CO2 washout during hyperventilation in man

Respiration Physiology (1969) 7, 163-172; North-Holland Publishing Company, Amsterdam

COz WASHOUT

DURING

HYPERVENTILATION

IN MAN1

GIORGIOBRANDI~ANDMARIECLODE Department of Medicine of the Royal Postgraduate Medical School, London W.12, U.K.

Abstract. Ventilation was regulated to produce a square-wave reduction of end-tidal Pco,, and the rate of CO2 elimination from the body stores was measured to define the “CO2 washout curve”. This was resolved into two exponentials whose half-times were 1.12 and 13.4 min respectively. The “non metabolic COz” was 3.17 ml~kg%um Hgl. The fast COZ pool was 24% of the total. The relative size of the two components and their time constants suggest that they originate from well perfused organs (brain, gastro-intestinal tract, etc.) and poorly perfused tissues (resting skeletal muscle, connective tissue) respectively. If this holds true no rate-limiting factor besides blood flow need be postulated. Slower adjustments, which might take place at cellular or cell membrane level, were not detected with this technique. CO2 stores

COZ washout curves

There have been many previous studies of the CO, combining capacity of the whole body. The underlying procedure for these measurements is to estimate the difference between CO2 output and production while the body Pco2 is lowered by hyperventilation, or increased either by breathing CO2 mixtures or by breatholding. Although the results show that the CO2 combining capacity per unit mass of the body is lower than the value for blood (4.6 ml per litre per mm Hg), the values quoted range from 0.5 to 3.6 ml per kg body weight per mm Hg. The range of values reported are obviously related to the equilibration time allowed after the change of alveolar Pcoz, but the physiological mechanism is uncertain. The circulation obviously limits the CO2 exchange in the organs and tissues when blood Pco2 is altered and, as elaborated by FARHI and RAHN (1960), this is commonly regarded a sufficient explanation for the data. Other workers, however, have suggested that there may be some other rate-limiting mechanism such as CO2 exchange across cell membranes or the rate of hydratation and dehydratation of CO, in intracellular fluid (FOWLE and CAMPBELL,1964; CHERNIACKet al., 1966). We have examined the problem by measuring CO2 washout when a square-wave Accepted for publication 3 April 1969. 1 This investigation was supported in part by the British Heart Foundation. 2 NATO Research Fellow. Present address: Istituto di Fisiologia dell’Universit8, 163

Modena,

Italy.

164

GIORGIO

BRAND1 AND MARIE CLODE

reduction in Pcol was produced by voluntary hyperventilation. Although hyperventilation has often been used in the past it has not been regulated to produce a square wave change in alveolar Pco2. Such a square wave enabled an analysis of data in a similar manner to that applied to inert gas washout. This is discussed later, but some preliminary considerations will show the advantage of this approach. CO,

WASHOUT

Consider flow (Q) assumed changes constant; and :

FROM AN IDEAL COMPARTMENT

OF THE BODY

an ideal compartment of the body; suppose it is homogeneous for blood and tissue CO2 pressure (Ptco,). If the dissociation curve of this tissue is to be linear, changes of concentration can be calculated from pressure (dCt co2= b,dPt,o,). This compartment is in equilibrium when PtCo2 is under these conditions CO2 output (dco2) equals CO2 production @gob,)

where b,, is the slope of the dissociation curve of blood for CO*, assumed to be linear in the range under consideration, and PV& is the mean CO2 pressure of the blood leaving the compartment at steady state. If the P co2 gradient along the diffusion path is very small, PVZ, is not only constant but also equal to Pt,,. Now consider the unsteady state. Suppose there is a square wave drop in P~co~ to a new steady level. PO,,, will not be immediately lowered to the new PQ value, but will remain higher in proportion to the rate at which CO2 is released by the tissues (irEi;). 51::; equals the product of the mass of the compartment (M) and the rate of change of concentration, which in accord with our premises, is proportional to the rate of change of pressure. Therefore :

(2)

d&o

i’;;;=-M+,2.

dt

Adding this amount of CO2 to the blood would increase the CO2 pressure from PVc%p,2 to a higher variable value PV,,,. (3)

- M . b, %.!A

= Q* b,, * (PVcol - PV&).

