(hemolysis, hematocrite and cryoscopic method) and have supported his

Transcription

1 THE ACTION OF CARBON DIOXIDE ON SALT AND WATER DISTRIBUTION IN BLOOD. BY GENKO MUKAI, M.D. (From the Institute of Physiology, University College, London.) THE osmotic behaviour of the red blood corpuscles has been much debated since Hamburger (1883) pointed out that the amount of water in the corpuscles was determined by the law of osmotic pressure. Many investigators have carried out experiments by the most diverse methods (hemolysis, hematocrite and cryoscopic method) and have supported his view, as long as the semipermeability of the membrane was preserved. In 1894, Limbeck(l) found that the corpuscles absorbed water from plasma, when carbon dioxide was passed through a sample of blood. If the same law holds good also in this case, the osmotic pressure of the corpuscles must increase more than that of plasma. The results so far obtained by the A determination render this doubtful, and at the suggestion of Prof. Bayliss I have investigated the question. All experiments were performed with defibrinated dog's blood, which was drawn from a cannula in the carotid artery of a dog under chloroform and ether. Special care was taken to use the blood as soon as possible. A. CHANGES PRODUCED IN BLOOD BY CARBON DIOXIDE. The distribution ofsalts and water. It is well known that carbon dioxide has a great effect upon the osmotic pressure of blood. The mere addition of C02 raises the osmotic pressure considerably more than the molar concentration of the added substance would explain. This effect is generally understood as a result of a complex reaction of C02 with the salts of the blood. As they are separated by a semipermeable membrane, corpuscles contain different salts from those of plasma, both in quality and quantity, which is responsible for the different osmotic effects of C02 on both sides of the membrane. Experiments were made therefore to determine the distribution of salts and water in the blood at various tensions of 002. Exp. I. Dog's defibrinated blood was divided into two portions. Sample A was exposed to the air. Sample B was equilibrated with CO2 in a large flask which contained almost pure CO2. The following were determined:

2 SALT AND WATER IN BLOOD. 357 (1) The volume percentage of the corpuscles. The blood samples were centrifuged beneath liquid paraffin until the reading became constant. (2) The solid. A known volume of blood and of serum was weighed, dried at about 100 '-110 C. until the weight became constant. (3) Ash as sulphate. The same quantity of diluted sulphuric acid was added to 4 c.c. of the samples and then dried and ashed to constant weight. (4) Chlorine content. After removing the protein from blood and serum by my method1, the chlorine contents were determined by Volhard's method. 100 c.c. blood* 4 c.c. serum r A A --A Before C02 After C02 Before After treatment treatment C02 C c.c. treat- treat- Cor- Cor- Sample blood ment ment Serum puscles Serum puscles I. Volume c.c Weight gm Water,, Solid,, Ash,, NaCl,, II. Volume c.c Weight gm Water,, Solid,, Ash,, NaCl,, III. Volume c.c Weight gm Water,, Solid,, Ash,, NaCl,, * The total amounts of NaCl and ash present in serum and corpuscles were calculated from the percentage of both components of blood. It will be noted that under the influence of C02: (1) The corpuscles increase in size bv taking up water from serum. (2) The percentages of the solid and ash increase in serum. (3) The total amount of ash as sulphate remains the same in serum as a whole, when allovance is made for the loss of water, which shows clearly that cations do not pass from corpuscles into serum, and that there is no interchange of base between them. This result is identical with that of Giirber(2) and Hoeber(3). (4) As Hamburger(4) pointed out, the total amount, as well as concentration, of chlorine decrease in serum which must have entered into corpuscles. These changes have been proved to be reversible and parallel in response to the variations of C02 tension (Hamburger(s), Limbeck(5), Haggard, Howard and Y. Henderson(6), Fridericia(7)). 1 This will be published shortly in the Biochemical Journal.

