactate, which may also increase CO2 output. The mechanism underlying both changes is thought to be an increase in glycolysis due to a pH-linked activation of the rate-limiting enzyme phosphofructokinase (PFK) in liver (13) and red blood cells (25). The possible effects of these changes on the assumptions made in calculating CO:! washed out of stores by hyperventilation are impossible to calculate with present information. However, it seems likely that in situations where the changes in PCO~and pH are less than those used in the previous in vitro and animal studies, the effects on metabolism will be insignificant, especially when related to the high body metabolism in exercise. Previous studies of CO2 storage capacity have not examined the linearity of the whole-body CO* dissociation curve. As the dissociation curve in blood is nonlinear, it would be surprising if the whole-body curve were linear. Farhi and Rahn (9) made an extensive theoretical analysis based on an electrical analog model. They assumed that the slope of the CO2 dissociation curve in blood and tissues was constant in the physiological range while realizing that departures from linearity outside this range would affect calculation of CO2 storage capacities. In support of this they pointed out that one of the highest reported values for the whole-body CO2 dissociation curve, 3.8 ml. kg-’ *TOW’ (21), might be accounted for by the low final PCO~ of 15 Torr following the hyperventilation used in the washout of COZ. An important difference wiIl exist between rest and exercise with respect to the venous blood pool and tissues. At rest, hyperventilation may lead to both venous and arterial PCO~ reaching
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CO2 STORES
IN
815
EXERCISE
levels at which the CO2 dissociation curve is steep, whereas in exercise the increased venoarterial PCO~ difference will tend to maintain venous and tissue Pcoz within the flatter portion of the curve. Extrapolation of our data (Fig. 3) to the left would indicate a CO2 storage capacity calculated during hyperventilation experiments at rest ( Pvco, falling from 45 to 30 Torr) of about 3.2 ml. kg-’ Torr-‘, which is consistent with results of experiments at rest lasting longer than 20 min (1, 3). Our finding of a lower CO2 storage capacity at higher levels of exercise suggest that the relationship between PCO~ and body CO2 stores is not linear and tends to flatten at high Pcoz. Thus the ability to store CO2 may fall with increasing PVco, during heavy exercise due to a progressive flattening of the in vivo CO2 dissociation curve. Another factor that presumably will be important at higher levels of exercise than we studied is the fall in
plasma and muscle HCOs concentrations accompanying lactic acid accumulation. Although a fall in HCOs and pH will have little effect on the slope of the CO2 dissociation curve (22), the absolute CO2 stores for a given Pvco, will fall. Thus in exercise of progressively increasing power, a diminishing CO2 storage capacity may lead to a progressive increase in CO2 excretion if large increases in Pvco,, and thus of tissue PCO~ and H+ ion concentration, are to be avoided. This mechanism may play an important part in the relative increase in ventilation that is observed above the so-called “anaerobic threshold” (24) associated with a progressive increase in CO2 output and plasma lactate concentration. This study was supported Research Council of Canada. Received
16 May
1978; accepted
by Grant in final
MA-4234 form
from
28 November
the
Medical
1978.
