Part 13

June 13: 450 cc urine, 500 cc water, 300 grams meat.

June 14: Weight, 7.9 kilos; 1.2 grams caffein, 500 cc water, 300 grams meat, 490 cc urine.

June 15: Weight 7.8 kilos, 500 cc water, 300 grams meat, 550 cc urine.

June 16: Weight 8.0 kilos, 1.2 gram caffein, 500 cc water, 300 grams meat, bone dust added to check diarrhea.

June 17: 500 cc water, 300 grams meat, 450 cc urine, diarrhea continues, bone dust added.

June 18. Weight 7.8 kilos, 1.3 gram caffein, 300 grams meat, 500 cc water, 300 cc urine.

June 19: Dog very thirsty, drank 1 liter of water and ate 350 grams of meat; 960 cc urine passed during the past 24 hour.

June 21: Weight 7.5 kilos, 1.5 grams caffein given at 10 a. m. At 2 p. m. convulsions and death. This dog received a total of 21.15 grams caffein in 25 doses during a period of 35 days, which amounts to an average of 85 mg per kilo daily.

_Dog 19._ _Female fox terrier._

May 13: Weight 6.4 kilos. Diet consisted of rice, 100 grams; sugar, 100 grams; and a sufficient quantity of meat and cracker meal to give flavor to the food. Examination of the urine showed a trace of albumin but no sugar. The urine was acid to litmus. Two days later the urine was alkaline to litmus. There was still a small amount of albumin but no sugar.

May 17: 0.5 gram caffein was given by mouth. Examination of the urine collected the next day still showed the presence of albumin and the absence of reducing substances. The dog had tremors. Caffein was, therefore, not administered.

May 19: 0.5 gram caffein was given by mouth.

May 20: 0.75 gram caffein was fed at 12.45 p. m. The dog vomited during the night and tremors were observed the next morning. The urine collected was examined for albumin and sugar, but neither was found.

May 21: 12 noon, 0.75 gram caffein was fed. The dog weighed 6 kilos, which therefore represented a loss of 0.4 kilo. Grew abnormally thirsty and lost appetite, but no other symptoms of caffein poisoning were observed.

May 22: The dog was again given 0.75 gram caffein at 12 noon. The examination of the urine for albumin and sugar gave negative results. The dog died at 4.15 p. m. The fatal dose for this dog was therefore 0.125 gram caffein per kilo, and the total amount of caffein ingested in six days amounted to 3.25 grams, or 0.54 gram per day, which makes 90 mg per kilo.

_Dog 21._ _White female bull._

This dog was kept on a diet exclusively of meat, and was given water ab libitum. From 0.5 to 0.6 gram of caffein was administered daily for seven days; the doses were then increased and were given at longer intervals. No symptoms of the effects of caffein were observed until a dose of 1.5 gram was fed, when diarrhea was noticed on the next day. In the following record the details of the experiment are given:

+---------+---------- Date. | Weight. | Caffein. ----------+---------+---------- | _Kilos._| _Grams._ June 7 | 12.5 | 0.5 June 8 | 12.5 | .5 June 9 | 12.5 | .5 June 10 | 12.3 | .6 June 11 | 12.3 | .6 June 12 | 12.3 | .6 June 13 | 12.3 | .6 June 14 | 12.3 | .8 June 16 | 12.7 | 0.8 June 18 | 12.9 | 1.0 June 21 | 13.4 | 1.2 June 24 | 13.3 | 1.5 June 25 | (1) | .0 June 27 | 13.5 | 1.5 June 30 | 13.5 | 1.75 ----------+---------+----------

(1) Diarrhea.

July 2: 11.30 a. m., 2.0 gram caffein fed by mouth; 1.30 p. m., tetanus, dog died. The total amount of caffein fed to dog No. 21 out of the 25 days of the experiment was 14.45 grams, or an average of 578 mg per day, which amounts to about 42 to 43 mg per kilo of body weight.

