(From the Physiological Laboratory, Groningen, Holland.)

6I2. I27.3 THE CARRIAGE OF CARBON DIOXIDE BY BLOOD. BY M. N. J. DIRKEN AND H. W. MOOK. (From the Physiological Laboratory, Groningen, Holland.) INTRODUCTION. THE opinion held by B ohr, B ayliss and others of the existence of a direct union of CO2 with heemoglobin, has quite recently again been placed in the foreground by the two papers on "carbheemoglobin" of 0. M. Henriques [1928, 1929]. In a series of experiments on the velocity with which gases are taken up by fluids the present writers [1930] were able to show that in the first 0 005 sec. of contact between gas and fluid the same quantity of carbon dioxide is absorbed by water, serum or haemoglobin solution. From this they concluded that in this very short period no CO2 is combined either by serum or by haemoglobin. The next step was to try to determine the time required for the union of CO2 with the various constituents of the blood, and for this purpose the principle, made use of by Hartridge and Roughton [1923], was incorporated in the apparatus used in the previous experiments. Thus the fluid to be examined could be mixed with another one saturated with CO2 and, after some other alterations had been made in the instrument, it was possible to measure either the CO2 tension or the H-ion concentration of the mixed fluid at any moment from about 0-05 sec. up to about 4 sec. after the moment of mixing. These readings were compared with measurements taken when the state of equilibrium had established itself. (a) METHODS. Measurement of the C02 tension of the fluid shortly after addition of CO2. A general view of the apparatus in its final form is shown in Fig. 1, while a more detailed illustration of its principal parts is given in Fig. 2. The reagents to be mixed are contained in the four syringes a, a (each of 20 c.c. capacity) and are ejected with constant velocity by a clockwork mechanism (not shown) which, by pulling the wire c, presses down the

374 M. N. J. DIRKEN AND H. W. MOOK. metal disc b. The fluids come in contact with each other in the mixing chamber d (diameter 2 mm.) and, as they do not enter radially, a strong Fig. 1. rotatory motion is set up, which ensures a thorough mixing. The mixed fluid leaves the chamber by the openingf, passes through g and h, falls in a fine jet (diameter 1-2 mm.) through the gas chamber k and leaves this by opening I (diameter 1-5 mm.). When the rate of flow has been correctly adjusted the gas can readily be prevented from leaving the gas chamber through I by keeping the level of the fluid in tube 8 exactly up to this point during the experiment. This can be done by regulating by means of the rack and pinion m (Fig. 1), the height of the suction tube n, which is connected to a filter pump. Reliable results can only be expected if the jet of liquid in the gas chamber has the appearance of a slender glass

CARBON DIOXIDE IN BLOOD. 375 rod, and this has to be obtained by adjusting the velocity of the flow of liquid. For this reason it was impossible to use the same rate of flow in all experiments, and, therefore, the observed periods vary in the various experiments. In entering the gas chamber the fluid comes in contact with an atmosphere of pure C02, saturated with water vapour, with which the gas chamber has been washed out before each experiment through the tubes q -d and r. After closing stopcock o and connecting the gas chamber with the capillary tube p, the absorption of gas by the fluid can be measured by noting the movement of a drop of kerosene. Now it was found that under identical conditions the gas absorption in a given time depends solely upon the tension of the gas in the liquid as can be demonstrated by the following experiment. If C02-saturated water is mixed with C02-free water, so that the degree of k saturation in the resulting fluids is o, v 20, 40, 60 and 80 p.c., the amount of CO2 taken up by each of these mixtures in 100 seconds by running them through -' the apparatus appears to be: 0 p.c., 0-975 c.c.; 20 p.c., 0-778 c.c.; 40 p.c., 0-572 c.c.; 60 p.c., 0-373 c.c.; 80 p.c., 0-173 c.c. Fig. 3, drawn from these Fig. 2. data, shows a linear function between the volume absorbed and the gas tension1. Identical experiments were conducted on several other fluids, the only variation consisting in bringing the mixture up to the required gas tension in a tonometer in contact with gas of known CO2 tension. These observations show that for serum and Hb solution, too, the result is a straight line, which is not so remarkable, as our earlier experiments [1930] 1 That the straight line does not pass exactly through the point of 100 p.c. saturation, is explained by the fact that in this particular experiment the water used in making the mixture happened to be not fully saturated with CO2.