REFERENCES 1. BRANDI, G., AND M. CLODE. CO2 washout during hyperventilation in man. Respir. Physiol. 7: 163-172, 1969. 2. CAIN, S. M. Increased oxygen uptake with passive hyperventilation of dogs. J. AppZ. Physiol. 28: 4-7, 1970. 3. CHERNIACK, N. S., AND G. S. LONGOBARDO. Oxygen and carbon dioxide gas stores of the body. PhysioZ. Reu. 50: 196-243, 1970. 4. CHERNIACK, N. S., G. S. LONGOBARDO, AND A. P. FISHMAN. Transients in carbon dioxide stores. In: Carbon Dioxide and Metabolic Regulations, edited by G. Nahas and K. E. Shaefer. New York: Springer-Verlag, 1974, p. 324-338. 5. CLODE, M., T. J. H. CLARK, AND E. J. M. CAMPBELL. The immediate CO2 storage capacity of the body during exercise. CZin. Sci. 32: 161-165, 1967. 6. DENISON, D., R. H. T. EDWARDS, G. JONES, AND H. POPE. Direct and rebreathing estimates of the 02 and CO2 pressures in mixed venous blood. Respir. Physiol. 7: 326-334, 1969. 7. EDWARDS, R. H. T., AND M. CLODE. The effect of hyperventilation on the lactacidaemia of muscular exercise. CZin. Sci. 38: 269-276, 1970. 8. EFFROS, R. M., R. S. Y. CHANG, AND P. SILVERMAN. Acceleration of plasma bicarbonate conversion to carbon dioxide by pulmonary carbonic anhydrase. Science 199: 427-429, 1978. 9. FARHI, L. E., AND H. RAHN. Dynamics of changes in carbon dioxide stores. Anesthesiology 2 1: 604-614, 1960. 10. FORSTER, R. E. Can alveolar PCO~ exceed pulmonary end-capillary COZ? No. J. AppZ. Physiol.: Respirat. Environ. Exercise Physiol. 42: 323-328, 1977. 11. FOWLE, A. S. E., AND E. J. M. CAMPBELL. The immediate carbon dioxide storage capacity of man. CZin. Sci. 27: 41-49, 1964. 12. FOWLE, A. S. E., C. M. E. MATTHEWS, AND E. J. M. CAMPBELL. The rapid distribution of ‘Hz0 and “COe in the body in relation to the immediate carbon dioxide storage capacity. CZin. Sci. 27: 51-65, 1964. 13. GEVERS, W., AND E. DOWDLE. The effect of pH on glycolysis in vitro. CZin. Sci. 25: 343-349, 1963.
14. GRIMBY, G., E. HAGGENDAL, AND B. SALTIN. Local xenon 133 clearance from the quadriceps muscle during exercise in man. J. AppZ. Physiol. 22: 305-310, 1967. 15. GURTNER, G. H. Can alveolar PCO~exceed pulmonary end-capiIlary COZ? Yes. J. AppZ. PhysioZ.: Respirat. Environ. Exercise Physiol. 42: 323-326, 1977. 16. HILL, E. P., G. G. POWER, AND R. D. GILBERT. Rate of pH changes in blood plasma in vitro and in vivo. J. AppZ. Physiol.: Respirat. Environ. Exercise Physiol. 42: 928-934, 1977. 17. JONES, N. L., E. J. M. CAMPBELL, R. H. T. EDWARDS, AND D. G. ROBERTSON. CZinicaZ Exercise Testing. Philadelphia, PA: Saunders, 1975. 18. JONES, N. L., E. J. M. CAMPBELL, R. H. T. EDWARDS, AND G. WILKOFF. Alveolar to blood PCO~ difference during rebreathing in exercise. J. AppZ. Physiol. 27: 356-360, 1969. 19. JONES, N. L., E. J. M. CAMPBELL, G. J. R. MCHARDY, B. E. HIGGS, AND M. CLODE. The estimation of mixed venous blood during exercise. CZin. Sci. 32: 311-327, 1967. 20. KHAMBATTA, H. J., AND S. F. SULLIVAN. Carbon dioxide production and washout during passive hyperventilation alkalosis. J. AppZ. PhysioZ. 37: 665-669, 1974. 21. LILLEHEI, J. P. AND B. BALKE. Studies OfHyperuentiZation. USAF School of Med. Rept. 55-62, 1955. 22. MCHARDY, G. J. R. The relationship between the differences in pressure and content of carbon dioxide in arterial and venous blood. CZin. Sci. 32: 299-309, 1967. 23. SHEPHARD, R. J. The carbon dioxide balance-sheets of the body: Their determination in normal subjects and in cases of congenital heart disease. J. Physiol. London 129: 142-158, 1955. 24. WASSERMAN, K., B. J. WHIPP, S. M. KOYAL, AND W. L. BEAVER. Anaerobic threshold and respiratory gas exchange during exercise. J. AppZ. Physiol. 35: 236-243, 1973. 25. ZBOROWSKA-SLUIS, D. T., AND G. A. KLASSEN. Carbon dioxide mediated glycolysis. II. Respir. Physiol. 19: 162-175, 1973.
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