As it was assumed that venous blood and tissues are in complete equilibrium (PVCo2=Ptco2), the relative changes must also be equal (dPtco, =dPcco,). If this is so, eq. (3) can be solved. Integrating and rearranging gives: (4)

7;IFg;= v&

* e - kt

where V,!& is the initial volume rate of the CO2 released by the tissue as a consequence of the drop in pressure. The rate constant (k) is equal to the ratio between the CO, carrying power of the blood (0 -b,,) and the capacity of the compartment for CO2 (M-b,).

CO,

(5)

WASHOUT

DURING

HYPERVENTILATION

IN MAN

165

k = @b,, -KK/

If these assumptions are valid the whole body may be represented as a number of homogeneous compartments, each giving an exponentially decaying washout curve. Under these conditions, i.e. after a square wave change of alveolar PcoI, all these compartments are in parallel. In fact as long as PAco2 is maintained constant, changes occurring in one compartment do not affect the others. The difference between the use of a step function or a square wave change in Pco2, and the usual procedure of a step function or square wave of alveolar ventilation must be emphasized. When COZ body stores are reduced by a step increase of alveolar ventilation different parts of the body are partially in series with each other; PACT, in this case is a variable which depends on the amount of CO2 released by all the compartments. In this condition no tissue or organ, however well perfused or quickly equilibrating with blood, may reach a steady state as long as COZ is being released by other tissues. Method

Five CO, washout curves were determined in a 32 years old male subject (weight 78 kg; height 186 cm). The experiments took place in the afternoon, at least three hours after a normal meal, and lasted from 70 to 90 min. To avoid the acid base changes of the recovery from anaerobic exercise the subject remained at rest for about one hour before the experiment. During the experiment he was sitting and remained as still as possible. The CO2 output from body stores (ir,!?:;) was calculated by substracting the CO, production (knob,) from the total COZ output. DETERMINATION

OF CO,

PRODUCTION

(i7c”02) DURING

HYPERVENTILATION

is difficult to determine %‘Fooz accurately. Other authors (VANCE and FOWLER, 1960) determined the CO2 output at steady state and assumed that COZ production remained the same during hyperventilation. In our experiments it was soon evident that the metabolic rate, as indicated by the oxygen uptake, was not constant but varied irregularly within about 10% of the control value. Any small changes that might occur in the 0, body stores could not account for the oscillations, which were more likely to be due to alterations in posture and muscular tone. On the assumption that the metabolic substrate is the same throughout each experiment, in spite of extensive changes in CO2 pressure, we determined the respiratory quotient (R) after a steady state of alveolar Pco2 was maintained - as accurately as possible - for at least 20 min. The steady state was obtained by recording end-tidal Pco2 and asking the subject to correct deviations from the preset level as soon as they appeared. This was achieved in two ways, either by the subject being in position to watch the end-tidal Pcol record, or being told by the operator when the Pco2 rose above or It

166

GIORGIO BRAND1 AND MARIE CLODE

dropped below the preset level and counteracting this by deeper and shallower breathing respectively. The metabolic R.Q. was determined from gas analysis of 3 to 5 samples of expired air collected throughout the 20 min. The mean of the last two values was taken to represent the metabolic R and used to calculate COz production during hyperventilation. 3::; was calculated from the following equation

(6)

ps. co2

DETERMINATION

=

7$

co=-R.Q.fioz-

OF THE CO, WASHOUT

CURVE

After the last sample for R determination was collected, the subject was asked to hyperventilation to reach, as quickly as possible, a low predetermined end-tidal P co2. Since the desired change was obtained in a few seconds, the reduction in Pco2 approximated a step change. Thereafter the subject maintained constant, as strictly as possible, the end-tidal Pco2 in the same way as in the previous R.Q. measurement. GAS EXCHANGE MEASUREMENTS