3 358 G. MUKAI. Exp. II. The dog's blood was divided into several portions. After exposing it to the given tension of CO., each sample was centrifuged under liquid paraffin and then the volume percentage of corpuscles, the chlorine concentration of serum and the percentage of the solid in serum were determined. The effect of increased C02 tension upon the salt and water distribution in blood. 4 c.c. of serum In serum C02 tension Weight Solid % of % of Vol. % of mm. gm. gm. solid NaCl corpuscles (1) Exposed to the air (2) 143 4* * (3) 225 4* (4) * (5) *0 (6) The capacity of serum to carry C02. There is a close relation between the salts and the C02 contents of serum as the following experiments show. Exp. III. Defibrinated dog's blood was divided into two portions, which were treated as follows: Sample A (blood). Sample B (serum). The serum was obtained by centrifuging. Both samples were divided again into six portions and each was equilibrated respectively with the air and air containing 75, 112, 153, 186 and 223 mm. of C02 at room temperature. All portions of sample A were then centrifuged under liquid paraffin and serum was separated. The amount of C02 in the serum was determined by Van Slyke's method. The effect of increased C02 tension upon the C02 carrying power of serum. c.c. C02 content in 100 c.c. of serum Serum Serum C02 mm. sample A sample B Difference Exposed to the air 29* 29* * After exposing to the air, serum of sample B was obtained. Exp. IV. The same blood was used. The blood was exposed to the air containing CO2 at 186 mm., and was divided into two portions. Sample A (blood). Sample B (serum). The serum was separated from corpuscles. Both samples were divided again into three portions and then were equilibrated with C02 at various decreased tensions. Sample B' was a portion of the serum of sample A, which was separated at 153 mm. of C02, and was treated like sample B. The effect of decreased C02 tension upon the C02 carrying power of serum. c.c. C02 content in 100 c.c. of serum Serum Serum Serum C02 mm. sample A* sample B sample B' * Serum of sample A was obtained after exposing to the decreased tension of C02.

4 SALT AND WATER IN BLOOD. From these data we see the influence of corpuscles on the capacity of serum to carry CO2. Both samples A and B change their C02 contents in accordance with variations of tension; but the former is more liable to such change than the latter. This different behaviour of serum is attributed to the varying distribution of salts and water in blood under CO2 treatment. As long as serum is in contact with corpuscles and these alterations are reversible, serum contains a definite quantity of C02 at a given tension, no matter whether the tension is decreased or raised to that degree. (Compare sample A in Exp. III with sample A in Exp. IV.) After separation, serum is free from the influence of the corpuscles and carries CO2 by its own capacity. At zero tension of CO2 there is practically no trace of it in blood, but the serum, which has been previously separated from corpuscles, still contains a certain quantity. This remainder of C02 is found to be alkali carbonate and variable in quantity in proportion to tbe tension at which the serum is obtained. The fact shows clearly that there is an actual change in the total amount of diffusible alkali in serum as the result of treating blood with CO2 (Loewy and Zuntz(9), Hamburger(1o)). It might be explained by an interchange of either acid or basic radicals between corpuscles and serumr. While there is no evidence of alkali interchange, the cblorine passes to and fro between them in response to variation of C02 tension. Thus alkali of neutral salts (NaCl, KCI, etc.) is released' in serum to form alkali bicarbonate, if the tension is raised, and vice versa. Girber(11), Van Slyke and Cullen(l2) and Fridericia(7) have proved by cbloride analysis that the amount of HCI formed from NaCl and passing into corpuscles corresponds to the greater part of the amounts of alkali bicarbonate produced in plasma. Thus the influence of corpuscles upon the capacity to carry C02 of serum mav be regarded as aresult of changed distribution of anions in blood. The distribution of C02 between corpuscles and serum. Carbon dioxide in blood is approximately equally distributed between corpuscles and serum at every tension of C02. There is, however, a small difference between them. Exp. V. Defibrinated dog's blood was equilibrated with C02 at various tensions. C02 content in 100 c.c. of blood and serum C02 mm. Blood c.c. Serum c.c. Difference

