Catalysis of CO2 reactions lung carbonic anhydrase ROBERT A. KLOCKE Department of Medicine,
State University
by
of New
KLOCKE, ROBERT A. Catalysis of CO, reactions by lung carbonic anhydrase. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 44(6): 882-888, 1978. - Steady-state carbon dioxide excretion was studied in isolated bloodless lung preparations perfused with bicarbonate solutions. Addition of acetazolamide produced a prompt, significant decrease in the volume of excreted CO, under all conditions studied. Excreted CO, was derived from two sources: CO, dissolved in the permsate and CO, produced by dehydration of bicarbonate in the pulmonary capillary. The relative quantity of these two sources was determined by measurement of the simultaneous excretion of acetylene. Determination of the rate of CO, production permitted the calculation of capillary blood volume, mean capillary transit time, and the degree of catalysis of CO, reactions by carbonic anhydrase present in the lung. Contamination of perfusate with blood carbonic anhydrase was ruled out by measuring hemoglobin concentration and carbonic anhydrase activity in pulmonary venous drainage. Comparison of steady-state CO, production during control conditions and carbonic anhydrase inhibition indicated that bicarbonate in plasma has access to sufficient lung carbonic anhydrase to catalyze the CO, hydration-dehydration reaction by a factor of five.
York at Buffalo,
Buffalo,
New York 14214
to overwhelming catalysis of the CO, hydration-dehydration reaction. For these reasons, we have studied steady-state CO, excretion in an isolated, bloodless lung preparation perfused with bicarbonate solutions which were not recirculated. This approach mimics normal CO, exchange and avoids transients in chemical concentrations that may occur with nonsteady-state methods. The magnitude of carbon dioxide excretion under these circumstances is a function of the amount of CO, dissolved in the perfusate and the production of CO, from the dehydration of bicarbonate ion. Comparison of steady-state carbon dioxide excretion in the presence and absence of acetazolamide indicates that the bicarbonate in plasma has access to sufficient lung carbonic anhydrase to catalyze the rate of the CO, hydrationdehydration reaction by a factor of five. METHODS
Male New Zealand white rabbits weighing 1.0-2.0 kg were anesthetized with approximately 50 w/kg of pentobarbital sodiu m and the lungs isolated by use of the technique of Steinberg, Basset, and Fisher (23) with acetazolamide; capillary blood volume; mean capillary transit minor modifications. The animal was first heparinized time with 10,000 units of heparin. Lungs were washed free of blood by infusing the perfusate into the inferior vena cava with the portal vein clamped and the vessels to the DESPITE CONSIDERABLE RECENT INTEREST in puhonary head and upper limbs transected. If portions of the carbon dioxide exchange, perplexing questions regard- lungs did not clear of blood, the specimen was discarded. ing CO, exchange still remain unanswered. Reports After cannulation of the pulmonary artery, the lungs from multiple laboratories have demonstrated that al- were suspended from the arterial cannula in a waterveolar partial carbon dioxide pressure (Pco,) can exceed jacketed chamber maintained at 37°C (Fig. 1). Both that of arterial blood (22), but there is no unanimity ventricles were transected and pulmonary venous efconcerning the explanation of this paradox (9, 11, 16). fluent from the left ventricle was collected from the Furthermore, despite theoretical (20) and in vitro evi- bottom of the chamber. Flow was quantitated by timed dence (10) that localization of carbonic anhydrase (car- direct measurement. Since the lungs were suspended bonic dehydratase, EC 4.2.1.1) to the red blood cell from the pulmonary artery, the left ventricle was eleinterior causes considerable delay in attainment of vated above the lung apices and the entire pulmonary chemical equilibrium in blood, in vivo this delay either vasculature was in zone 3 conditions (27). Pulmonary is seen only when plasma carbonic anhydrase activity is artery pressure was monitored continuously using the inhibited (3) or is smaller in magnitude than predicted tip of the arterial cannula as a reference point; the (13). Finally, even though carbonic anhydrase may play specimen was discarded if vascular pressure did not a role in secretion of fetal lung fluid (l), it has no remain stable. Weight gain due to fluid accumulation apparent function in gas exchange (4). in this preparation is less than 5% of lung weight and Investigation of CO, exchange is complicated by dif- dry-to-wet weight ratios are constant within the limits ficulty in controlling physiological parameters and the of measurement error (23). Perfusate was equilibrated simultaneous occurrence of multiple chemical and dif- with varying concentrations of C02, 20% 02, balance N, fusive processes. In addition, intraerythrocytic carbonic and stored in two reservoirs maintained at 37OC. In anhydrase may mask some aspects of gas exchange due some experiments, 5% C,H, was included in the gas 882
0021~8987/78/0000-OOOO$Ol.
25 Copyright
0 1978 the American
Physiological
Society
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CATALYSIS
OF
CO,
REACTIONS
BY
LUNG
CARBONIC
In8phd Vent ibtion
Expttd Ventibth
883
ANHYDRASE
t
a step change in chamber CO, while monitoring endtidal Pco2 and &on. The diffusing capacity of the pleural surface was calculated and subsequent measurements of Vco2 were corrected for gas loss through the pleural surface. The CO, diffusing capacity for the pleural surface averaged 0.020 t 0.005 (SD) ml. min+ Torrl and the correction of vco., for pleural diffusion was usually less than 10% of its value. Correction for C,H2 loss through the pleura was made on the basis of the relative solubilities and molecular weights of the two gases. Hemoglobin concentration in the pulmonary venous drainage was calculated from the optical density of the fluid at 412 nm, an isobestic point for oxyhemoglobin and methemoglobin. Carbonic anhydrase activity in the effluent perfusate at 37°C was measured by a modification of the technique of Kernohan et al. (15). Perfusate was reacted in a stopped-flow apparatus with 50-90 mM HCl in a ratio of 9.7: 1.0, respectively, to reduce the pH to 6.4-6.7. The subsequent dehydration reaction and accompanying change in pH was monitored at 420 nm using 0.001% p-nitrophenol as an indicator. If catalysis of the reaction is modest, the kinetics are described by l
L-w---
Pulmonary
1
r-----d
I i c Venous
Broinoge
FIG. 1. Experimental apparatus used to perfuse isolated lungs. PA and Mass- Spec represent artery and mass spectrom-pulmonary eter, respectively. Reservoir C contains the control buffer; reservoir A contains the same buffer plus 250 mg. 1-l acetazolamide. Both reservoirs were immersed-in a 37°C water bath and contents of either could be delivered to perfusion pump by rotation of valve located upstream to pump. Heart is not shown.
mixture. Contents of both reservoirs were identical in composition, including pH and Pco2, except for the addition of acetazolamide (250 mg 1-l) to one reservoir. All perfusate contained 20 mM phosphate for buffering. Sodium bicarbonate concentration was adjusted to meet the desired experimental conditions and sodium chloride added to bring the total anion concentration to 150 mM. Spot checks of perfusate temperature throughout the system, including pulmonary venous drainage, indicated maintenance bf temperature within t l°CI Lungs were ventilated with 5-ml tidal volumes of room air at a frequency of approximately 60/min with a rodent respirator. Positive end-expiratory pressure of 1.0 cmH,O was used to prevent lung collapse. Expired ventilation passed through a Z&ml mixing chamber; timed samples were collected in a l-liter anesthesia bag and volume measured with a calibrated syringe. Mixed expired (FEDS,) and end-tidal (FETED., ) CO, fractions were analyzed on-line by mass spectrometry (model MGA-1100, Perkin-Elmer Corp.). End-expiratory gas concentration was assumed equal to alveolar CO,: Carbon dioxide output (Vco,) was calculated from mixed expired CO, concentration and expired gas volume. In experiments employing acetylene, 0.5-ml samples of mixed expiratory gas were analyzed by gas chromatography (model 100, Carle Instruments) using a 15-cm silica gel column for separation of CO, and C,H,. Preliminary experiments indicated that a small quantity of CO, diffused through the pleural surface into the lung chamber and did not appear in the expired ventilation. This quantity was directly proportional to the differences between -end-tidal Pco2- and chamber Pco2, i.e., the diffusing capacity of the pleural surface was constant. Administration of acetazolamide did not alter the pleural diffusing capacity. Initially we prevented loss of CO, through the pleura by adjusting chamber Pco2 to the same level as end-tidal Pco2. In later studies we found it more convenient to calibrate the system at the start of the experiment by producing
d[COzl -_f(k ---_____ IH+I[KHCO,,-I _ )., rco,l, -1 dt A
l
(1)
where h,. and k, are the velocity constants of the dehydration and hydration reactions, K, is the ionization constant of carbonic acid, taken to be 3.4 x 10e4 (Zl), and f describes the degree of catalysis. If the reaction is not catalyzed, f is equal to 1.0. The rate of change of pH under these circumstances will also depend on the concentration of the phosphate in the reaction mix. Phosphate buffering can be directly related to the change in CO, concentration by mass balance since production of each molecule of carbon dioxide consumes one hydrogen ion. The observed pH changes were recorded on 0.5-in. magnetic tape (model 7600, Honeywell, Inc.) and the initial 50% of the reaction was compared to data generated for different values of f using a forward Euler integration. The sum of squares was minimized using quadratic interpolation until the best fit for f was obtained. Each value of Itz\. represented the mean of the rate constants from three to four actual reactions. RESULTS
The mass spectrometer tracing of mixed expired CO&, in a representative experiment is illustrated in Fig. 2: Because ventilation was held constant, CO, concentration is directly proportional to Vco2. Perfusion at the beginning of the experiment was 47 ml min. After a steady state of CO, excretion was reached, perfusion was subsequently increased to 77 and 100 ml. min1 and steady-state CO, excretion rose with each increment in flow. At that point, the control perfusate was replaced with the perfusate containing acetazolamide (arrow, Fig. 2). Carbon dioxide elimination dropped precipitously by one-third from the control level even though ventilation, perfusion, and perfusate pH, Pco2, and bicarbonate concentration remained constant. At all l
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884
R. A.
1.5
KLOCKE
Acetarolamide 2.5
.