Expired gas was collected in a Tissot spirometer. Carbon dioxide in the expired and end-tidal gas was measured with an infrared COz analyser (URAS,CPI) through which gas was sampled at the rate of 600 ml/mm. The apparatus was calibrated with COz mixtures which had been analysed with the Lloyd-Haldane apparatus, and the CO2 concentrations were recorded on a Mingograph 81 recorder. Oxygen concentration was measured with a paramagnetic 0, analyser (SERVOMEX, DCL). The apparatus was set with nitrogen and pure oxygen and was calibrated with room air and one other mixture (15%-17q/, 0,) that had been analysed on the Lloyd-Haldane chemical analyser. Technical details and accuracy of gas analysis have been presented elsewhere (JONES et al., 1967). Results

Figure 1 is typical of all the results and shows the COz washout curve obtained in one of the experiments. VF.;, calculated according to eq. (6), is plotted against time. If Vo2 were constant, in order to maintain ~~~~~ steady at its new level, the curves of R (the overall respiratory exchange ratio) and of alveolar ventilation should have the same characteristics as that of the COz elimination. Furthermore, if the cardiac output (Q) were constant and the CO, dissociation curve of venous blood were linear in this range, the time course of the Pco2 of mixed venous blood would also be similar. The scales of R and VA shown on the right side of the figure and calculated from the mean oxygen uptake, facilitates comparison of 0%; with the metabolic CO2 production and provide an idea of the respiratory manoeuvre which was necessary to produce the P,, step change. Figure 1 shows that SE:; fell rapidly in the first 5 min and decayed more slowly after this. This is illustrated more clearly in fig. 2 where log 0::; is plotted against time. The scatter in the points of the experiments was large during the last 20 min

CO2 WASHOUT DURING HYPERVENTILATION

IN MAN

167

1.0

R

- B.S. Vcoe

VA (I/mln)

ml/min++mmHg

5-

.

0.6 I -

-60

4--50

0.6 .

-40 30.4 -30

. 2-

.

0.2

.20 ...

G ,-

......_ r,_ ‘“~a-N........ t ..,. ~........” ...y.* ........w I“--~ #......+..,... IO

0

20

30

40

50

60

minutes

Fig. 1. One example of “CO2 washout curve”. End-tidal Pco2 was suddenly lowered, at time zero, from 42 to 23 mm Hg and kept at that level. The rate of COa removal from body stores (ordinate.) is rapidly decaying with time. The quantity of COs involved can be better appreciated from the R scale and the alveolar ventilation required to maintain PC+ constant (scales on the right). The dotted line indicates the slow component.

. .

60 minutes

Fig. 2. Semilog replot of the data of fig. 1. The straight line describes the slow component.

168

GIORGIO BRAND1 AND MARIE CLODE

of the experiment when 3::; was very small compared to the metabolic CO, production. Analytical error as well as small unavoidable variation of end-tidal P,, therefore became more critical. Furthermore, changes in metabolic rate, although corrected for by using eq. (6), would produce changes in tissue P,, unless there was a coincidental change in blood flow with an opposing effect. TABLE

1

Kineticsof CO2 washout due to square-wavechange in PCO,in a normal subject at rest. Initial rate

Half time

Time constant

CO9,volume vB.S. co2

9B.S.

tt

k

ml~mi~.k~rnrnHg

min

ljmin

mlikgmm Hg

0.47 0.41 0.33

1.12

0.616

0.76

1.15

0.601

0.68

1.20

0.575

0.57

0.51

1.10

0.628

0.81

co20

Fast component:

Experiment 1 Experiment2 Experiment3 Experiment4 Experiment5 Mean S.D. Slowcomponent: Experiment 1 Experiment2 Experiment 3 Experiment4 Experiment5 Mean SD. Total S.D.