5 360 G. MUKAI. At low tension of C02, blood carries less of it than tbe corresponding serum. The difference between them becomes smaller and smaller until they contain the same quantity at 170 mm. of C02. At tension higher than 170, blood contains more CO2 than its serum. This result can be explained by the unequal distribution of CQ2 between corpuscles and serum at each tension except 170 mm. In other words, the increase of C02 content in corpuscles is always greater than that in serum, although both increase in proportion to the CO2 tension. If the tension of C02 is decreased, this relation between corpuscles and serum becomes the reverse, the corpuscles lose C02 more than serum. It is of interest to compare the effects of the decreased C02 tension upon the blood and the separated serum. The serum which has been separated from corpuscles loses less CO2 than that which has not (see Exp. IV). Blood is most sensitive to the alternation of CO2 tension and loses C02 in the highest degree. These unequal effects of decreased tension must be borne in mind when determining the freezing point of blood. The higher the tension of C02, at which the serum is separated from corpuscles, the greater the difference of escaping C02 from blood and separated serum. The mechanism by which the corpuscles carry C02. There is a remarkable fact in Exp. V wbich shows that the quantity of C02 in corpuscles changes in proportion to the variation of the C02 tension. This would seem to contradict the result, which might be expected from the interchange of acid radicals. The explanation of this is that proteins are amphoteric bodies and that the isoelectric points of heemoglobin, serum glob"ulin and semum albumin are different. Thus hiemoglobin releases all its alkali at CH , while the serum proteins set free only a small part of it. On the other hand, the corpuscles contain about 30 p.c. of hlemoglobin, which is a very high concentration compared with the protein in serum (about 8 p.c.). As the quantity of the newly produced bicarbonate depends on the amount of alkali-protein, and the rate of its increase on the isoelectric point of protein, the corpuscles have a stronger and quicker capacity to form bicarbonate than serum (Zuntz (15), Hamburgerlo)). If carbon dioxide is led through the blood, these capacities of botb corpuscles and serum will undergo a change. By means of acid radical interchange, the serum gets more available alkali to combine with C02, while the corpuscles lose it in just the same degree, i.e. corpuscles lose just what the serum gains, and the C02 carrying power of the blood remains uninfluenced by this interchange of acid radicals. The relation can be seen in the following experiment.

6 SALT AND WATER IN BLOOD. 361 Exp. VI. Dog's blood was divided into two portions. Sample A (not hssmolyzed). Sample B (hwmolyzed by freezing and thawing). Both samples were equilibrated with CO, at various tensions and analyzed for CO2 content. C.C. C02 content in 100 c.c. of Hoemolyzed Not haemolyzed C02 mm. blood blood Difference *5 66* * Blood was ha2molyzed and then exposed to C02. This shows also that (1) haemolysis itself has no effect upon the amount of CO2 in whole blood, (2) the chlorine seems to be an agent, which regulates the distribution of C02, CH and the osmotic pressure of blood, so that they are approximately equal on both sides of the corpuscles. B. THE INCREASE OF THE MOLAR CONCENTRATION OF BLOOD UNDER C02 TREATMENT. When blood is treated with CO2, both corpuscles and plasma increase in osmotic pressure. If C02 is led through a sample of plasma, which has been separated from corpuscles, the increase of osmotic pressure is decidedly less than that which has not. Exp. VII. Defibrinated dog's blood was divided into two portions. From one part, serum was obtained by centrifuging. Sample A (whole blood). Sample BR(serum). Both samples were exposed to the air containing 40, 510, 600 mm. of C02 at room temperature and then the A of serum determined. A of the serum A of the serum CO2 mm. of A 'C. of B 'C. 40-0*605-0* * The difference of osmotic pressure is due to the alteration of the capacity to carry tj2 of serum. From this it is evident that a close relation exists between the osmotic pressure and the CO2 carrying power -the amount of C02-of blood. On the other hand, it has been proved that corpuscles take up CO2 more than the corresponding serum at every tension of C02 and absorb water from serum. If C02 were Mi one form in blood, then the explanation of osmosis into corpuscles would be quite clear. Corpuscles are free to change in size by taking and giving up water from the surrounding fluid. Thus corpuscles are supposed always to have the same osmotic pressure with surroundings. According to the osmotic