376 M. N. J. DIRKEN AND H. W. MOOK. have shown that in the short time of contact with the gas no C02 is combined either by serum or by haemoglobin. 09 0*9 O*8 070- o0 03 042 X 0 10 20 30 40 50 60 70 p.c. saturation 80 90 100 Fig. 3. If there exists a simple connection between the velocity of C02 absorption and the gas tension of the fluid, then it must be possible to deduce, by the aid of the diagram, the concentration of C02 in a solution from the amount of carbon dioxide taken up by it in a given time. To try this two of the syringes of the apparatus were filled with C02-free and the other two with C02-saturated water, so that the degree of saturation ought to be 50 p.c. The actual gas absorption measured with the apparatus was 0 473 c.c. per 100 sec., which according to the diagram corresponds to 50 p.c. saturation. In calculating the concentration of C02 in solution from the velocity of gas absorption it is necessary to construct a diagram for each fluid to be examined. This, however, can easily be done, as we know the result to be a straight line, and, therefore, it is sufficient to determine the amount of C02 taken up per 100 seconds by the C02-free fluid and to connect this point with the point for 100 p.c. saturation, where the gas absorption = 0.

(b) CARBON DIOXIDE IN BLOOD. Measurement of the ph of the fluid a short time after the addition of C02. The ph of the mixed fluid was measured electrometrically: for this purpose the gas chamber with its tubes was replaced by a small vessel shown in Fig. 4. 4~~~~~~ 377 Fig. 4. Fig. 5, A short piece of glass tube (internal diameter 2-5 mm.) was cemented to the outflow tube (h, Fig. 2) of the apparatus and the electrode (a, Fig 4) introduced in this tube so that the outflowing fluid covered it completely and no C02 was lost to the atmosphere; b is the agar tube of a standard calomel electrode, tube c is connected with a filter pump and keeps the fluid level in the vessel down to the glass part of the electrode.. The E.M.F. is measured in the usual way with a potentiometer. In most of the experiments to be mentioned quinhydrone has been used with a bright platinum electrode; in the experiments, however, on haemoglobin we used the antimony electrode, to be described by Brinkman. Both electrodes were controlled at the beginning and at the end of each set of experiments by measuring the ph of a known buffer solution. The reaction time of both was short enough for our requirements and the readings were quite reproducible. The effect due to movement of the fluid along the antimony electrode actually used in our experiments happened to be small and constant and was measured to be about 0 5-1 millivolt. As Dr Roughton suggested to us, the possibility existed that the electrode does not give the ph of the average fluid moving past it, but of a relatively stagnant film in immediate contact with the electrode. The question of this stagnant film has been discussed by him in his paper on the measurement of temperature of rapidly moving fluids [1930],