0.67

1.06

0.651

1.03

0.478

1.12

0.614

0.77

0.127

0.05

0.029

0.17

0.13 0.15

12.5

0.053

2.45

12.2

0.057

2.61

0.11

14.5

0.048

2.29

0.12

13.5

0.051

2.35

0.11

14.3

0.048

0.124 0.017

13.4

0.051

2.29 2.39

0.0038

0.13

1.03

0.602

3.17

0.123

0.18

By extrapolating the experimental points after 8 min to zero time, two components were identified. From the graphical analysis of these washout curves the half times of the two components, and the initial values of 9%; have been determined (table 1). Derived data are also presented in this table. The total CO2 output from fast and slow exchanging pools were calculated by integration (Vs$ = Vzz;Jk). The results of all experiments were similar and all data were derived by graphical analysis in the same way. Discussion CO2 DI~OCIATION

CURVE FOR THE! WHOLE BODY

From the first attempts to determine the CO* dissociation curve of the whole body it was realized that the time allowed for the tissues to equilibrate to a lower or higher

CO, WASHOUT DURING HYPERVENTILATIONIN MAN

169

Pco2 was critically important. From experiments on cats, SHAW and MESSER (1930) estimated that 45 to 140 min (average 102 min) were required to bring about equilibration. FARHI and RAHN (1960) estimated that several hours were necessary to obtain a steady state when a step change in ventilation was produced. In the present instead of controlling ventilation, the experiments, setting a steady level of Pco2 time required for equilibration was reduced, for with arterial PcoZ constant any transfer of CO, from one area to another was eliminated. The degree of underestimation due to insufficient time for equilibration, is clearly shown by fig. 1. Other variables, besides the time allowed for equilibration should be considered. Previous measurements on another human subject using essentially the same technique gave a lower estimate (about 259’0 lower) for the CO2 combining capacity of the body (BRANDI et al., 1962), even after correction for the incomplete equilibration. Individual characteristics, such as body size, muscle mass and fat mass may be at the basis of this difference. VANCE and FOWLER (1960) have reported a value of 2.05 in man with hyperventilation lasting 60 min. SCHAEFER and ml.mm Hg-‘.kg-’ ALVIS (1951) reported a value of about 1 ml-mm Hgg ’ *kg-’ after 33 min hyperventilation. Only LILLEHEI and BALKE (1955) found a higher value than the present estimate. Experiments in animals show also the same degree of variability. In conclusion, although differences in the reported values are likely to be due to different degrees of equilibration, other variables, such as the relative size of muscle tissue need also be considered. It seems unlikely that with the techniques used here, small individual differences can be detected, but by using a step change in Pco2 it is possible to approach a satisfactory steady state in a relatively short time and therefore minimize the intervention of other factors, such as the renal compensatory mechanisms. THE RATE OF COZ WASHOUTRELATEDTO BLOODFLOW/VOLUMERATIOSOF MAJORORGANS AND TISSUES The rate of CO2 washout may be limited by circulation and/or some other processes such as diffusion of the physically dissolved CO, or the transfer of positive (K+, Na+) and negative (Cl-, lactate-) ions which are likely to be displaced in the CO2 adjustments between extracellular and the intracellular fluids; these transfers may be rate-limiting. According to the circulatory hypothesis the fast component of the washout curve would represent well perfused organs (brain, kidney, liver, etc.) the slow one would represent muscle, connective tissue, etc. To detect the contributions of each organ to the overall washout curve would require sampling of venous blood from each organ, as FARHI et al. (1962) have done for nitrogen. Another factor for a quantitative compartmental analysis is the CO, combining capacity of these organs; but the slope of the CO, dissociation curve of intracellular fluid of most organs and tissues is not known with sufficient confidence to estimate the size of these CO, pools. If, as a first approximation, the same CO, storage capacity is assumed for the two spaces considered, the fast component would represent about 24”,& of the body weight.

