Carl A. Dragstedt

Observations on a Physiologically Active Substance Appearing During Anaphylactic Shock

We have previously reported 1 observations on the appearance of a physiologically active substance in the thoracic duct lymph and the inferior vena cava blood of dogs during anaphylactic shock. It was reported that this substance had the property of contracting the smooth muscle of the guinea pig intestine, and that this contraction was observed equally well in the intestine of guinea pigs immunized to the antigen used in sensitizing the dogs. In this respect the nature of the active substance appears to be somewhat different from the “hepatic anaphylatoxin” of Manwaring and his coworkers. 2 Preliminary observations on the nature of the active substance occurring in the thoracic duct lymph and the inferior vena cava blood show it to have the following characteristics. The active substance is dialyzable, indicating that it has a small molecular size. Using a 3-compartment electro-dialyzer the active substance concentrates in the basic compartment showing it to be of basic nature. The intensity of physiological activity and the Pauly reaction are parallel and the physiological activity is neutralized after condensation with diazotized sulphanilic acid. This indicates that the activity is associated with an imidazole compound. Pharmacological observations with the active substance indicate that it depresses the blood pressure of an etherized, atropinized cat; and while contracting the smooth muscle of the guinea pig intestine has no effect on the intestine of the mouse. In this respect it resembles histamine. 3 A characteristic wheal is produced in human skin by endermal inoculation of an active dialysate. Additional chemical and pharmacological investigations are in progress, but it may be stated that at the present time we have made no observations which are incompatible with the assumption that the active substance may be histamine.

Role of Histamine in Circulatory Effects of Rattlesnake Venom (Crotalin)

Essex and Markowitz 1 have made an extensive study of the physiologic action of crotalin and have pointed out a number of similarities to the action of histamine. Feldberg and Kellaway 2 have recently shown that crotalin has the property of liberating histamine into the perfusate when it is added to the per fusion fluid with which the lungs of cats or guinea pigs are being perfused. Their studies demonstrate a mechanism whereby the characteristic action of histamine can be superimposed upon the direct effects of the venom. The extent to which the added action of histamine influences the action of the venom in intact animals is not indicated by these experiments. In a series of studies on anaphylactic shock 3 and peptone shock 4 in the dog, we have been able to show that the degree of the vascular reaction is correlated with and dependent upon the amount of histamine liberated from the dogs tissues in these reactions. We were, therefore interested in determining whether histamine is liberated when venom is administered to the intact dog and whether the amount of histamine liberated is adequate to account for the degree of the vascular effect produced. Crotalin∗ (from Crotalus atrox) was injected intravenously into 5 anesthetized dogs, in doses of 0.5 to 1.3 mg. per kilo, and samples of blood and thoracic duct lymph were collected during the ensuing reaction and tested for histamine. All of the reactions were severe, 2 being fatal within 20 minutes. Histamine was found in the blood in 2 experiments and also in the lymph in one of these. The identification of histamine was based upon the presence in extracts made by Codes 5 method for the determination of histamine, of a physiologically active substance which lowered the blood pressure of the atropinized cat, contracted the guinea pig intestine, and was inactivated by diazotized sulfanilic acid.