2.0
x co2 1.0 iico,
A
A
Ratio Ah A
f A A
0.5 i, =
47
77
100
101
77
levels of flow VCO~was substantially less during perfusion with the solution containing acetazolamide, indicating that the production of CO, via the dehydration reaction was catalyzed by carbonic anhydrase in the control situation. This observation was a consistent finding in a series of 102 paired comparisons of Vcob, in 29 sets of isolated lungs at a variety of flow rates (Fig. 3). Control VCO~ was significantly greater than acetazolamide Vco,, (P < 0.001) under all conditions. Mean hemoglobin concentration in the pulmonary venous drainage at maximum flow in 27 experiments was 3.1 x 10m7M during the control period and 2.0 x lop7 M during carbonic anhydrase inhibition. Assay of carbonic anhydrase activity by stopped-flow spectrophotometry indicated that there was no significant difference in enzyme activity between control and acetazolamide venous effluent in 18 paired samples. The apparent dehydration rate constant averaged 60.0 t 19.4 (SD) and 64.2 t 24.4 (SD) s-l, respectively, for the control and acetazolamide solutions. This represents a catalysis factor, f, of 1.09 and 1.17, both not significantly different than 1.0. We attempted to calculate the quantity of CO, produced by the dehydration reaction from VCO~, the rate of perfusion and the initial and end-capillary (i.e., endtidal) Pco2. Assuming an ideal lung, the sum of CO, dissolved in perfusate entering the lu ng and that produced in the capillaries must equal the sum of co 2 leaving the lung in the venous drainage and via the expired ventilation. Carbon dioxide production calculated in this manner varied widely, at times having negative values. Sample computations indicated that calculated CO, excretion was -dependent upon the assumption of an ideal lung. Because the majority of dissolved COOpresent in the perfusate is excreted in our preparation, the presence of small shunts invalidates a mass balance approach, particularly at high flow rates. Accordingly, simultaneous excretion of carbon dioxide and an inert gas, acetylene, was used to quantitate the degree of catalysis of CO&,reactions. Carbon dioxide in the-expired ventilation is derived from two sources. A portion of the dissolved CO, in equilibrium with hydrogen and bicarbonate ions enters the alveolar space as the perfusate reaches the pulmo-
4
1.u
57
FIG. 2. Mass spectrometer tracing of mixed expired CO, concentration. Perfusion pH was 7.39, Pco? 38 Torr, and [HCO:,- J 23.5 mM. Lung was perfused with control solution initially. At arrow, source of perfusate was changed to reservoir containing acetazolamide. Rate of perfusion (ml. min I) is indicated below CO, tracing.
A
0.
Q Wmin) 3. Ratio of total expired CO, elimination determined during control and acetazolamide perfusion as a function of the rate of perfusion. Absolute difference between control and acetazolamide perfusion rates was 3.5 2 5.1 (SD)% of the average of the two flows. Experimental conditions: n pH 7.7, [HCO,, J 23.5 mM; l pH 7.1, [HCO,, 1 23.5 mM; D pH 7.4, [HCO,; 1 23.5 mM; A pH 7.1, [HCOJ 11.5 mM. FIG.
nary capillary. This loss of CO, disrupts chemical equilibrium, favoring dehydration of bicarbonate to molecular CO&,. Further CO, is produced and excreted, the amount depending on chemical reaction rates and capillary transit time. The quantity of CO, produced in the capillary is equal to the difference between measured total CO2 excretion and the amount of the initial dissolved CO, which is excreted. The latter is calculated from the inert gas data. In the nonhomogeneous lung, the steady-state fractional excretion of two gases dissolved in the perfusate will be identical if their solubilities are the same (7). The fractional clearance of an inert gas contained in perfusate entering an alveolus is given by PIPA - ---~VA/Q _____PI h + VA/Q
(2)
where PI is the pressure of the inert gas in the perfusate and h is the Ostwald solubility coeffkient (7). PA and VA/Q are the respective alveolar pressure and ventilation-to-perfusion ratio of that alveolus. Because k for carbon dioxide (0.624 ml ml+ atm+) (19) and acetylene (0.984 ml ml-l atm-l) (25) in an aqueous media at 37°C are slightly different, their relative excretions will vary somewhat, particularly at low VA/Q. The presence of units with low VA/Q in a nonhomogeneous lung would introduce some difference in CO, and C,H, excretions. Because lungs in our experiments were obtained from normal animals and overall VA/Q was high (range 0.95.8), differences in fractional excretion of the two gases should be small. A first approximation to correct for the small difference in fractional excretion can be made using the overall VA/Q of the whole lung. To obtain the overall VA/Q for the lungs, alveolar ventilation was calculated from l
l
l
l
(3)
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CATALYSIS
OF
CO,
REACTIONS
BY
LUNG
CARBONIC
and perfusion, &, was directly measured. Writing Eq. 2 for both CO, and C&H, and using measured acetylene elimination in expired ventilation (%$H~), the expected excretion of dissolved CO, is ir CO, exI-,ectecl
=
v C2H,
k0.
’
l
PF c, [I,
pr-0,
x c, 11,
h *-
l
+
VA@
hco2+ WQ-,
885
ANHYDRASE
1
(4)
The term in the brackets represents the adjustment for I differing solubilities and averaged only 1,133 t 0.055
and unequivocal. The differences are much greater than could be attributed to possible experimental error in CO, measurements even if the small correction for diffusion of CO, through the pleura had not been taken into account. In our treatment of these data, we have assumed that CO, crosses the alveolar-capillary membrane instantaneously, i.e., the membrane diffusing capacity (Dmco,) is infinite. Hyde et al. (14) attempted
(SD).
Simultaneous excretion of CO, and C&H, was measured in 23 paired control and acetazolamide experiments in isolated lungs from six animals. Details of the experiments are given in Table 1. The results from a single series of paired experiments are shown in Fig. 4 for both control conditions and carbonic anhydrase inhibition. The shaded area between the expected and observed CO, elimination curves represents the quantity of CO, produced in the pulmonary capillary. As noted in Fig. 4, this quantity is substantially greater at all flow rates when the action of carbonic anhydrase is not inhibited. Results from the other experiments in the series were similar without exception. Mean pulmonary artery pressures during perfusion in these experiments were 10.4 t 1.3 (SD) and 12.1 t 1.5 (SD) Torr at flow rates of 106 and 157 ml. minP, respectively. DISCUSSION
The difference between CO, excretion during perfusion with control and acetazolamide solutions is striking
ObsmNd Expected
8 (cchin) FIG. 4. Total observed (A) and expected (A) CO, excretion in expt 5 during perfusion with control (upper panel) and acetazolamide (lower paneZ) solutions. Shaded area between two curves is equal to quantity of CO, produced in pulmonary capillaries.