170

GIORGIOBRAND1ANDMARIECLODE

This is probably an overestimate of the weight of well perfused organs. As indicated by eq. (5) the slope of the CO2 dissociation curve of the tissues (b,) besides determining the size of the pool, also determines the rate of exchange. This must be considered when comparison is made between the time constants and the normal values of blood flow/tissue volume ratios of organs and tissues. The limits within which circulation alone can explain our results can be approximately estimated. Let us consider the slow component; as a first premise suppose it is determined by skeletal muscle. By the 133Xe clearance technique and by venous occlusion plethysmography, the perfusion rate at rest was found to be 1.6 to 3 ml*min- l* 100 g-l (LASSEN,LINDBJERGand DAHN, 1965). By solving eq. (5) with these figures and the mean rate constant (k=0.0514) of the slow component, i.e. assuming the circulatory limitation only, the following values for the ratio b,/b,,, were obtained: 0.58 and 0.32. In other words, other rate limiting factors beside circulation should only be postulated if the slope of the COZ dissociation curve of muscle was less than 58;; or 3296 of the corresponding value in blood. Determinations in isolated but structurally undamaged muscle gave a value from l/3 to l/2 for the above ratio (FENN, 1929). Recently reported values are even higher (RAHN, 1962). That circulation alone can account for our results is furthermore favoured by the possibility that less well perfused tissues than muscle (connective and subcutaneous tissue) are also included in the slow component. Using a rebreathing procedure in which Pco2 was raised progressively in 3-4 min, FOWLE and CAMPBELL(1964) concluded that limitation of distribution of CO, by blood flow could not account alone for their results and suggested that exchange of CO, between intra- and extra-cellular fluid may be a rate limiting process. They also found that “C labelled CO, was rapidly distributed in a large mass of CO, in a relatively small volume of water which they suggested was the extra-cellular space. Further studies are required to explain why our data are compatible with limitation of COZ distribution by blood flow whereas those of FOWLE et al. are apparently not. Perhaps the difference is referable to the fact that in our study Pcol was lowered and the effects on intracellular fluid may not be the same (ADLER, ROY and RELMAN, 1965). In further studies of “COZ kinetics, MATTHEWSet al. (1968) were still unable to satisfactorily explain their finding on the assumption that circulation is the only rate limiting factor. A similar problem has been encountered in the study of inert gas elimination from the body. By comparing the washout curves of several inert gases (NZ, Xe, He and Kr) having different physical characteristics, e.g. molecular weight and diffusibility, JONES(1950) obtained reasonable evidence that diffusion was not the limiting factor. This conclusion was also supported by the agreement between the time constants of the components into which the washout curves were resolved and the blood flow/ tissue volume ratios of the major organs and tissues. Insufficient carbonic anhydrase activity in the muscle has been proposed as a rate limiting factor (FOWLE and CAMPBELL,1964; CHERNIACKand LONGOBARDO, 1966). Incomplete equilibrium between muscle and blood would certainly result in