7 362 G. MUKAI. theory, the increase of water in the corpuscles under C02 treatment must be the result of increased internal concentration of substances to which the membrane is impermeable. The freezing point determination seems to be the only method which gives a direct experimental answer to the enquiry as to whether the osmotic pressures are equal on both sides of the corpuscles, i.e. whether the law of osmosis is perfectly applicable to blood corpuscles. Hamburger(l6) carried out such measurements and obtained results which showed that the corpuscles had a less osmotic pressure than the plasma, both in the normal condition and in the case of blood treated with C02. He insisted, however, that the law of osmosis was applicable and ascribed the deviations to the adsorptive phenomenon of crystalloids after baemolysis. Some authors assume the swelling pressure of the protein, which compensates the osmotic negative pressure of the corpuscles, and some go so far as to state that the regulation of the amount of water between corpuscles and plasma is solely determined bv the protein and not by the salts. Such ideas are contradicted by the following experiments. Cryoscopic method. By the freezing and thawing method I haemolyzed the blood and repeated the experiment of Hamburger. Exp VIII. After exposing the blood to the air for one hour, I determined the A of both haemolyzed blood and serum with Beckmann's apparatus. The A of normal blood A of haemolyzed Sample blood 0 C. A of serum 0 C. Difference 0 C. 1-0* O O O * Exp. IX. After exposing the blood to carbon dioxide, I divided it into two parts: One part was htsmolyzed under liquid paraffin. The other was centrifuged under liquid paraffin, which separated serum from corpuscles. The A of both h*emolyzetl blood and serum were determined. The A of blood treated with CO2 A of haemolyzed Sample blood 0 C. A of serum 0 C. Difference C A ± O O0674 -Q * These results are identical with those of Ham burger.

8 SALT AND WATER IN BLOOD There are, however, some possible sources of error in these experiments: 1. Hamburger attributed the difference of the A between baemolyzed blood and serum to an adsorption of crystalloids by the colloids of the corpuscles. This might be so, but he did not satisfactorily explain why the osmotic difference was so great in the case of blood treated with Co2. 2. There is an opportunity of CO2 escaping from the fluid, wbich changes the A of it. The first reading of the freezing point is always lower than the second. The difference between the first and second readings is found to be enormous when the determination is made after C02 treatment. But serum, as a rule, is less liable to change its A than haemolyzed blood. Ham birger(17) has pointed out that the A of serum can be determined without separation from corpuscles unless it contains much C02. In the latter case it must be separated from the corpuscles, otherwise the A of this serum reads less than it ought. These facts seem to coincide with what I noticed in Exps. IV and V. By exposure to the air, the blood loses very quickly a considerable amount of 002, while the separated serum only loses a small quantity. The error caused by escaping C02 is magnified by the stirting and the long exposure, which cannot be avoided in the cryoscopic method. The higher the concentration of C02, the greater the error. As far as the amount of C02 is concerned, haemolysis has no effect upon the blood, but the tension of C02 bas great effect. For this reason I decided to adopt another method of determining molar concentration, which prevents the escape of C02 from the fluid. Barger's microscopical method. In 1904 Barger(ls) published a method, which seems most suitable for this purpose: A solution of unknown molar concentration is compared with a standard solution. A series of drops are collected from the two solutions alternately into a glass capillary tube. After sealing both ends of tube, the length of the drops is measured with a micrometer under the microscope. If the vapour pressure is equal in both solutions, they have the same molecular concentration and there is no change in the length of the drops. In order to use this method with blood, some special conditions are necessary. Since haomoglobin is apt to crystallize out, the blood should be diluted to a known degree with its own serum. The standard solution must bave the same hydrogei-ion concentration and the same tension of carbon dioxide as the blood or serum. Otherwise the latter will lose

9 364 G. MUKAI. carbon dioxide and its osmotic pressure fall. The blood in the capillary is haemolyzed by freezing and thawing after sealing. Two methods were used. In the first, alternate drops of blood and corresponding serum were compared. In the second, the blood and the serum were separately compared with solutions of sodium chloride of known strength. The second method is exposed to a slight error owing to the possibility of unequal loss of CO2 from blood and from serum. All samples of blood and serum were kept under liquid paraffin. Directly before use, I took out a small portion of them in crucibles, taking care to avoid mixing them with paraffin. It was reasonable to suppose that some carbon dioxide escaped from the fluid during the manipulation. To restrict these errors to the minimum, I took drops of equal length as quickly as I could and tried to leave equal air spaces between them. Exp. X. Normal blood. The volume change of drops in Barger's method, at 15 C., in the first 24 hours. (Ocular micrometer 0-1 mm. No. 2 and Leitz objective No. 2.) By the second method s, \~~~~~~ By the first method a b a b a Sample 1. Result a =b Standard NaCl solutiohi p.c (a) The blood was compared A\ a 8 a s a ± a =0-961 % NaCl Sample 2. Result a = b 0 990>a>0-982 % Many other experiments gave the same results. a indicates blood which is diluted with the same quantity of serum. b indicates serum. (b) The serum was compared b 8 b 8 b ± b=0-961 % NaCl ±0 ± >b>0-982 % 8 indicates standard NaCl solution. The length is indicated in one-tenth of one degree. These data show that: (1) The molar concentration of the normal blood is the same inside and outside the cells. (2) The difference of molar concentration, which corresponds to the A of 0005 C., can be easily distinguished by this method.