378 M..N. J. DIRKEN AND H. W. MOOK_ and in order to get information on this point we have copied his control experiments. On mixing 0-06 N bicarbonate (ph 8.27) with 0-006 N hydrochloric acid, carbonic acid is formed, as will be shown later on (Table II), at such a rate that the reaction is finished in about 4 seconds, starting with great velocity and slowing up towards the end. If, therefore, stagnant films around the electrode have any appreciable effect on the readings their influence must be felt most at the beginning of the reaction, when the ph changes are greatest. The readings were taken 0*06 sec. after the mixing firstly with the electrode in a glass tube of 2-5 mm. internal diameter. This was then replaced by a tube of 1 05 mm. internal diameter, and again measurements made. Although the stagnant film in the second case must have been a great deal thinner than in the first, owing to the much greater velocity with which the fluid was flowing past the electrode, and the measurements moreover were done at a moment in which the ph of the fluid was still varying greatly, no difference was found, as is shown by the following readings of the potentiometer: Wide tube (2.5 mm. int. diam.): 90, 84, 82, 82 millivolts. Narrow,, (1.05,, ): 82, 86 It must, therefore, be regarded as improbable that a significant error is introduced in our experiments by stagnant films around the electrode. (c) Method of varying the time interval between the beginning of the reaction and the moment of observation. It is obvious that, at a given rate of flow of the fluid, the volume of the tubing between the mixing chamber and the gas chamber determines the time taken by each particle of fluid to travel from one to the other. At least this would be so if the fluid moved as a solid mass, but, when the rate of flow remains below the critical velocity, an axial flow develops if straight tubes are used. This being the case, in our apparatus the route to be travelled by the fluid was made as tortuous as possible. This was done by drilling holes of 2 mm. diameter (Fig. 5 a, a) in a brass disc and connecting these with grooves of 1 mm., care being taken not to let the fluid enter axially, so that in each 2 mm. hole a strong rotatory motion is set up. This disc can be clamped between the discs g and h (Fig. 2), and by turning disc g before screwing on the clamps the amount of holes and therefore the distance to be travelled by the fluid can be varied. The effectiveness of this arrangement was tested by the following experiments. The mixing chamber was replaced by a metal three-way stopcock and

CARBON DIOXIDE IN BLOOD.379 the syringes by two glass bulbs, which were each connected to a tube of the stopcock, the third tube of which was very short (1.5 mm.), so that the fluid on leaving the stopcock immediately entered the hole (Fig. 2). Of the two glass bulbs one contained water and the other 10 p.c. KOH, both fluids under pressure of the same column of mercury, and thus by turning the stopcock either one or the other of these fluids could be moved through the apparatus with practically equal velocity. The further set-up was as illustrated in Fig. 1, but the capillary tube p was replaced by a very sensitive membrane manometer carrying a small mirror. Therefore it was possible by optical registration methods to note exactly the moment when, on turning the stopcock, the jet of water in the gas chamber was changed into a jet of alkali by the acceleration of the rate of CO. absorption. As we knew from our earlier experiments [1930] that the CO2 absorption at the surface of a fluid is practically instantaneous and, moreover, that the velocity of CO2 absorption of KOH solutions varies with the strength of the solution, it was possible to deduce from the resulting curves exactly the moment of change from water to alkali, and also if this chging over takes place gradually or abruptly. The stopcock was turned by releasing a spring, the moment of changing from one vessel to the other being registered by an electrical signal. In order to make the sensitivity of the recording system as great as possible, a specially narrow gas chamber was constructed and the amount of tubing between the gas chamber and the manometer kept as small as possible, tube r (Fig. 1) being omitted completely. Fig. 6 A shows Fig. 6A. a typical curve, time is in 1/5 second. At a the stopcock is turned, no variation in the rate of gas absorption resulting until the KOH appears in the jet, and shortly afterwards the new rate of gas absorption is installed. The time for changing from one rate to the other varies with the

380 M. N. J. DIRKBN AND H. W. MOOK. distance between stopcock and gas chamber, but even when it takes the alkali 4 seconds to reach the jet the change is completed within 0-1 sec., thereby introducing an error in the time measurements not larger than 3 p.c. For comparison purposes the curve is given too (Fig. 6 B) when a 11 1g1 I11 IN1 Fig. 6 B. straight capillary tube of 1 m. length and 1 mm. diameter is placed between stopcock and gas chamber. The changing over takes place much more gradually, showing that a strong axial flow has been developed. In this way a good many curves were obtained with varying volume of tubing between stopcock and gas chamber. The results of the experiments with the disc described above justify the calculation of the time taken by a particle of fluid to reach the jet from the volume of the tubing between the stopcock and the gas chamber and the rate of flow of the fluid. The latter has been determined during each experiment by noting the time required for a given displacement of the pistons of the syringes, which had been calibrated for this purpose. (d) Measurement of the CO2 tension and the ph half an hour after the mixing. In some experiments measurements of the CO2 tension and the ph were done in order to find out if any alterations took place a long time after the mixing. In this case the reagents, after being mixed in the apparatus, are expelled from tube h (Fig. 2) in a narrow glass tube beneath a layer of paraffin oil. Half an hour later all four syringes are filled with this mixture, and either the concentration of the CO2 in solution or the ph is measured with the apparatus in the usual way.

