Westminster Hospital Medical School.) connection with the general problem of blood clotting, we have investigated. (Received June 18, 1935.

Transcription

1 173 6I2. II5.3 CHANGES IN THE COAGULABILITY OF THE BLOOD PRODUCED BY CITRIC ACID AND SOME OF ITS DECOMPOSITION PRODUCTS. BY DAVID DE SOUZA AND F. D. M. HOCKING. (From the John Burford Carlill Laboratories, Westminster Hospital Medical School.) (Received June 18, 1935.) FROM a survey of the literature relating to the production of hypercoagulability of the blood by the injection of citrates, it appears that the results have been obtained from experiments with sodium citrate. Neither citric acid nor any other of its compounds seems to have been much used. As a knowledge of the action of the citric acid molecule and its decomposition products might be of value not only per se but also in connection with the general problem of blood clotting, we have investigated the effects of the intramuscular injection of some of these substances on the alkali reserve and the coagulability of the blood. METHODS. Cats, anaesthetized by intraperitoneal injection of nembutal, 60 mg. per kg. body weight, were the subjects of the experiments. The substances to be tested were given by injection into the muscles. The methods of carrying out the experiments and the estimations have been described in detail in a previous paper [de Souza and Hocking, 1934]. RESULTS. In the first place it was necessary to find out whether the changes produced in the blood by intramuscular injection of sodium citrate were due entirely to the citric acid molecule or were influenced by the presence of sodium in it. For this purpose a series of experiments was carried out, in which citric acid was injected instead of sodium citrate. A single injection of citric acid, g. per kg. body weight, was given, that

2 174 D. DE SOUZA AND F. D. M. HOCKING. being the amount equivalent to sodium citrate g. per kg. body weight used in the previous investigation. A typical result is shown in Exp. 1. Exp. 1. Intramuscular injection of citric acid. Alkali reserve Coagulation time There was an increase in the alkali reserve from 56 to a maximum of 61 in 25 min., and a diminution in the coagulation time from 3X5 to a minimum of 2X8 in 15 min., with an indicated return towards the control in the former case and an almost complete return in the latter, 45 min. after the injection. The changes are much smaller than those with sodium citrate but are in the same direction. While the citric acid undergoes a certain amount of oxidation, it is evident that the presence of sodium in the citrate molecule leads to a greater increase in the alkali reserve, and this may account for the larger effect of the sodium citrate on coagulability. The action of ammonium chloride in diminishing the alkali reserve and increasing the coagulation time has already been described by us [1934]. Prof. Lovatt Evans suggested that it would be of interest to compare with this the action of ammonium citrate. This has been done in experiments on the same plan as the preceding. The amount of ammonium citrate injected was 0'118 g. per kg. body weight. Exp. 2 is an example. Bxp. 2. Intramuscular injection of ammonium citrate. Time after injection Alkali reserve Coagulation time * The alkali reserve increased from 52 to a maximum of 59 in 25 min. and remained practically at that level for the next 20 min. The coagulation time fell from 3x8 to a minimum of 2x5 in 15 min., returning to 3*3 in the next 10 min. and remaining there for the rest of the experiment. The result is like that from citric acid. The ammonia seems to have been removed from the citrate, perhaps to take part in the formation of urea, leaving the citric acid which has been oxidized to some extent.

3 CITRIC ACID AND BLOOD CLOTTING. Attention was directed next to some of the decomposition products of citric acid. At present no evidence is available as to which, if any, of those examined are formed from the breakdown of citric acid in the body. The action of acetone-dicarboxylic acid (CH2COOH.CO.CH2COOH) was first studied. Added to blood in a paraffined test-tube it converts the hemoglobin into acid haematin and acid hamatoporphyrin and causes the formation of a gel, which looks somewhat granular and does not undergo syneresis. Its sodium compound, formed by dissolving the acid in the calculated amount of NaOH solution, does not affect the haemoglobin and has been found to prevent the clotting of blood up to 48 hours. In the injection experiments the quantity of acetone-dicarboxylic acid put into the muscle was 0 07 g. per kg. body weight, that being the amount which would be obtained from g. of citric acid, equivalent to g. of sodium citrate taken as a standard. When the sodium compound was used it was freshly prepared from this amount of acetonedicarboxylic acid. Exp. 3. Exp. 3 shows the effects of the acid. Intramuscular injection of acetone-dicarboxylic acid. Alkali reserve Coagulation time 3.3 1F * The alkali reserve was not affected. The coagulation time diminished from 3.3 to a minimum of 1-8 in 5 min. then rose to 3 0 in the next 20 min. and remained near that value for the rest of the experiment. This kind of result was obtained in three experiments, but in one other the coagulation time increased after the injection from 3.3 to 4-5 in 15 min. and kept about that level for the next 20 min. It is notable that in this case the addition of the acid to the blood in vitro did not produce the usual granular-looking gel. Instead of this, the blood became thick and treacly and was still in this condition after 48 hours. Exp. 4 gives the type of result whenthe sodium compound was injected. Exp. 4. Intramuscular injection of sodium acetone-dicarboxylate. Alkali reserve Coagulation time