1. Conditions and results in acetylene-carbon dioxide experiments -____I ________
TABLE
Perfusate
CO, Production
Expt No. [HCO:,-
1, mM
&, ml +min. 1
Control, ml * min. 1
(STPD)
Acetazolamide, mlemin 1
vc, ml
t, 7 S
f
End-Capillary Equilibrium,
%
PH
Pco2, Torr
1
7.47
31
23.5
44 102 156
0.94 1.20 1.09
0.68 0.39 0.35
5.9 3.5 3.3
7.1 2.0 1.3
1.6 4.2 4.1
44 30 20
2
7.47
31
23.5
28 66 106 159
0.99 1.28 1.39 1.23
0.54 0.57 0.59 0.41
4.4 4.7 5.2 3.9
9.4 4.3 2.9 1.5
2.4 3.1 3.3 4.0
59 41 33 22
3
7.11
35
11.5
39 62 106 158
1.66 1.90 2.06 2.12
0.47 0.62 0.60 0.19
2.9 4.1 4.2 1.4
4.1 3.7 2.5 0.5
5.4 4.4 4.9 15.9
58 44 33 26
4
7.11
35
11.5
39 70 107 158
1.53 1.65 1.53 1.45
0.46 0.29 0.25 0.18
2.9 1.8 1.7 1.3
4.5 1.6 0.9 0.5
4.5 7.6 7.6 8.7
45 32 22 14
5
7.07
79
23.5
46 70 106 156
2.83 3.28 3.87 4.13
0.73 0.99 0.96 0.73
2.1 3.0 3.0 2.4
3.0 2.6 1.7 0.9
6.3 5.3 6.6 9.4
61 55 49 42
6
7.07
79
23.5
40 71 109 158
3.01 3.48 3.96 3.83
1.20 1.35 1.03 1.11
3.9 4.4 3.4 3.9
6.3 3.8 1.9 1.5
3.9 3.9 6.0 5.1
71 57 49 37
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886
R. A. KLOCKE
to measure the overall pulmonary diffusing capacity for CO, using an isotope technique, b ut could not arrive at an accurate result since pulmonary CO, diffusing capacity (DqTo ) was so great. They concluded that it must be in excess’of 200 ml. min l Torr * in the human. Therefore, the Dm,,., must be even greater since it is only one of several- processes which limit CO, movement between blood and alveolus. Thus, our assumption of an infinite Dm()., has little effect on our results, and a finite Dmo would, if anything, tend to reduce the difference between control and acetazolamide data since more CO, was excreted in the former case. The possibility of contamination of the perfusate with blood is of greater concern since, if this were the case, the demonstrated changes in Vco., would be trivial. Lungs with gross, visible contamination resulting from incomplete clearance of blood during preparation were discarded without performing any measurements. Hemoglobin concentration in venous emuent was minimal in our experiments, averaging only 3.1 x lO-7 M under control conditions. This quantity of hemoglobin indicates that the perfusate at most was contaminated with red blood cells in a ratio of (3.1 x lo- 7)/0.02 = l/64,516 assuming an intraerythrocytic concentration of 0.02 M hemoglobin monomer. Since the red blood cell contains sufficient carbonic anhydrase to catalyze the CO, hydration-dehydration reaction by a factor of 13,000 (15), this contamination would increase the rate of CO, reactions by only 13,000/64,516 or 20%. This is well below the approximately 500% catalysis demonstrated in our experiments. The possibility that carbonic anhydrase from lung tissue was released into the perfusate is also unlikely because the dehydration rate constant measured in the pulmonary drainage collected under control conditions was 60.0 s-l. This was not significantly different from the natural rate constant of 55.0 ssl at 37°C under similar conditions (5). In addition, there was no significant difference in the rate constant measured in the eMuent from paired control and acetazolamide experiments. Hence the increased production of co, seen in the control experiments did not resu.lt from contamination of the perfusate with enzyme Al though some ions such as phosphate may catalyze the hydration-dehydration reaction, phosphate concentration was so small that catalysis would barely be detectable if present (21). If phosphate catalysis did occur, it should be identical in both control and acetazolamide solutions because phosphate concentration was held constant. Although acetazolamide has been reported to affect some processes which are independent of carbonic anhydrase (l), its effect on CO*, exchange is limited to inhibition of carbonic anhydrase activity. Thus, it appears that carbonic anhydrase contained in lung tissue is responsible for the increased CO, production observed. Comparison of acetylene and carbon dioxide excretion permits accurate determination of the quantity of CO-, produced in the pulmonary “capillary,” i.e., any vessel in which CO, exchange occurs between perfusate and alveolar gas. Knowing CO, production during carbonic anhydrase inhibition and initial conditions in the perfusate prior to any gas exchange, mean pulmonary l
capillary volume and transit time can be calcul .ated. In the presence of acetazolamide, the rate of CO, production is given by Eq. 1 with f equal to 1.0. As the perfusate enters the capillary initial [H+] and [HCO:; ] are the same as those in the perfusate reservoir, but intravascular PCO~ drops abruptly to the alveolar level. Carbon dioxide subsequently produced via the dehydration reaction enters the alveolar space because Pco., remains fixed at the alveolar level during capillary transit under these steady state conditions. The initial rate of CO, production, d[CO,]/dt, is calculated using the uncatalyzed velocity constants, Itz, (55.0 s- l> and h,, (0.13 s-l) (5), and initial ionic concentrations. Endcapillary d[CO,]/dt is calculated in the same manner but requires knowledge of end-capillary [H+] and [HCO:,J. Since generation of one molecule of CO, utilizes a single molecule of both hydrogen and bicarbonate, the quantity of these two ions consumed is equal to the amount of CO, produced. Knowing CO*, production and perfusion rate, the change in perfusatebicarbonate concentration is calculated. For example, in the first experiment in Table 1, the CO, produced during carbonic anhydrase inhibition is 0.68 ml mint (3.0 x lo-:’ mol min). Flow rate is 50 ml. rnin’ (0.05 1 min) so [HCO:,J in the capillary decreased by 3.0 x 10-“/O/0.05= 6.0 x lop4 mol *l-l. S’mce an equal quantity of hydrogen ions is supplied by the phosphate buffer in the proteinfree perfusate, end-capillary pH, and therefore [H+], can be calculated from the initial pH, phosphate concentration, the pK of phosphoric acid, and hydrogen ion consumption. Initial and end-capillary d[ COJdt are averaged to obtain the mean rate of CO, production, d[CO,l/dt. Averaging is valid only if the rate of CO, production decreases linearly along the capillary. Using a modified Euler technique, we computed instantaneous d[CO,]/dt along the capillary from Eq. 1 for all our experimental conditions. Largely due to the presence of the phosphate buffer, the change in d[COJdt was linear throughout capillary transit, justifying the method of computation. Since all CO, produced in the capillary appears in the expired ventilation, after converting all quantities to common units, the pulmonary capillary volume, Vc, is equal to l
l
l
Vc = Vco,l(d[Cm/dt) Knowing the perfusion rate and the pulmonary lary volume, mean capillary transit time, t,, is t, = VCIQ
(5) capil(6)
Vc in 23 observations averaged 3.36 t 1.21 (SD) ml (Table 1). This agrees quite well with Vc predicted from the morphometric data of Weibel (26). From his measurements of alveolar surface area and the relationships between surface area and capillary volume, we calculated that our l-2 kg animals would have capillary volumes between 2.6 and 4.7 ml. Vc was independent of flow rate; this probably is a reflection of the capillary bed being maximally dilated due to the zone 3 conditions present in our experiments. As expected & was markedly dependent on the rate of perfusion (Table 1). Mean capillary tran .sit averaged 1.0 s at flows of approximately 160 ml m.in- l, the expected ca.rdiac output for a
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CATALYSIS
OF
CO,
REACTIONS
BY
LUNG
CARBONIC
ANHYDRASE