10 SALT AND WATER IN BLOOD. Exp. XI. Blood was treated with CO2. After establishing equilibrium I separated the serum from corpuscles and measured the molar concentration. The volume change of drops in Barger's method, at 15 C., in the first 24 hours. (Ocular micrometer 0*1 mm. No. 2 and Leitz objective No. 21) By the second method By the first method Standard NaCl '_ a b A a b a p.c. 1X ± ± ±0 ±0 ± Sample 1. Result a b ± * ± ±0 ± Sample 2. Result a=b solution (a) The blood was compared a 8 a 8 a ± %>a> % ±0 +1 ± a= % (b) The serum was compared b 8 b 8 b ±0 +4 ± % >b> % ± % >b > % Many other experiments gave the same results. a indicates blood which is diluted with the same quantity of serum. b indicates serum. 8 indicates standard NaCl solution. The length is indicated in one-tenth of one degree. 365 There was no change in the length of the drops by the first method. The molar concentration of both blood and serum must be the same within the limits of accuracy of this method. A small difference between (a) and (b), which corresponds to the A of C., was due partly to the unavoidable error caused by the escape of C02. From this result it is safe to say that the molar concentration of the corpuscles is almost equal to that of the serum, at the most, the difference between them is below that which corresponds to a A of C. The difference of the molar concentration between corpuscles and serum, which is obtained by the cryoscopic method, is due chiefly to the loss of C02. C. OSMOSIS OR DIFFUSION AFTER DEATH? It is said that the cells obey the osmotic law while the semipermeability of the membrane is preserved. As soon as this semipermeability is destroyed which occurs soon after death, the cells no longer follow this law and the salts diffuse through the membrane until their concentration becomes the same on both sides of the membrane. It may be asked whether the corpuscles were alive or not in the foregoing experiments. Unfortunately it is impossible to discriminate between the living

11 366 G. MUKAI. and dead corpuscles. For this purpose muscle is more suitable and I have made experiments on this. Isolated frog's muscle seems to be isotonic with 0.7 or 0 75 p.c. of NaCI solution. It takes a long time to reach osmotic equilibrium and there is a tendency to accumulate metabolic products in the muscles, so that the muscles when active have a stronger osmotic pressure than when inactive. This was proved by experiment with fatigued muscle, which required more concentrated solution to keep its original weight. Otherwise this muscle took water from the surrounding medium and the weight reached a maximum about two to three hours later (Cooke (19), Fletcher(20)). For this reason I compared both sartorii of a frog and weighed them two or three hours later after immersing them in the fluid. Exp. XII. M. sartorii of frogs were immersed in dog's blood serum, which was diluted with water so that the serum became isotonic with 07 p.c. NaCl solution. After weighing, I passed carbon dioxide through the serum, which contained one group (a) of these sartorii, and observed the change in weight and irritability to electric stimulant. Experiments were carried out by Overton's method (23). Weight after immersing Percentage of increase in weight after (hours) 3 hours in m Sample serum, gm. D. 1 1* 2 3 4i a D. 9-3 D. 0 1 b 0* ±0 ±0 - ±0 ±0 ±OD D. 15 2a D. 8 ±OD. 0 2b D D a * D. 6 3b 0* ±0 ±0 - ±OD. 10 Muscle (a) was treated with CO3. Muscle (b) was, for the purpose of contrast, not so treated. D. indicates the maximum distance (cm.) of an induction coil, at which the muscle contracted. D. 0 means dead. + means increase. - means decrease. It is evident from this that muscles increase in weight after treatment with CO2. The muscle lives after more than six hours of C02 treatment. Experiment in a solution, which contained 0 5 p.c. NaCl and p.c. NaHCO3 (isotonic with 0 7p.c. NaCl solution), gave nearlythe same result. Exp. XIII. The same experiment was made in a 0 7 p.c. NaCl solution. Weight after immersing Percentage of increase in weight after (hours) 1 hour in,ak_ Sample NaCl sol., gm. D a D lb D D.0 2 a D b D D. 0 3 a D b 0* D D.0 a treated with C02. D. as in Exp. XII.