CARBON DIOXIDE IN BLOOD. 381 (e) Fluids. The haemoglobin solutions used in our experiments were made by the ether method and, after preparation, diluted with 0 9 p.c. NaCl solution to the original concentration of the blood. Ox-blood was used throughout, which was defibrinated by hand immediately on bleeding the animal, then filtered and a small amount of sodium fluoride added to prevent glycolysis. Two syringes of the apparatus were filled with the hiemoglobin solution, the other two contained a 0 9 p.c. NaCl solution, saturated with C02. In this way the original fluid is diluted 100 p.c. For studying the C02 carriage by blood it was thought advisable to carry out the experiments on undiluted blood. The erythrocytes, therefore, of the defibrinated blood were separated from the serum by centrifuging, the latter was then saturated with C02 in a tonometer, and afterwards in the apparatus mixed again with the erythrocytes. In this manner it was possible to study the alterations in C02 tension and ph in undiluted blood. DISCUSSION OF RESULTS. When N/20 lactic acid is added to a 0 128 M phosphate solution of ph 7-76 equilibrium is reached within 0 09 sec. (Table I (a)). If, however, water saturated with C02 instead of the lactic acid is added to the phosphate solution, a very slow reaction begins which does not reach its final value even in 10 secs. (Table I (b) and (c)). Even at a temperature of 38.5 C. the reaction after 4 secs. is still at some distance from its equilibrium (Table I (d)). TABLE I. Concen- Time tration after of C02 the in somixing lution Reagents (sec.) (p.c.) ph (a) Phosphate +lactic acid 009 6x91 (temp. 150 C.) 00-6-91 (b) Phosphate +water (CO2 sat.) 0-09 7.74 (temp. 150 C.) 3*87 7-64 00 7.14 (c) Phosphate +water (CO2 sat.) 0-32 42 (temp. 170 C.) 4-4 38 9.3 34-13-4 27 00 9 (d) Phosphate +water (CO2 sat.) 0-098 - 7.44 (temp. 38.50 C.) 2-15 - 7 31 4-3 7-24 00 7X10 PH. LXX. 25

382 M. N. J. DIRKEN AND H. W. MOOK. Since the reaction of acids with phosphate solution is very rapid, whereas anhydrous CO2 is combined slowly, we have to assume, as other investigators have done, that H2C03, once formed, will be combined readily enough by the buffer solution but that the hydration of the C02 (002 + H20 -+ H2CO3) must be slow. The reaction H2CO3 -+ C02 + H20 was studied by adding 0-006 N acid to an 006 N bicarbonate solution (ph about 8.2) at room temperature and at 390 C., the results showing that this reaction, although much faster than its reverse, still takes seconds to be completed even at body temperature (Table II). TABLE II. TimLe after (a) (c) Reagents Bicarbonate (ph=8-27) +hydrochloric acid (temp. 15 C.) Bicarbonate (ph =8 29) +lactic acid mp 13-5' C.) Bicarbonate (ph-=817) + hydrochloric acid (temp. 39.50 C.) the mixing (2 ;ec.) ph 0l088 5 70 0 296 613 00-513 6'60.773 6-90 1 05 7-02 4 03 7-32 00 7-37 0*092 5-22 0*276 5 70 0*617 6-51 1 *82 7*12 4 03 7-34 00 7.45 01095 6-87 0-32 7-10 2,.7 7-20 4.4 7-24 00 7-26 Identical results were obtained with serum as is shown in Table III, TABLE III. Reagents (a) Serum (ph =7-87) +0 9 p.c. NaCl (C02 sat.) (b) (c) Serum (ph=7-63)+0-9 p.c. NaCl (C02 sat.) (temp. 15 C.) Serum (ph-=780) +0 004 N lactic acid (temp. 150 C.) Time after the mixing 0057 sec. 30 min. 0 09 sec. 403, 00 0 09 sec. 054,, 2-08,, 00 Concentration of C02 in solution (p.c.) 43* 29 ph 7-60 7*51 6-93 7-40 7-52 7-56 7-56 * The NaCl solution used in this experiment was not fully saturated with 002, therefore 43 p.c. was about the maximum that could be expected after mixing the fluids, showing that little or no C02 had disappeared.