Absorption of Drugs from the Esophagus

While studying the effect of acute esophageal obstruction in the dog, 1 it was noted that the production of a closed loop of the esophagus was invariably fatal. Although post mortem evidence indicated that death was due to an aspiration pneumonia or to rupture of the loop with subsequent pleuritis and mediastinitis, the possibility of toxin absorption from the esophageal loop was considered. The following experiments were performed to ascertain the absorptive ability of the esophagus under various conditions. Dogs were used. Under sodium pentobarbital anesthesia, a closed loop of the esophagus was prepared by ligating the esophagus in the neck and in the thorax just above the diaphragm. The substances to be tested were introduced into the lumen of the esophageal loop and the absorption estimated either by chemical tests of the urine and saliva, or by observation of the characteristic physiological effects. Five experiments were performed in each group. Potassium iodide (in 10% solution), sodium ferrocyanide (in 10% solution), and sodium nitrite (in 5% solution) were all slowly but definitely absorbed under the conditions of these experiments. Histamine hydrochloride (in doses up to 3 mg. per kilo), strychnine sulphate (in doses up to 5 mg. per kilo), cocaine hydrochloride (in doses up to 15 mg. per kilo), ephedrine hydrochloride (in doses up to 10 mg. per kilo), nicotine (in doses up to 2 mg. per kilo), and epinephrine hydrochloride (in doses up to 10 mg. per kilo) were not sufficiently absorbed to produce characteristic effects. Phenolsulphonphthalein, methylene blue, and antipyrine were not sufficiently absorbed during the course of 2 hours to be detected in the urine. Distention of the esophageal loop with air pressure up to 60 mm. of mercury after introduction of the above drugs which were not normally absorbed, caused various reflex disturbances of respiration and blood pressure, but did not apparently facilitate absorption.

Effect of Iron on Hemoglobin Regeneration in Gastrectomized Dogs

We have previously shown 1 that the gastrectomized dog has a markedly reduced capacity to regenerate hemoglobin when maintained at anemic levels on various diets. The average production capacity is approximately 10% of that of normal dogs and is around 0.2 gm. of hemoglobin per day on a meat diet. It was also noted that liver feeding or the administration of liver extract 343 (Lilly) is comparatively ineffectual in increasing the level of hemoglobin production. Liver extract 55 (Lilly), however, caused a definite increase in hemoglobin production. As this preparation contains added iron, the present studies were directed to determining the effect of the oral administration of iron. Incidentally, the character of the blood findings during the period of anemia and of iron therapy were followed. The same 4 dogs used in the previous study were used, the gastrectomy operation thus preceding the present experiments by from 2 to 3 years. The technique of study was similar to that used previously. The dogs were maintained at an anemic level (e. g., hemoglobin levels of 6 to 9 gm. per 100 cc.) by intermittent bleeding, the hemoglobin production for a given period being determined as the total hemoglobin removed by the bleedings plus or minus the gain or deficit in total circulating hemoglobin during the same time. This is the method developed by Whipple and his coworkers. The hemoglobin determinations were made with a Sahli-Hellige Haemometer and the blood volume determinations by the method of Keith and Rowntree as modified by Whipple. One dog (No. 12) was spontaneously anemic and was not subjected to bleedings. After several consecutive periods of standardization on a meat diet alone, iron in the form of Blauds pills (Pills of Ferrous Carbonate), ferric ammonium citrate, or saccharated iron oxide was administered orally and the hemoglobin production during the iron regime determined.

Relationship Between Peptone Shock and Anaphylactic Shock

We have recently reported that the intravenous injection into dogs of a solution of peptone produces shock indirectly by leading to the liberation of a vasodepressor substance, identified as histamine, from the tissues. 1 Thus the mechanism of peptone shock is apparently similar to that of anaphylactic shock. 2 Biedl and Kraus 3 pointed out the remarkable similarities between these conditions. They also reported that peptone shock desensitizes an animal to anaphylactic shock and vice versa. They concluded that the fundamental mechanism concerned in these reactions was identical. They interpreted the reactions as indicating that peptone contains some active substance, such as Popielskis vasodilatin, which can produce shock directly, and that in the anaphylactic experiment a mother substance is formed as the result of the sensitization which then discharges this active peptone constituent when the shocking injection of antigen is made. Subsequent investigators have had variable results in attempting to influence an anaphylactic reaction by the prior administration of peptone. Hill and Martin 4 in their review summarize the reports as follows: “In short, there is some, but not striking evidence that peptone may inhibit shock within certain defined limits.” In view of our demonstration that both peptone shock and anaphylactic shock are reactions that are brought about indirectly by the liberation of histamine from the tissues of the shocked animal, the relationship between these reactions has considerable significance. For example, the question whether desensitization is related to a depletion or exhaustion of the mobilizable store of histamine in the tissues is subject to direct approach by such a study. The present investigation, therefore, was undertaken in the effort to determine whether or not one reaction would desensitize to the alternative reaction.