12 SALT AND WATER IN BLOOD. 367 The change in weight was so small in this experiment, that during life there was no appreciable effect of C02. After death there was a great difference between them; muscle treated with C02 decreased in weight, while the other increased greatly. This behaviour of dead muscle occurs to a greater or leser degree in all experiments. Exp. XIV. Muscle in 6 1 p.c. cane sugar solution (isotonic with 0 7 p.c. NaCi). The muscle died very quickly, after two hours it could not be contracted by electric stimulation. Weight after immersing Percentage of increase in weight after (hours) I hourin the Sample solution, gm. 2k 3k a lb a 2 b a b a treated with CO.. D. as in Exp. XII. It is evident from these experiments that: (1) the living muscles increase in weight by treatment with C02 and reach the maximum about two to three hours later; (2) as long as they are alive, they keep their weight in an isotonic salt solution; (3) soon after death there appears a change in their weight, although they are kept in the same solution. This behaviour of the dead muscle finds its explanation in the physical property of muscle proteins., Muscle contains several kinds of protein gels. Some of them are characterized by their strong capacity for swelling and some of them by their power of spontaneous coagulation after death which is regarded as a phenomenon of dehydration. When a muscle is treated with C02, lactic acid is formed very rapidly and accumulates in it. On the one hand, this tends to cause some of the proteins to take up water, and thus the living muscles increase in weight for two to three hours. On the other hand, it tends to cause coagulation, i.e. dehydration. In a muscle treated with C02, the coagulation may occur almost immediately after death, so that soon after death there is a decrease of the osmotic pressure of muscle. A proof of this is that a muscle left in sugar solution till it is dead decreases in weight only after adding some quantity of acid (CO2). We have seen that muscle continues to live in serum over six hours after C02 treatment. I infer that the same is the case with blood corpuscles. The following conclusions may then be drawn: (1) the corpuscles increase in size by osmosis under C02 treatment, (2) the law of osmosis is perfectly applicable in the case of blood treated with C02,

13 368 G. MUKAI. i.e. water enters into the corpuscles until the osmotic pressure becomes equal on both sides of the membrane. It may be argued against the result obtained by Barger's method that the corpuscles may change their osmotic concentration after deathhaemolysis-just like muscle. This may be possible to a certain extent, because there is a sudden production of lactic acid in corpuscles after death (Mellanby and Thomas(21)). Thus increased acidity may alter the degree of swelling of protein particles and may decompose any bicarbonate present in corpuscles. It is difficult to state how far this is actually the case. The main constituent of corpuscles-haemoglobin-is a hydrophobic colloid which cannot change its degree of swelling. Of course other protein particles may change their swelling but this factor will be negligibly small on account of their low concentration. The factor of decomposing bicarbonate must be assumed in haemolyzed blood. This error, if there is any, is included in the difference between corpuscles and serum obtained by Barger's method, but it is very small as shown in Exp. VI. D. THE CAUSE OF OSMOSIS OF THE BLOOD CORPUSCLES UNDER CO2 TREATMENT. It remains to discuss the cause of osmosis of the corpuscles under CO2 treatment. The foregoing experiments indicate that two factors come into play: (1) imbibition of colloid particles, and (2) change in salt distribution. Imbibition of colloid particles. When blood is treated with C02, it increases in acidity. Consequently the hydrophilic colloids both in serum and corpuscles may change in swelling. The red blood corpuscles are a mixture of hydrophilic (protein of stroma and lecithin) and hydrophobic (haemoglobin and cholesterin) colloids. Among them the protein of stroma is of great importance, which exists about 3-5 p.c. in corpuscles and is known as "fibrin like" in character. On the other hand, serum contains a very small quantity (6-7 p.c.) of hydrophilic colloids. Owing to their low concentration, the amount of water absorbed by these colloids must be small. If this factor plays any part in osmosis, it may be explained by the difference in the amount of water absorbed by colloids on both sides of the membrane, and it must be very small. Change in salt distribution. Exp. V shows that the corpuscles take up CO2 more than the corresponding serum at every tension of CO2- Exps. I and II show that the chlorides also increase in the corpuscles in proportion to CO2 tension. From these facts it is evident that the