mammal of this size (19). From paired data obtained during perfusion of the same lungs with the control solution at identical flow rates, the degree of catalysis of the dehydration reaction can be calculated with the same general approach. However, instead of assuming f to be 1.0 and calculating Vc as with the acetazolamide data, the process is reversed and f is calculated to fit CO, production during control perfusion. Except for a single value of 15.9 (Table l), calculated values off grouped around a value of 5 (mean of 5.6, median of 4.9), significantly greater than the uncatalyzed factor (P < 0.001). Scatter in the data, particularly in a given animal, is predominantly due to difficulty in measurement of CO, production during carbonic anhydrase inhibition. This relatively small value is the difference between two larger quantities, the actual excretion and that expected on the basis of the inert gas excretion (Fig. 4). Small errors in calculated CO, production during enzyme inhibition are magnified since this parameter appears in the denominator off, a ratio grater than 1.0. Despite catalysis of the CO, dehydration reaction, chemical equilibrium was not reached in the pulmonary capillary in any of our experiments. The CO, production which would occur at equilibrium for a given alveolar PCO~ was calculated from Eq. 1 by setting d[COJdt equal to zero and solving for the values of [H+] and [HCOJ which satisfied this relationship. A unique solution could be obtained by trial and error in each case since consumption of bicarbonate ion and pH are related through the phosphate buffering system. The fraction of equilibrium attained was calculated from the actual measured production and the calculated equilibrium CO, production. As noted in Table 1, even in those experiments conducted at low flow rates and prolonged capillary transit times, end-capillary chemical reactions were still far from the equilibrium value. Under more physiological conditions, e.g., flows of approximately 160 ml min-l, the dehydration reaction reached only 14-42% of its equilibrium value. Although accurate for the present experiments, these calculations must be interpreted cautiously in a physiological sense since the speed of the CO, reactions are critically dependent on experimental conditions. For example, experiments performed at physiological PCO~ reached a much lower degree of equilibrium at maximum flow (14-26%) than those conducted at elevated Pco., (37-42%). In addition, the chemical system in our experiments is relatively simple; no red blood cells are present so that transmembrane ionic movements and the Haldane effect are absent. Thus, if equilibrium is not attained in our experiments, it seems unlikely that complete equilibrium would be attained in the plasma in vivo in the presence of these complicating factors which would tend to prolong the approach to equilibrium. A more quantitative answer requires not only sophisticated kinetic calculations, but also a better understanding of the localization and action of the carbonic anhydrase which is catalyzing the dehydration reaction. The site of carbonic anhydrase activity in the lung cannot be deduced from the present experiments, but Fain and Rosen (6) have demonstrated carbonic anhyl
887
drase activity associated with the pulmonary capillary endothelium of reptiles. However, the specificity of the histochemical technique used in these experiments has been challenged (17), and to date there are no reported studies using more specific techniques. If these findings are corroborated in the mammalian lung, it would seem likely that plasma CO, reactions may be catalyzed by enzyme localized to the endothelium. However, an alternate possibility exists which would also explain our findings. If bicarbonate is able to leave the vascular space and enter the pulmonary interstitium, enzyme in lung tissue could catalyze CO, reactions. This postulate would also require similar movement of hydrogen ion (or alternatively movement of hydroxyl ion in the opposite direction) because a source of hydrogen ions is needed for the dehydration reaction. In experiments with short-term transients interstitial buffers could serve as a proton source. However, these buffers would be depleted in steady-state experiments and COO production would decline with time, a finding not observed in the present work. Evidence for ionic movement out of the vascular space is controversial. Chinard and coworkers (2) studied bicarbonate distribution following bolus injection into the pulmonary circulation and concluded that this ion was confined to the vascular space. However, it is unlikely that the bolus technique has sufficient sensitivity to detect the small amounts of bicarbonate movement that would be necessary to explain our data. Taylor and Gaar (24) have demonstrated that the capillary endothelium is quite permeable to large uncharged solute molecules, but did not investigate ionic movement across the endothelium. Pore size calculated from their measurements are sufficiently large to raise the possibility of bulk fluid movement across the endothelium. Olver (18) has measured movement of ions between the vascular and alveolar spaces in the fetal lung. He concluded that the alveolar epithelium provides the principal barrier to this movement, and that the capillary endothelium is relatively permeable to ions. Thus, as postulated by Feisal et al. (8) on the basis of their work employing injection of a bicarbonate bolus into the pulmonary circulation, this ion may have a volume of distribution greater than the capillary volume. It is not clear which of these two alternatives, endothelial localization of carbonic anhydrase or movement of bicarbonate out of the vascular space, is more likely. Enhancement of the rate of CO, reactions in lung tissue may play a role in two puzzling observations noted in recent years. Multiple investigators have observed that alveolar Pco2 is greater than arterial Pco? under some circumstances (22). This has been attributed to movement of bicarbonate into the lung interstitium (16), increased CO, production near the capillary endothelium (ll), and continued CO, reactions occurring after blood has left the capillary (9). The present observations are supportive, but are certainly not definitive evidence, for the first two possibilities. Gurtner et al. (12) have calculated that a IO-fold enhancement of the natural rates of the dehydration reaction could explain the observed negative arterial-alveolar gradients.
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888
Forster and Crandall (10) have postulated that slow pH changes occur in plasma due to restriction of blood carbonic anhydrase to the erythrocyte interior, but measurements in dogs and cats from two laboratories indicate pH changes are less than those predicted (13; personal communication, E. D. Crandall). The present data suggest that catalysis of CO, reactiolrs outside the red blood cell during capillary transit would diminish the expected pH changes occurring after blood has left the lung. Although our data would qualitatively support such observations, a more precise quantitative
R. A.
KLOCKE
approach requires delineation of the actual site and mechanism of catalysis of CO, reactions in lung tissue. The author appreciates the technical assistance of Mrs. Anne Coe, Mrs. Gail Murawski, and Mr. Charles Soles, and the editolial assistance of Mrs. Marsha Barber. Dr. Aron Fisher kindly permitted the author to visit his laboratory to learn the techniques involved in perfusion of isolated lungs. Acetazolamide was supplied as Diamox by Lederle Laboratories. This work was presented at the Fall meeting of the American Physiological Society (Physiologist 20: 52, 1977) and supported by Grant HL-15194 from the National Heart, Lung and Blood Institute. Received
for publication
3 October
1977.
REFERENCES 1. ADAMSON, T. M., AND B. P. WAXMAN. Carbonate dehydratase (carbonic anhydrase) and the fetal lung. In: Lung Liquids. New York: American Elsevier, 1976, p. 221-234. 2. CHINARD, F. P., T. ENNS, AND M. F. NOLAN. Contributions of bicarbonate ion and of dissolved CO, to expired CO, in dogs. Am. J. Ph.ysiol. 198: 78-88, 1960. 3. CRANDALL, E. D., A. BIDANI, AND-R. E. FORSTER. Postcapillary changes in blood pH in vivo during carbonic anhydrase inhibition. J. Appl. Physiol.: Respirct. Environ. Exercise Physiol. 43: 582490, 1977. 4. DUBOIS, A. B. Significance of carbonic anhydrase in lung tissue. In: COz: Chemical, Biochemical and Physiological Aspects. Washington, D.C.: NASA, 1969, NASA SP-188, p. 257-259. 5. EDSALL, J. T. Carbon dioxide, carbonic acid, and bicarbonate ion: physical properties and kinetics of interconversion. In: CO,: Chemical, Biochemical, and Physiological Aspects. Washington, D.C.: NASA, 1969, NASA SP-188, p. 15-27. 6. FAIN, W., AND S. ROSEN. Carbonic anhydrase activity in amphibian and reptilian lung: a histochemical and biochemical analysis. Histochem. J. 5: 519-528, 1973. 7. FARHI, L. E. Elimination of inert gas by the lung. Respiration Physiol. 3: l-11, 1967. 8. FEISAL, K. A., M. A. SACKNER, AND A. B. DUBOIS. Comparison between the time available and the time required for CO, equilibration in the lung. J. Clin. Invest. 42: 24-28, 1963. 9. FORSTER, R. E. Can alveolar PCO~ exceed pulmonary end-capillary CO,? No. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 42: 326-328, 1977. 10. FORSTER, R. E., AND E. D. CRANDALL. Time course of exchanges between red cells and extracellular fluid during CO, uptake. J. Appl. Physiol. 38: 710-718, 1975. 11. GURTNER, G. H. Can alveolar Pcoz exceed pulmonary endcapillary CO,? Yes. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 42: 324-326, 1977. 12. GURTNER, G. H., S. H. SONG, AND L. E. FARHI. Alveolar to mixed venous Pco., difference under conditions of no gas exchange. Respiration Physiol. 7: 173-187, 1969. 13. HILL, E. P., G. G. POWER, AND R. D. GILBERT. Rate of pH changes in blood plasma in vitro and in vivo. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 42: 928-934, 1977. 14. HYDE, R. W., R. J. M. PUY, W. F. RAUB, AND R. E. FORSTER. Rate of disannearance of labeled carbon dioxide from the lungs
15.
16.
17.
18.
19. 20. 21.
22.
23.
24.
25.
26.