CARBON DIOXIDE IN BLOOD. acid such as lactic acid being combined fairly rapidly, whereas anhydrous CO2 takes seconds to disappear. We may conclude from these experiments that the absorption of anhydrous C02 is rather slow if the CO2 is to be combined as a bicarbonate, in which case the formation of H2CO3 from C02 and H20 is essential. On mixing C02-saturated water with a haemoglobin solution we find, however, a very rapid disappearance of the C02, equilibrium being reached in not much over 0*05 sec. (Table IV), while experiments on whole blood give practically identical results. TABLE IV. Concen- Time tration of after C02 in the solution Reagents mixing (p.c.) ph (a) Hb solution (ph=7.40) +09 p.c. NaCl 0-051 sec. 13 (002 sat.) 30 min. 12 (temp. 150 C.) (b) Hb solution (ph =7.40) +0 9 p.c. NaCl 0-076 sec. - 652 (C02 sat.) 30 min. - 6-52 (temp. 150 C.) (c) Erythr. +serum (CO2 sat.) 0-083 sec. 22 (temp. 150C.) 30 min. 20 383 For the reason given above this C02 cannot be combined as bicarbonate and must therefore be bound to the haemoglobin as anhydrous C02 in some complex form as the "carbhaemoglobin" suggested by Henriques [1928, 1929], unless we ascribe to haemoglobin the function of catalyst to the reaction C02 + H20 -- H2CO3. In their recent paper van Slyke and Hawkins [1930] suggest a catalytic acceleration by the cell contents of the reaction HC0O3-+ H+ = H2CO3. As far as we know there is no reason to suspect this part of the C02 absorption of needing any acceleration, and, in accordance with Faurholt's data, our own experiments given above make it much more probable that the hydration of C02 is slow, and if therefore heamoglobin or some other cell contents act as catalyst it most probably influences the velocity of the reaction C02 + H20 = H2003. SUMMARY. An apparatus is described with which it is possible to measure either the ph or the C02 tension of a fluid after C02 has been added at various moments up to about 4 seconds. 25-2

384 M. N. J. DIRKEN AND H. W. MOOK. The reaction of acids with phosphate solution is found to be very rapid, whereas anhydrous C02 reacts very slowly. This suggests that the hydration of C02 is responsible for the retardation. In the same sense the dehydration of H2C03, although much more rapid than its reverse reaction, must be regarded as slow, which can be deducted by comparing the velocity with which acids combine with bicarbonate and phosphate. If haemoglobin is present anhydrous C02 disappears from the fluid just as rapidly as any acid. From this it is concluded either that there must exist some direct combination of anhydrous C02 with haemoglobin as suggested by Henriques, or else that a catalytic acceleration of the reaction C02 + H20 -. H2C03 by haemoglobin must occur. REFERENCES. Brinkman, R. (1930). Handb. biol. Arb. Met. Abt. m, Teil A2. Dirken, M. N. J. and Mook, H. W. (1930). Biochem. Z. 219, 452. Hartridge, H. and Roughton, F. J. W. (1923). Proc. Roy. Soc. A, 104, 376. Henriques, 0. M. (1928). Biochem. Z. 200, 1. Henriques, 0. M. (1929). Ergebn. Phy8iol. 28, 625. Roughton, F. J. W. (1930). Proc. Roy. Soc. A, 126, 439. van Slyke, D. and Hawkins, J. A. (1930). J. Biol. Chem. 87, 265.