14 SALT AND WATER IN BLOOD. essential cause of osmosis must be some compounds of C02 and chlorine. There are many forms of these compounds, but in general they can be divided into two groups; organic and inorganic. The former group is combined with protein and exiists only in a reaction more acid than the isoelectric point of protein. The isoelectric point of haemoglobin is about CH and can be easily reached by treating blood with 150 mm. of C02 (Hasselbalch(22)). On the contrary the isoelectric points of serum proteins have a very high CH and are difficult to be reached. Thus at lower tension than 150 mm. of C02, blood combines with C02 and chlorine only in such forms as alkali carbonate, alkali bicarbonate, carbonic acid and alkali chlorides. Consequently the cause of osmosis below 150 mm. of C02 must be attributed to these salts and acid. If the tension of C02 is raised more than 150 mm., hawmoglobin can combine directly with C02 and chlorine, and the further increase of water in corpuscles is determined chiefly by the dissociation of these organic compounds. SUMMARY. 1. By treating blood with C02, the corpuscles increase in chlorine and water, but there is no evidence of cation interchange between corpuscles and serum. 2. At every tension of C02, corpuscles take up more C02 than the corresponding serum. 3. Carbon dioxide is carried partly by increased diffusible alkali and partly by protein in blood, the quantities of which depend solely on the tension of C02-CH. 4. The error involved in the cryoscopic method is chiefly caused by the escape of C By means of Barger's method, which is able to determine the molar concentration without losing C02, it has been proved that the law of osmosis is perfectly applicable in the case of blood treated with C From the results obtained by the experiments with muscle, it may be inferred that the corpuscles are alive for more than six hours after C02 treatment and take up water by osmosis, not by diffusion. 7. The cause of osmosis is due to the increased C02 and chlorine in corpuscles. I take this opportunity of thanking Prof. W. M. Bayliss, F.R.S. for his kindly help and advice during the progress of this work. 369 PH. LV. 24

15 370 G. MUKAI. REFERENCES. (1) v. Limbeck. Arch. f. exp. Pathol. u. Pharm. 35. p (2) Gurber. Sitzb. d. med. physik. Gesells. zu Wurzburg, 25 Feb (3) Hoeber. Arch. f. d. ges. Physiol p (4) Hamburger. Zts. f. Biol. 28. p (5) v. Limbeck. Arch. f. exp. Pathol. u. Pharm. 35. p (6) Haggard, Howard and Henderson, Y. J. Biol. Chem. 45. p (7) Fridericia. Ibid. 42. p (8) Hamburger. Arch. f. (Anat. u.) Physiol. p ; Zts. f. Biol. 28. p (9) Loewy u. Zuntz. Arch. f. d. ges. Physiol. 58. p (10) Hamburger. Arch. f. (Anat. u.) Physiol. p (11) Gurber. Maly's Jahresb. Tierchem. 25. p (12) Van Slyke and Cullen. J. Biol. Chem. 30. p (13) Rona u. Gyorgy. Bioch. Zts. 56. p (14) Hamburger. Ibid. 86. p (15) Zuntz. Beitr. z. Physiol. des Blutes, Diss. Bonn (16) Hamburger. Arch. f. (Anat. u.) Physiol. p (17) - Cntrlb. f. Physiol. p (18) Barger. Trans. Chem. Soc. 85. p (19) Cooke. J. of Physiol. 23. p (20) Fletcher. Ibid. 30. p (21) Mellanby and Thomas. Ibid. 54. p (22) Hasselbalch u. Warburg. Bioch. Zts. 86. p (23) Overton. Pfluiger's Arch. 92. p