27.
of humans during breathholding: a method for studying the dynamics of pulmonary CO, exchange. J. Clin. Invest. 47: 15351552, 1968. KERNOHAN, J. C., W. W. FORREST, AND F. J. ROUGHTON. The activity of concentrated solutions of carbonic anhydrase. Biochim. Biophys. Acta 67: 31-41, 1963. LAZLO, G., T. J. H. CLARK, H. POPE, AND E. J. M. CAMPBELL. Differences between alveolar and arterial P,,,, during rebreathing experiments in resting human subjects. Respiration Physiol. 12:36-52, 1971. MUTHER, T. A critical evaluation of the histochemical methods for carbonic anhydrase. J. Histochem. Cytochem. 20: 319-330, 1972. OLVER, R. E. Ion transport and water flow in the mammalian lung. In: Lung Liquids. New York: American Elsevier, 1976, p. 199-220. Respiration and Circulation, edited by P. L. Altman. Bethesda, Md.: Fed. Am. Sot. Exptl. Biol., 1971, p. 17-18, 320-321. ROUGHTON, F. J. W. Recent work on carbon dioxide transport by the blood. Physiol. Rev. 15: 241-296, 1935. ROUGHTON, F. J. W. Transport of oxygen and carbon dioxide. In: Handbook of Physiology. Respiration. Washington, DC.: Am. Physiol. Sot., 1964, sect 3, vol. I, chapt. 31, p. 767-826. SCHEID, P., J. TEICHMANN, F. ADARO, AND J. PIIPER. Gas-blood CO, equilibration in dog lungs during rebreathing. J. Appl. Physiol. 33: 582-588, 1972. STEINBERG, H., D. J. P. BASSETT, AND A. B. FISHER. Depression of pulmonary 5-hydroxytryptamine uptake by metabolic inhibitors. Am. J. Physiol. 228: 1298-1303, 1975. TAYLOR, A. E., AND K. A. GAAR. Estimation of equivalent pore radii of pulmonary capillary and alveolar membranes. Am. J. Physiol. 218: 1133-1140, 1970. WAGNER, P. D., P. F. NAUMANN, AND R. B. LARAVUSO. Simultaneous measurement of eight foreign gases in blood by gas chromatography. J. Appl. Physiol. 36: 600-605, 1974. WEIBEL, E. R. Morphometric estimation of pulmonary diffusion capacity. V. Comparative morphometry of alveolar lungs. Respiration Physiol. 14: 26-43, 1972. WEST, J. B. Topographical distribution of blood flow in the lung. In: Handbook of Physiology. Respiration. Washington, DC.: Am. Physiol. Sot., 1964, sect. 3, vol. II, chapt. 58, p. 1437-1451.
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State University
by
of New
KLOCKE, ROBERT A. Catalysis of CO, reactions by lung carbonic anhydrase. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 44(6): 882-888, 1978. - Steady-state carbon dioxide excretion was studied in isolated bloodless lung preparations perfused with bicarbonate solutions. Addition of acetazolamide produced a prompt, significant decrease in the volume of excreted CO, under all conditions studied. Excreted CO, was derived from two sources: CO, dissolved in the permsate and CO, produced by dehydration of bicarbonate in the pulmonary capillary. The relative quantity of these two sources was determined by measurement of the simultaneous excretion of acetylene. Determination of the rate of CO, production permitted the calculation of capillary blood volume, mean capillary transit time, and the degree of catalysis of CO, reactions by carbonic anhydrase present in the lung. Contamination of perfusate with blood carbonic anhydrase was ruled out by measuring hemoglobin concentration and carbonic anhydrase activity in pulmonary venous drainage. Comparison of steady-state CO, production during control conditions and carbonic anhydrase inhibition indicated that bicarbonate in plasma has access to sufficient lung carbonic anhydrase to catalyze the CO, hydration-dehydration reaction by a factor of five.
York at Buffalo,
Buffalo,
New York 14214
to overwhelming catalysis of the CO, hydration-dehydration reaction. For these reasons, we have studied steady-state CO, excretion in an isolated, bloodless lung preparation perfused with bicarbonate solutions which were not recirculated. This approach mimics normal CO, exchange and avoids transients in chemical concentrations that may occur with nonsteady-state methods. The magnitude of carbon dioxide excretion under these circumstances is a function of the amount of CO, dissolved in the perfusate and the production of CO, from the dehydration of bicarbonate ion. Comparison of steady-state carbon dioxide excretion in the presence and absence of acetazolamide indicates that the bicarbonate in plasma has access to sufficient lung carbonic anhydrase to catalyze the rate of the CO, hydrationdehydration reaction by a factor of five. METHODS
Male New Zealand white rabbits weighing 1.0-2.0 kg were anesthetized with approximately 50 w/kg of pentobarbital sodiu m and the lungs isolated by use of the technique of Steinberg, Basset, and Fisher (23) with acetazolamide; capillary blood volume; mean capillary transit minor modifications. The animal was first heparinized time with 10,000 units of heparin. Lungs were washed free of blood by infusing the perfusate into the inferior vena cava with the portal vein clamped and the vessels to the DESPITE CONSIDERABLE RECENT INTEREST in puhonary head and upper limbs transected. If portions of the carbon dioxide exchange, perplexing questions regard- lungs did not clear of blood, the specimen was discarded. ing CO, exchange still remain unanswered. Reports After cannulation of the pulmonary artery, the lungs from multiple laboratories have demonstrated that al- were suspended from the arterial cannula in a waterveolar partial carbon dioxide pressure (Pco,) can exceed jacketed chamber maintained at 37°C (Fig. 1). Both that of arterial blood (22), but there is no unanimity ventricles were transected and pulmonary venous efconcerning the explanation of this paradox (9, 11, 16). fluent from the left ventricle was collected from the Furthermore, despite theoretical (20) and in vitro evi- bottom of the chamber. Flow was quantitated by timed dence (10) that localization of carbonic anhydrase (car- direct measurement. Since the lungs were suspended bonic dehydratase, EC 4.2.1.1) to the red blood cell from the pulmonary artery, the left ventricle was eleinterior causes considerable delay in attainment of vated above the lung apices and the entire pulmonary chemical equilibrium in blood, in vivo this delay either vasculature was in zone 3 conditions (27). Pulmonary is seen only when plasma carbonic anhydrase activity is artery pressure was monitored continuously using the inhibited (3) or is smaller in magnitude than predicted tip of the arterial cannula as a reference point; the (13). Finally, even though carbonic anhydrase may play specimen was discarded if vascular pressure did not a role in secretion of fetal lung fluid (l), it has no remain stable. Weight gain due to fluid accumulation apparent function in gas exchange (4). in this preparation is less than 5% of lung weight and Investigation of CO, exchange is complicated by dif- dry-to-wet weight ratios are constant within the limits ficulty in controlling physiological parameters and the of measurement error (23). Perfusate was equilibrated simultaneous occurrence of multiple chemical and dif- with varying concentrations of C02, 20% 02, balance N, fusive processes. In addition, intraerythrocytic carbonic and stored in two reservoirs maintained at 37OC. In anhydrase may mask some aspects of gas exchange due some experiments, 5% C,H, was included in the gas 882
0021~8987/78/0000-OOOO$Ol.
25 Copyright
0 1978 the American
Physiological
Society
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CATALYSIS
OF
CO,
REACTIONS
BY
LUNG
CARBONIC
In8phd Vent ibtion
Expttd Ventibth
883
ANHYDRASE
t
a step change in chamber CO, while monitoring endtidal Pco2 and &on. The diffusing capacity of the pleural surface was calculated and subsequent measurements of Vco2 were corrected for gas loss through the pleural surface. The CO, diffusing capacity for the pleural surface averaged 0.020 t 0.005 (SD) ml. min+ Torrl and the correction of vco., for pleural diffusion was usually less than 10% of its value. Correction for C,H2 loss through the pleura was made on the basis of the relative solubilities and molecular weights of the two gases. Hemoglobin concentration in the pulmonary venous drainage was calculated from the optical density of the fluid at 412 nm, an isobestic point for oxyhemoglobin and methemoglobin. Carbonic anhydrase activity in the effluent perfusate at 37°C was measured by a modification of the technique of Kernohan et al. (15). Perfusate was reacted in a stopped-flow apparatus with 50-90 mM HCl in a ratio of 9.7: 1.0, respectively, to reduce the pH to 6.4-6.7. The subsequent dehydration reaction and accompanying change in pH was monitored at 420 nm using 0.001% p-nitrophenol as an indicator. If catalysis of the reaction is modest, the kinetics are described by l
L-w---
Pulmonary
1
r-----d
I i c Venous
Broinoge
FIG. 1. Experimental apparatus used to perfuse isolated lungs. PA and Mass- Spec represent artery and mass spectrom-pulmonary eter, respectively. Reservoir C contains the control buffer; reservoir A contains the same buffer plus 250 mg. 1-l acetazolamide. Both reservoirs were immersed-in a 37°C water bath and contents of either could be delivered to perfusion pump by rotation of valve located upstream to pump. Heart is not shown.