CO2 WASHOUTDURINGHYPERVENTILATION IN MAN

171

a reduction of the rate of washout, but the factors determining the rate of the unaccelerated reaction (ROUGHTON, 1964) are not sufficiently known for muscle to evaluate this factor quantitatively. A more likely possibility is that active processes, besides physical diffusion, may determine the rate and also the direction of CO, exchanges. This hypothesis has been related to the fact that HCO;, unlike other diffusible ions (for example Cl-), are not partitioned across the membrane of the muscle fibre according to the transmembrane resting potential (CALDWELL,1954). Although a low membrane permeability to HCO; and H+, with the bulk of COz changes taking place through dissolved CO,, would explain the HCO; and H+ imbalance, this hypothesis is not supported by the experimental findings Of ADLERet al. (1965) and WADDELLand BUTLER(1959). We cannot comment on this: it should be pointed out, though, that the hypothetical active processes may also be rapidly readjusting when Pcoz is changed. In this condition distribution by circulation would still be the rate-limiting factor. The discrepancy between our results and those of FOWLE and CAMPBELL(1964) may be simply due to the different direction of Pco2 change. References ADLER, S., A. ROY and A. S. RELMAN(1965). Intracellular acid-base regulation. II. The interaction between COZ tension and extracellular bicarbonate in the determination of muscle cell pH. J. Clin. Invest. 44: 21-30. BRANDI, G., L. COVACEV,V. MINI and G. TORELLI(1962). Curva di dissociazione per il CO2 dell’organismo “in toto”. Boll. Sot. It. Biol. Sper. 38: 971-974. CALDWELL,P. C. (1954). An investigation of the intracellular pH of crab muscle fibres by means of micro-glass and micro-tungsten electrodes. J. Physiol. (London) 126: 169-180. CHERNIACK,N. S., G. S. LONGOBARDO, I. STAWand M. HEYMANN(1966). Dynamics of carbon dioxide stores changes following an alteration in ventilation. J. Appl. Physiol. 21: 785-793. FARHI, L. E. and H. RAHN (1960). Dynamics of changes in carbon dioxide stores. Anesthesiology 21: 604-614. FARHI, L. E., T. HOMMA,D. BERGERand D. BUSBY(1962). Tissue NZ washout in the whole animal and in individual organs. Physiologist 5: 138. FENN, W. 0. (1929). The carbon dioxide dissociation curve of nerve and muscle. Am. J. Physiol. 85 : 207-223. FOWLE, A. S. E. and E. J. M. CAMPBELL(1964). The immediate carbon dioxide storage capacity of man. Clin. Sci. 27: 41-49. JONES, H. B. (1950). Respiratory system: Nitrogen elimination. In: Medical Physics, Vol. 2, edited by 0. Glasser. Chicago Ill., The Year Book Publ., pp. 855-871. JONES, N. L., E. J. M. CAMPBELL,G. J. R. MCHARDY, B. E. HIGGS and M. CLYDE (1967). The estimation of carbon dioxide pressure of mixed venous blood during exercise. Clin. Sci. 32: 311-327. LASSEN,N. A., I. F. LINDBJERGand I. DAHN (1965). Validity of the Xenon’s3 method for measurement of muscle flow evaluated by simultaneous venous occlusion plethysmography. Circulation Res. 16: 287-293. LILLEHEI,J. P. and B. BALKE (1955). Studies of hyperventilation. U.S.A.F. School of Aviation Medicine Reports No. 55-62. MATTHEWS,C. M. E., G. LASZLO,E. J. M. CAMPBELL,P. M. KIBBYand S. FREEDMAN (1968). Exchange of llCOz in arterial blood with body COz pools. Respir. Physiof. 6: 29-44.

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H. (1962). The gas stores of the body, with particular reference to carbon dioxide. In: Man’s Dependence on the Earthly Atmosphere, edited by K. E. SCHAEFER.New York, The Macmillan

RAHN,

CY. ROUGHTON,F. J. W. (1964). Transport of oxygen and carbon dioxide. In: Handbook of Physiology. Section 3. Respiration. Vol. I, edited by W. 0. Fenn and H. Rahn. Washington D.C., American Physiological Society, pp. 767-825. SCHAEFER,K. E. and H. J. ALVIS (1951). Effect of inhalation of low oxygen concentration (19% Or. in Nz) over a period of 33 minutes on respiration, pulse rate, arterial oxygen saturation and oxygen uptake. Naval Medical Research Laboratory, Report No. 175, 10-76. SHAW, A. L. and A. MESSER(1930). The carbon dioxide capacity of the body and the rate at which the body comes into equilibrium with changes in the alveolar carbon dioxide tension. Am. J. Physiol.

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VANCE,J. W. and W. S. FOWLER(1960). Adjustement of stores of carbon dioxide during voluntary hyperventilation. Dis. Chest. 37: 304-313. WADDELL,W. J. and T. C. BUTLER(1959). Calculation of intracellular pH from the distribution of 5,5-dimetyl-2,4-oxazolidinedione (DMO). Application to the skeletal muscle of the dog. J. Clin. Invest. 38 : 720-729.