4 176 D. DE SOUZA AND F. D. M. HOCKING. Here there was an increase in the alkali reserve from 55 to 60 in 15 min., showing a tendency to rise slightly in the next 30 min. The coagulation time diminished in 5 mi. from 4*0 to 2-8 and rose again to 4*0 in the next 10 min., varying a little round 3-8 for the remaining 30 min. The lessening of the coagulation time seems always to occur in the first 5 min., with a return to the control value 10 min. later. As acetone is a product of the breakdown of acetone-dicarboxylic acid, a series of similar experiments was carried out with it. The amount injected was 0x125 g. per kg. body weight, about 4x5 times the quantity equivalent to g. of sodium citrate. There was no effect on the alkali reserve or on the coagulation time. The other decomposition product of citric acid examined was aconitic acid (CH2COOH.C.COOH:CHCOOH). The addition of this to blood in a paraffined test-tube leads to the formation of a granularlooking gel which resembles that due to acetone dicarboxylic acid and does not undergo syneresis. The haemoglobin is converted into acid hiematin. The sodium compound prevents coagulation and does not change the hiemoglobin. As the quantity of the acid corresponding to g. of sodium citrate is 0-08 g., that was the amount per kg. body weight injected into the muscles. Exp. 5 shows the result of an injection. Exp. 5. Intramuscular injection of aconitic acid. Time after injection Alkali reserve Coagulation time *8 There was practically no change in the alkali reserve. The coagulation time diminished from 3 0 to 2-3 in 5 min., then rose above the control level to 3-5 in the next 10 min., keeping about 3-8 for the rest of the time. In every experiment in which aconitic acid was injected the clotting time eventually exceeded the control. Sometimes it did so in the first 5 min., at others there was a previous diminution, as in Exp. 5. The sodium compound was freshly prepared from aconitic acid in the same way as sodium acetone-dicarboxylate from its acid. The results of its injection were very consistent and are seen in Exp. 6. Exp. 6. Intramuscular injection of sodium aconitate. Alkali reserve Coagulation time *5 2-5

5 CITRIC ACID AND BLOOD CLOTTING. 177 Time is expressed in minutes, the alkali reserve in c.c. of CO2 per The result is like that of injecting sodium citrate. The alkali reserve rose from 47 to 59 in 35 min., and the clotting time fell from 3-5 to 1-5 in 15 min., rising again to 2*5 in the next 10 min. DISCUSSION. In discussing the production of hypercoagulability of the blood by intramuscular injection of sodium citrate de Souza and Hocking [1934] pointed out that, while it might be due in part to the increase in the alkali reserve, there was some other factor besides this, possibly a small amount of citrate absorbed unchanged and circulating in the blood. The likelihood of its being a decomposition product of the citrate molecule was borne in mind and is strengthened by the experiments described here. Citric acid, as shown in Exp. 1, increases the alkali reserve and the coagulability of the blood, but not to the same extent as sodium citrate. The difference must be due to the presence of sodium in the citrate molecule, which thus supplies its own base for the formation of carbonate, whereas in the oxidation of citric acid to carbonate, base has to be obtained from the body. The greater rise in the alkali reserve accounts for the greater effect on coagulability. Ammonium citrate acts like citric acid (Exp. 2). As in the case of ammonium chloride, the ammonia is removed and perhaps goes to form urea, but here the alkali reserve is not diminished but increased, as the citric acid moiety is oxidized with the formation of carbonate. With regard to the decomposition products of citric acid no evidence has been found as to which are formed when citric acid is broken down in the body. Those studied here, acetone-dicarboxylic acid and aconitic acid, are known to be derived from citric acid when it is heated or acted upon by sulphuric acid. When injected into the muscles neither of them affects the alkali reserve. Acetone-dicarboxylic acid, however, may increase the coagulability of the blood as in Exp. 3, or in exceptional circumstances diminish it. Aconitic acid always diminishes the coagulability, sometimes after first increasing it, as in Exp. 5. The effects of the presence of sodium in the molecule are shown by the injection of the sodium compounds of the acids, which always cause a rise in the alkali reserve and an increase in the coagulability of the blood as in Exps. 4 and 6.

6 178 D. DE SOUZA AND F. D. M. HOCKING. The interesting fact emerges that these two acids, obtained from the breakdown of citric acid, can alter the coagulability of the blood without affecting the alkali reserve. The further decomposition product, acetone, in the amount injected in these experiments, has no effect on either. SUMMARY. 1. Intramuscular injections of citric acid or ammonium citrate increase the alkali reserve and the coagulability of the blood. 2. Intramuscular injections of acetone-dicarboxylic acid or aconitic acid do not affect the alkali reserve, but may increase or diminish coagulability. 3. Intramuscular injections of sodium acetone-dicarboxylate or sodium aconitate increase the alkali reserve and the coagulability of the blood. 4. Intramuscular injections of acetone have no effect on alkali reserve or coagulability. REFERENCE. de Souza, D. and Hocking. F. D. M. (1934). J. Phy8iol. 83, 49.