mixture. Contents of both reservoirs were identical in composition, including pH and Pco2, except for the addition of acetazolamide (250 mg 1-l) to one reservoir. All perfusate contained 20 mM phosphate for buffering. Sodium bicarbonate concentration was adjusted to meet the desired experimental conditions and sodium chloride added to bring the total anion concentration to 150 mM. Spot checks of perfusate temperature throughout the system, including pulmonary venous drainage, indicated maintenance bf temperature within t l°CI Lungs were ventilated with 5-ml tidal volumes of room air at a frequency of approximately 60/min with a rodent respirator. Positive end-expiratory pressure of 1.0 cmH,O was used to prevent lung collapse. Expired ventilation passed through a Z&ml mixing chamber; timed samples were collected in a l-liter anesthesia bag and volume measured with a calibrated syringe. Mixed expired (FEDS,) and end-tidal (FETED., ) CO, fractions were analyzed on-line by mass spectrometry (model MGA-1100, Perkin-Elmer Corp.). End-expiratory gas concentration was assumed equal to alveolar CO,: Carbon dioxide output (Vco,) was calculated from mixed expired CO, concentration and expired gas volume. In experiments employing acetylene, 0.5-ml samples of mixed expiratory gas were analyzed by gas chromatography (model 100, Carle Instruments) using a 15-cm silica gel column for separation of CO, and C,H,. Preliminary experiments indicated that a small quantity of CO, diffused through the pleural surface into the lung chamber and did not appear in the expired ventilation. This quantity was directly proportional to the differences between -end-tidal Pco2- and chamber Pco2, i.e., the diffusing capacity of the pleural surface was constant. Administration of acetazolamide did not alter the pleural diffusing capacity. Initially we prevented loss of CO, through the pleura by adjusting chamber Pco2 to the same level as end-tidal Pco2. In later studies we found it more convenient to calibrate the system at the start of the experiment by producing
d[COzl -_f(k ---_____ IH+I[KHCO,,-I _ )., rco,l, -1 dt A
l
(1)
where h,. and k, are the velocity constants of the dehydration and hydration reactions, K, is the ionization constant of carbonic acid, taken to be 3.4 x 10e4 (Zl), and f describes the degree of catalysis. If the reaction is not catalyzed, f is equal to 1.0. The rate of change of pH under these circumstances will also depend on the concentration of the phosphate in the reaction mix. Phosphate buffering can be directly related to the change in CO, concentration by mass balance since production of each molecule of carbon dioxide consumes one hydrogen ion. The observed pH changes were recorded on 0.5-in. magnetic tape (model 7600, Honeywell, Inc.) and the initial 50% of the reaction was compared to data generated for different values of f using a forward Euler integration. The sum of squares was minimized using quadratic interpolation until the best fit for f was obtained. Each value of Itz\. represented the mean of the rate constants from three to four actual reactions. RESULTS
The mass spectrometer tracing of mixed expired CO&, in a representative experiment is illustrated in Fig. 2: Because ventilation was held constant, CO, concentration is directly proportional to Vco2. Perfusion at the beginning of the experiment was 47 ml min. After a steady state of CO, excretion was reached, perfusion was subsequently increased to 77 and 100 ml. min1 and steady-state CO, excretion rose with each increment in flow. At that point, the control perfusate was replaced with the perfusate containing acetazolamide (arrow, Fig. 2). Carbon dioxide elimination dropped precipitously by one-third from the control level even though ventilation, perfusion, and perfusate pH, Pco2, and bicarbonate concentration remained constant. At all l
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884
R. A.
1.5
KLOCKE
Acetarolamide 2.5
.
2.0
x co2 1.0 iico,
A
A
Ratio Ah A
f A A
0.5 i, =
47
77
100
101
77
levels of flow VCO~was substantially less during perfusion with the solution containing acetazolamide, indicating that the production of CO, via the dehydration reaction was catalyzed by carbonic anhydrase in the control situation. This observation was a consistent finding in a series of 102 paired comparisons of Vcob, in 29 sets of isolated lungs at a variety of flow rates (Fig. 3). Control VCO~ was significantly greater than acetazolamide Vco,, (P < 0.001) under all conditions. Mean hemoglobin concentration in the pulmonary venous drainage at maximum flow in 27 experiments was 3.1 x 10m7M during the control period and 2.0 x lop7 M during carbonic anhydrase inhibition. Assay of carbonic anhydrase activity by stopped-flow spectrophotometry indicated that there was no significant difference in enzyme activity between control and acetazolamide venous effluent in 18 paired samples. The apparent dehydration rate constant averaged 60.0 t 19.4 (SD) and 64.2 t 24.4 (SD) s-l, respectively, for the control and acetazolamide solutions. This represents a catalysis factor, f, of 1.09 and 1.17, both not significantly different than 1.0. We attempted to calculate the quantity of CO, produced by the dehydration reaction from VCO~, the rate of perfusion and the initial and end-capillary (i.e., endtidal) Pco2. Assuming an ideal lung, the sum of CO, dissolved in perfusate entering the lu ng and that produced in the capillaries must equal the sum of co 2 leaving the lung in the venous drainage and via the expired ventilation. Carbon dioxide production calculated in this manner varied widely, at times having negative values. Sample computations indicated that calculated CO, excretion was -dependent upon the assumption of an ideal lung. Because the majority of dissolved COOpresent in the perfusate is excreted in our preparation, the presence of small shunts invalidates a mass balance approach, particularly at high flow rates. Accordingly, simultaneous excretion of carbon dioxide and an inert gas, acetylene, was used to quantitate the degree of catalysis of CO&,reactions. Carbon dioxide in the-expired ventilation is derived from two sources. A portion of the dissolved CO, in equilibrium with hydrogen and bicarbonate ions enters the alveolar space as the perfusate reaches the pulmo-
4
1.u
57
FIG. 2. Mass spectrometer tracing of mixed expired CO, concentration. Perfusion pH was 7.39, Pco? 38 Torr, and [HCO:,- J 23.5 mM. Lung was perfused with control solution initially. At arrow, source of perfusate was changed to reservoir containing acetazolamide. Rate of perfusion (ml. min I) is indicated below CO, tracing.
A
0.
Q Wmin) 3. Ratio of total expired CO, elimination determined during control and acetazolamide perfusion as a function of the rate of perfusion. Absolute difference between control and acetazolamide perfusion rates was 3.5 2 5.1 (SD)% of the average of the two flows. Experimental conditions: n pH 7.7, [HCO,, J 23.5 mM; l pH 7.1, [HCO,, 1 23.5 mM; D pH 7.4, [HCO,; 1 23.5 mM; A pH 7.1, [HCOJ 11.5 mM. FIG.
nary capillary. This loss of CO, disrupts chemical equilibrium, favoring dehydration of bicarbonate to molecular CO&,. Further CO, is produced and excreted, the amount depending on chemical reaction rates and capillary transit time. The quantity of CO, produced in the capillary is equal to the difference between measured total CO2 excretion and the amount of the initial dissolved CO, which is excreted. The latter is calculated from the inert gas data. In the nonhomogeneous lung, the steady-state fractional excretion of two gases dissolved in the perfusate will be identical if their solubilities are the same (7). The fractional clearance of an inert gas contained in perfusate entering an alveolus is given by PIPA - ---~VA/Q _____PI h + VA/Q
(2)
where PI is the pressure of the inert gas in the perfusate and h is the Ostwald solubility coeffkient (7). PA and VA/Q are the respective alveolar pressure and ventilation-to-perfusion ratio of that alveolus. Because k for carbon dioxide (0.624 ml ml+ atm+) (19) and acetylene (0.984 ml ml-l atm-l) (25) in an aqueous media at 37°C are slightly different, their relative excretions will vary somewhat, particularly at low VA/Q. The presence of units with low VA/Q in a nonhomogeneous lung would introduce some difference in CO, and C,H, excretions. Because lungs in our experiments were obtained from normal animals and overall VA/Q was high (range 0.95.8), differences in fractional excretion of the two gases should be small. A first approximation to correct for the small difference in fractional excretion can be made using the overall VA/Q of the whole lung. To obtain the overall VA/Q for the lungs, alveolar ventilation was calculated from l
l
l
l
(3)
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CATALYSIS
OF
CO,
REACTIONS
BY
LUNG
CARBONIC
and perfusion, &, was directly measured. Writing Eq. 2 for both CO, and C&H, and using measured acetylene elimination in expired ventilation (%$H~), the expected excretion of dissolved CO, is ir CO, exI-,ectecl
=
v C2H,
k0.
’
l
PF c, [I,
pr-0,
x c, 11,
h *-
l
+
VA@
hco2+ WQ-,
885
ANHYDRASE
1
(4)
The term in the brackets represents the adjustment for I differing solubilities and averaged only 1,133 t 0.055
and unequivocal. The differences are much greater than could be attributed to possible experimental error in CO, measurements even if the small correction for diffusion of CO, through the pleura had not been taken into account. In our treatment of these data, we have assumed that CO, crosses the alveolar-capillary membrane instantaneously, i.e., the membrane diffusing capacity (Dmco,) is infinite. Hyde et al. (14) attempted
(SD).
Simultaneous excretion of CO, and C&H, was measured in 23 paired control and acetazolamide experiments in isolated lungs from six animals. Details of the experiments are given in Table 1. The results from a single series of paired experiments are shown in Fig. 4 for both control conditions and carbonic anhydrase inhibition. The shaded area between the expected and observed CO, elimination curves represents the quantity of CO, produced in the pulmonary capillary. As noted in Fig. 4, this quantity is substantially greater at all flow rates when the action of carbonic anhydrase is not inhibited. Results from the other experiments in the series were similar without exception. Mean pulmonary artery pressures during perfusion in these experiments were 10.4 t 1.3 (SD) and 12.1 t 1.5 (SD) Torr at flow rates of 106 and 157 ml. minP, respectively. DISCUSSION
The difference between CO, excretion during perfusion with control and acetazolamide solutions is striking
ObsmNd Expected
8 (cchin) FIG. 4. Total observed (A) and expected (A) CO, excretion in expt 5 during perfusion with control (upper panel) and acetazolamide (lower paneZ) solutions. Shaded area between two curves is equal to quantity of CO, produced in pulmonary capillaries.