Mechanism of Secretin Diuresis.

Owen and Ivy have shown that the intravenous injection of concentrated vasodilatin-free secretin preparations into dogs results in a typical moderate diuresis. Certain characteristics of the diuresis led us to suspect that it was related to, and probably dependent upon the secretory stimulation of pancreatic juice and bile. The following experiments give evidence that this is the case. Following pancreatectomy and ligation of the common bile duct, previously active preparations are without effect on the rate of urine flow. Secretin preparations cause no diuresis if the bile and pancreatic juice resulting from their injection are diverted from the intestine by cannulae. Reintroduction of the collected bile and pancreatic juice into the intestine is followed by the typical diuresis. Diuresis in the intact dog is therefore dependent on the secretion of pancreatic juice and bile, and the entrance of these secretions into the intestine where they may be reabsorbed. It may be noted that in the experiments in which Mellanby 1 reported that secretin had no diuretic effect, the pancreatic juice and bile were simultaneously collected by means of cannulae so that they could not enter the intestine.

Chondroitin in Canine Anaphylaxis

The accidental discovery of Crandall and Roberts 1 that the administration of chondroitin sulphuric acid to patients with migraine frequently results in marked relief has not been explained. Since both the physiological action of chondroitin and the pathogenesis of migraine are only imperfectly understood, the therapy is of course empirical. Migraine has, however, been considered by a number of investigators as an allergic manifestation. Also chondroitin has been shown to have a protective action against liver injury due to a variety of causes. 2 The relationship between the liver and the anaphylactic reaction in the dog is well known. Consequently it was hoped that a study of the effect of chondroitin administration upon the anaphylactic reaction in the dog might contribute some information to these various and possibly related problems. Chondroitin was administered to 13 horse-serum sensitized dogs in doses of 10 gm. per day. In 8 animals it was given during the last 10 days of the incubation period, and in 5 it was given for 10 days prior to the sensitizing injection and throughout the incubation period. The animals were anesthetized so that the degree of shock resulting from the assaulting dose of serum could be recorded by the blood pressure tracing. Definite shock occurred in all animals, 3 reactions being fatal, the remainder moderate to severe. The distribution of the various grades of severity of shock was identical with that in a large number of controls. 3 Consequently it is concluded that the administration of chondroitin does not influence in any way the anaphylactic reaction in the dog.

Demonstration of an “Anaphylactic Poison”

That the liver is practically indispensable for the development of anaphylactic shock in the dog has been indicated, principally by Manwaring and his colleagues, 1 Voegtlin and Bernheim, 2 and Simonds and Brandes. 3 Manwaring, 4 in addition, has reported that during anaphylactic shock in the dog, physiologically active substances, called by him “hepatic anaphylatoxins,” appear in the circulating blood so that by appropriate cross circulation experiments their presence can be demonstrated by the effects they produce in the recipient animal. A number of considerations led us to suspect that if transportable physiologically active substances are set free during anaphylactic shock in the dog, they might appear in the thoracic duct lymph. Petersen and Levinson 5 have noted the marked increase in the rate of flow of the thoracic duct lymph during shock. That this increase in flow is secondary to the shock phenomena occurring in the liver is indicated by the work of Simonds and Brandes, 6 who reproduced similar effects by mechanical obstruction of the hepatic veins. The points of similarity between the manifestations of anaphylactic shock and mechanical obstruction of the hepatic veins in the dog have been studied in considerable detail by Simonds and Brandes, and warrant the conclusion that a considerable proportion of the increased lymph flow occurring during shock is of hepatic origin. If this is the case, it would seem possible that substances such as the hepatic anaphylatoxins of Manwaring might appear in the lymph. As one of the most delicate methods of testing for the presence of certain smooth muscle stimulating substances, the isolated surviving strip of guinea pig intestine was used.