1. Conditions and results in acetylene-carbon dioxide experiments -____I ________
TABLE
Perfusate
CO, Production
Expt No. [HCO:,-
1, mM
&, ml +min. 1
Control, ml * min. 1
(STPD)
Acetazolamide, mlemin 1
vc, ml
t, 7 S
f
End-Capillary Equilibrium,
%
PH
Pco2, Torr
1
7.47
31
23.5
44 102 156
0.94 1.20 1.09
0.68 0.39 0.35
5.9 3.5 3.3
7.1 2.0 1.3
1.6 4.2 4.1
44 30 20
2
7.47
31
23.5
28 66 106 159
0.99 1.28 1.39 1.23
0.54 0.57 0.59 0.41
4.4 4.7 5.2 3.9
9.4 4.3 2.9 1.5
2.4 3.1 3.3 4.0
59 41 33 22
3
7.11
35
11.5
39 62 106 158
1.66 1.90 2.06 2.12
0.47 0.62 0.60 0.19
2.9 4.1 4.2 1.4
4.1 3.7 2.5 0.5
5.4 4.4 4.9 15.9
58 44 33 26
4
7.11
35
11.5
39 70 107 158
1.53 1.65 1.53 1.45
0.46 0.29 0.25 0.18
2.9 1.8 1.7 1.3
4.5 1.6 0.9 0.5
4.5 7.6 7.6 8.7
45 32 22 14
5
7.07
79
23.5
46 70 106 156
2.83 3.28 3.87 4.13
0.73 0.99 0.96 0.73
2.1 3.0 3.0 2.4
3.0 2.6 1.7 0.9
6.3 5.3 6.6 9.4
61 55 49 42
6
7.07
79
23.5
40 71 109 158
3.01 3.48 3.96 3.83
1.20 1.35 1.03 1.11
3.9 4.4 3.4 3.9
6.3 3.8 1.9 1.5
3.9 3.9 6.0 5.1
71 57 49 37
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886
R. A. KLOCKE
to measure the overall pulmonary diffusing capacity for CO, using an isotope technique, b ut could not arrive at an accurate result since pulmonary CO, diffusing capacity (DqTo ) was so great. They concluded that it must be in excess’of 200 ml. min l Torr * in the human. Therefore, the Dm,,., must be even greater since it is only one of several- processes which limit CO, movement between blood and alveolus. Thus, our assumption of an infinite Dm()., has little effect on our results, and a finite Dmo would, if anything, tend to reduce the difference between control and acetazolamide data since more CO, was excreted in the former case. The possibility of contamination of the perfusate with blood is of greater concern since, if this were the case, the demonstrated changes in Vco., would be trivial. Lungs with gross, visible contamination resulting from incomplete clearance of blood during preparation were discarded without performing any measurements. Hemoglobin concentration in venous emuent was minimal in our experiments, averaging only 3.1 x lO-7 M under control conditions. This quantity of hemoglobin indicates that the perfusate at most was contaminated with red blood cells in a ratio of (3.1 x lo- 7)/0.02 = l/64,516 assuming an intraerythrocytic concentration of 0.02 M hemoglobin monomer. Since the red blood cell contains sufficient carbonic anhydrase to catalyze the CO, hydration-dehydration reaction by a factor of 13,000 (15), this contamination would increase the rate of CO, reactions by only 13,000/64,516 or 20%. This is well below the approximately 500% catalysis demonstrated in our experiments. The possibility that carbonic anhydrase from lung tissue was released into the perfusate is also unlikely because the dehydration rate constant measured in the pulmonary drainage collected under control conditions was 60.0 s-l. This was not significantly different from the natural rate constant of 55.0 ssl at 37°C under similar conditions (5). In addition, there was no significant difference in the rate constant measured in the eMuent from paired control and acetazolamide experiments. Hence the increased production of co, seen in the control experiments did not resu.lt from contamination of the perfusate with enzyme Al though some ions such as phosphate may catalyze the hydration-dehydration reaction, phosphate concentration was so small that catalysis would barely be detectable if present (21). If phosphate catalysis did occur, it should be identical in both control and acetazolamide solutions because phosphate concentration was held constant. Although acetazolamide has been reported to affect some processes which are independent of carbonic anhydrase (l), its effect on CO*, exchange is limited to inhibition of carbonic anhydrase activity. Thus, it appears that carbonic anhydrase contained in lung tissue is responsible for the increased CO, production observed. Comparison of acetylene and carbon dioxide excretion permits accurate determination of the quantity of CO-, produced in the pulmonary “capillary,” i.e., any vessel in which CO, exchange occurs between perfusate and alveolar gas. Knowing CO, production during carbonic anhydrase inhibition and initial conditions in the perfusate prior to any gas exchange, mean pulmonary l
capillary volume and transit time can be calcul .ated. In the presence of acetazolamide, the rate of CO, production is given by Eq. 1 with f equal to 1.0. As the perfusate enters the capillary initial [H+] and [HCO:; ] are the same as those in the perfusate reservoir, but intravascular PCO~ drops abruptly to the alveolar level. Carbon dioxide subsequently produced via the dehydration reaction enters the alveolar space because Pco., remains fixed at the alveolar level during capillary transit under these steady state conditions. The initial rate of CO, production, d[CO,]/dt, is calculated using the uncatalyzed velocity constants, Itz, (55.0 s- l> and h,, (0.13 s-l) (5), and initial ionic concentrations. Endcapillary d[CO,]/dt is calculated in the same manner but requires knowledge of end-capillary [H+] and [HCO:,J. Since generation of one molecule of CO, utilizes a single molecule of both hydrogen and bicarbonate, the quantity of these two ions consumed is equal to the amount of CO, produced. Knowing CO*, production and perfusion rate, the change in perfusatebicarbonate concentration is calculated. For example, in the first experiment in Table 1, the CO, produced during carbonic anhydrase inhibition is 0.68 ml mint (3.0 x lo-:’ mol min). Flow rate is 50 ml. rnin’ (0.05 1 min) so [HCO:,J in the capillary decreased by 3.0 x 10-“/O/0.05= 6.0 x lop4 mol *l-l. S’mce an equal quantity of hydrogen ions is supplied by the phosphate buffer in the proteinfree perfusate, end-capillary pH, and therefore [H+], can be calculated from the initial pH, phosphate concentration, the pK of phosphoric acid, and hydrogen ion consumption. Initial and end-capillary d[ COJdt are averaged to obtain the mean rate of CO, production, d[CO,l/dt. Averaging is valid only if the rate of CO, production decreases linearly along the capillary. Using a modified Euler technique, we computed instantaneous d[CO,]/dt along the capillary from Eq. 1 for all our experimental conditions. Largely due to the presence of the phosphate buffer, the change in d[COJdt was linear throughout capillary transit, justifying the method of computation. Since all CO, produced in the capillary appears in the expired ventilation, after converting all quantities to common units, the pulmonary capillary volume, Vc, is equal to l
l
l
Vc = Vco,l(d[Cm/dt) Knowing the perfusion rate and the pulmonary lary volume, mean capillary transit time, t,, is t, = VCIQ
(5) capil(6)
Vc in 23 observations averaged 3.36 t 1.21 (SD) ml (Table 1). This agrees quite well with Vc predicted from the morphometric data of Weibel (26). From his measurements of alveolar surface area and the relationships between surface area and capillary volume, we calculated that our l-2 kg animals would have capillary volumes between 2.6 and 4.7 ml. Vc was independent of flow rate; this probably is a reflection of the capillary bed being maximally dilated due to the zone 3 conditions present in our experiments. As expected & was markedly dependent on the rate of perfusion (Table 1). Mean capillary tran .sit averaged 1.0 s at flows of approximately 160 ml m.in- l, the expected ca.rdiac output for a
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CATALYSIS
OF
CO,
REACTIONS
BY
LUNG
CARBONIC
ANHYDRASE
mammal of this size (19). From paired data obtained during perfusion of the same lungs with the control solution at identical flow rates, the degree of catalysis of the dehydration reaction can be calculated with the same general approach. However, instead of assuming f to be 1.0 and calculating Vc as with the acetazolamide data, the process is reversed and f is calculated to fit CO, production during control perfusion. Except for a single value of 15.9 (Table l), calculated values off grouped around a value of 5 (mean of 5.6, median of 4.9), significantly greater than the uncatalyzed factor (P < 0.001). Scatter in the data, particularly in a given animal, is predominantly due to difficulty in measurement of CO, production during carbonic anhydrase inhibition. This relatively small value is the difference between two larger quantities, the actual excretion and that expected on the basis of the inert gas excretion (Fig. 4). Small errors in calculated CO, production during enzyme inhibition are magnified since this parameter appears in the denominator off, a ratio grater than 1.0. Despite catalysis of the CO, dehydration reaction, chemical equilibrium was not reached in the pulmonary capillary in any of our experiments. The CO, production which would occur at equilibrium for a given alveolar PCO~ was calculated from Eq. 1 by setting d[COJdt equal to zero and solving for the values of [H+] and [HCOJ which satisfied this relationship. A unique solution could be obtained by trial and error in each case since consumption of bicarbonate ion and pH are related through the phosphate buffering system. The fraction of equilibrium attained was calculated from the actual measured production and the calculated equilibrium CO, production. As noted in Table 1, even in those experiments conducted at low flow rates and prolonged capillary transit times, end-capillary chemical reactions were still far from the equilibrium value. Under more physiological conditions, e.g., flows of approximately 160 ml min-l, the dehydration reaction reached only 14-42% of its equilibrium value. Although accurate for the present experiments, these calculations must be interpreted cautiously in a physiological sense since the speed of the CO, reactions are critically dependent on experimental conditions. For example, experiments performed at physiological PCO~ reached a much lower degree of equilibrium at maximum flow (14-26%) than those conducted at elevated Pco., (37-42%). In addition, the chemical system in our experiments is relatively simple; no red blood cells are present so that transmembrane ionic movements and the Haldane effect are absent. Thus, if equilibrium is not attained in our experiments, it seems unlikely that complete equilibrium would be attained in the plasma in vivo in the presence of these complicating factors which would tend to prolong the approach to equilibrium. A more quantitative answer requires not only sophisticated kinetic calculations, but also a better understanding of the localization and action of the carbonic anhydrase which is catalyzing the dehydration reaction. The site of carbonic anhydrase activity in the lung cannot be deduced from the present experiments, but Fain and Rosen (6) have demonstrated carbonic anhyl
887
drase activity associated with the pulmonary capillary endothelium of reptiles. However, the specificity of the histochemical technique used in these experiments has been challenged (17), and to date there are no reported studies using more specific techniques. If these findings are corroborated in the mammalian lung, it would seem likely that plasma CO, reactions may be catalyzed by enzyme localized to the endothelium. However, an alternate possibility exists which would also explain our findings. If bicarbonate is able to leave the vascular space and enter the pulmonary interstitium, enzyme in lung tissue could catalyze CO, reactions. This postulate would also require similar movement of hydrogen ion (or alternatively movement of hydroxyl ion in the opposite direction) because a source of hydrogen ions is needed for the dehydration reaction. In experiments with short-term transients interstitial buffers could serve as a proton source. However, these buffers would be depleted in steady-state experiments and COO production would decline with time, a finding not observed in the present work. Evidence for ionic movement out of the vascular space is controversial. Chinard and coworkers (2) studied bicarbonate distribution following bolus injection into the pulmonary circulation and concluded that this ion was confined to the vascular space. However, it is unlikely that the bolus technique has sufficient sensitivity to detect the small amounts of bicarbonate movement that would be necessary to explain our data. Taylor and Gaar (24) have demonstrated that the capillary endothelium is quite permeable to large uncharged solute molecules, but did not investigate ionic movement across the endothelium. Pore size calculated from their measurements are sufficiently large to raise the possibility of bulk fluid movement across the endothelium. Olver (18) has measured movement of ions between the vascular and alveolar spaces in the fetal lung. He concluded that the alveolar epithelium provides the principal barrier to this movement, and that the capillary endothelium is relatively permeable to ions. Thus, as postulated by Feisal et al. (8) on the basis of their work employing injection of a bicarbonate bolus into the pulmonary circulation, this ion may have a volume of distribution greater than the capillary volume. It is not clear which of these two alternatives, endothelial localization of carbonic anhydrase or movement of bicarbonate out of the vascular space, is more likely. Enhancement of the rate of CO, reactions in lung tissue may play a role in two puzzling observations noted in recent years. Multiple investigators have observed that alveolar Pco2 is greater than arterial Pco? under some circumstances (22). This has been attributed to movement of bicarbonate into the lung interstitium (16), increased CO, production near the capillary endothelium (ll), and continued CO, reactions occurring after blood has left the capillary (9). The present observations are supportive, but are certainly not definitive evidence, for the first two possibilities. Gurtner et al. (12) have calculated that a IO-fold enhancement of the natural rates of the dehydration reaction could explain the observed negative arterial-alveolar gradients.
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888
Forster and Crandall (10) have postulated that slow pH changes occur in plasma due to restriction of blood carbonic anhydrase to the erythrocyte interior, but measurements in dogs and cats from two laboratories indicate pH changes are less than those predicted (13; personal communication, E. D. Crandall). The present data suggest that catalysis of CO, reactiolrs outside the red blood cell during capillary transit would diminish the expected pH changes occurring after blood has left the lung. Although our data would qualitatively support such observations, a more precise quantitative
R. A.
KLOCKE
approach requires delineation of the actual site and mechanism of catalysis of CO, reactions in lung tissue. The author appreciates the technical assistance of Mrs. Anne Coe, Mrs. Gail Murawski, and Mr. Charles Soles, and the editolial assistance of Mrs. Marsha Barber. Dr. Aron Fisher kindly permitted the author to visit his laboratory to learn the techniques involved in perfusion of isolated lungs. Acetazolamide was supplied as Diamox by Lederle Laboratories. This work was presented at the Fall meeting of the American Physiological Society (Physiologist 20: 52, 1977) and supported by Grant HL-15194 from the National Heart, Lung and Blood Institute. Received
for publication
3 October
1977.
REFERENCES 1. ADAMSON, T. M., AND B. P. WAXMAN. Carbonate dehydratase (carbonic anhydrase) and the fetal lung. In: Lung Liquids. New York: American Elsevier, 1976, p. 221-234. 2. CHINARD, F. P., T. ENNS, AND M. F. NOLAN. Contributions of bicarbonate ion and of dissolved CO, to expired CO, in dogs. Am. J. Ph.ysiol. 198: 78-88, 1960. 3. CRANDALL, E. D., A. BIDANI, AND-R. E. FORSTER. Postcapillary changes in blood pH in vivo during carbonic anhydrase inhibition. J. Appl. Physiol.: Respirct. Environ. Exercise Physiol. 43: 582490, 1977. 4. DUBOIS, A. B. Significance of carbonic anhydrase in lung tissue. In: COz: Chemical, Biochemical and Physiological Aspects. Washington, D.C.: NASA, 1969, NASA SP-188, p. 257-259. 5. EDSALL, J. T. Carbon dioxide, carbonic acid, and bicarbonate ion: physical properties and kinetics of interconversion. In: CO,: Chemical, Biochemical, and Physiological Aspects. Washington, D.C.: NASA, 1969, NASA SP-188, p. 15-27. 6. FAIN, W., AND S. ROSEN. Carbonic anhydrase activity in amphibian and reptilian lung: a histochemical and biochemical analysis. Histochem. J. 5: 519-528, 1973. 7. FARHI, L. E. Elimination of inert gas by the lung. Respiration Physiol. 3: l-11, 1967. 8. FEISAL, K. A., M. A. SACKNER, AND A. B. DUBOIS. Comparison between the time available and the time required for CO, equilibration in the lung. J. Clin. Invest. 42: 24-28, 1963. 9. FORSTER, R. E. Can alveolar PCO~ exceed pulmonary end-capillary CO,? No. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 42: 326-328, 1977. 10. FORSTER, R. E., AND E. D. CRANDALL. Time course of exchanges between red cells and extracellular fluid during CO, uptake. J. Appl. Physiol. 38: 710-718, 1975. 11. GURTNER, G. H. Can alveolar Pcoz exceed pulmonary endcapillary CO,? Yes. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 42: 324-326, 1977. 12. GURTNER, G. H., S. H. SONG, AND L. E. FARHI. Alveolar to mixed venous Pco., difference under conditions of no gas exchange. Respiration Physiol. 7: 173-187, 1969. 13. HILL, E. P., G. G. POWER, AND R. D. GILBERT. Rate of pH changes in blood plasma in vitro and in vivo. J. Appl. Physiol.: Respirat. Environ. Exercise Physiol. 42: 928-934, 1977. 14. HYDE, R. W., R. J. M. PUY, W. F. RAUB, AND R. E. FORSTER. Rate of disannearance of labeled carbon dioxide from the lungs
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