Notwithstanding the diversity in the method of experimentation, there was a striking uniformity in some of the results obtained. All the experiments of the series showed absence of cumulative action of caffein. The experimental evidence presented indicates that moderately large doses may be given at intervals of about 24 hours without inducing any symptoms of nervous or any other disturbance. This is illustrated in the tests on dog 11, which were preliminary in character. In this subject 100 to 150 mg of caffein per kilo were ingested daily for several days without showing any changes. Later in the course of the experiment, after larger doses were given, mild symptoms only, such as tremors, were observed. Additional evidence of the absence of cumulative action of caffein was furnished by the results of the following experiments:

Dog 23 received 142 mg of caffein per kilo on three successive days. His general condition indicated that these amounts of caffein were toxic, but he survived. In another series of tests, made after he was allowed to rest a few days, he again failed to show any cumulation of the drug, as he survived this time a series of tests of longer duration than the first.

A much better illustration of the absence of cumulative action of the drug is furnished by the experiments on dog No. 20. In this case 100 to 125 mg of caffein per kilo, given on 10 consecutive days, did not cause any marked effects. Diarrhea and restlessness were the only symptoms observed. These experiments therefore show that the elimination and decomposition of caffein are apparently effected by the body within twenty-four hours or thereabouts.

Experiments on dog 19, however, form an exception--the third dose of 125 mg caffein per kilo having proved fatal. The very low protein content of the diet of this dog suggests itself as a possible cause of the lower resistance to caffein of this subject. But it may be observed that the same diet was furnished to dog 20, which stood such amounts of caffein much longer. The presence of a trace of albumin in the urine of dog 19 is likewise inadmissible as a cause of the difference in the toxicity of caffein in this dog, for the urine of dog 20 likewise contained a trace of albumin. The alkaline reaction of the urine, together with the fact that the first dose of only 60 mg of caffein per kilo induced symptoms of toxicity, suggests the presence of an abnormal condition which in all probability was the cause of the death of this subject under the conditions indicated.

In a large number of experiments on caffein performed in this laboratory it has been observed that symptoms due to caffein often disappeared when the administration of the same dose of the drug was continued. Thus dog 19 vomited when the amount of caffein was increased to 125 mg per kilo. When this amount was repeated the next day there was no vomiting. Similar observations were made on dogs 11 and 23, also on other dogs. No. 22 developed diarrhea at first; when the administration of caffein was resumed several days later, however, there was no diarrhea. In other experiments performed in this laboratory, symptoms of nervous irritability induced by caffein disappeared on continued treatment.

It was interesting, therefore, to inquire whether resistance to caffein would be increased by the continued administration of progressively larger amounts of the drug. When doses of 150 and over were fed, the intervals allowed were usually longer than 24 hours. Two and sometimes three days were permitted to elapse between two successive doses. This was done in order to allow time for recovery from possible changes induced by larger doses of caffein, and thus prevent the summation of effect. In the experiments considered, therefore, Nos. 11, 23, 20, and 19, the toxicity of caffein does not seem to be greater than in the experiments on acute caffein intoxication in the dog. It was thought, however, that the large initial doses or the quick change to large doses when the amounts used in the beginning were small, might have something to do with failure to induce a marked degree of tolerance. The experiment on dog 21 was therefore carried out by giving from 40 to 60 mg per kilo for eight days, and then increasing the dose, but tolerance could not be induced, as is shown in the protocol to the experiment.

SERIES B.

According to the studies of Chittenden,(16) low protein diet improves the general metabolism of the body, fatigue is diminished, and bodily vigor, therefore, correspondingly increased. The expectation is, therefore, justified that the defense of the organism against deleterious substances introduced into the body is much improved by such a diet, thus increasing its resistance to poisons. Hunt's experiment on this subject, also quoted by Chittenden, lends support to this view. He found that mice fed on carbohydrates chiefly, or on foods containing only a small amount of protein, were more resistant to acetonitril. It was interesting, therefore, to inquire whether the toxicity of caffein differs under similar conditions of diet.

A fixed diet of the same calorific value was provided for all dogs of this series, but the protein content for three of the animals was approximately one-third of the amount usually fed to dogs. Caffein was at first administered subcutaneously, but all the dogs on a low protein diet developed abscesses at the site of injection, while none of those on high protein diet showed a local reaction. Feeding by mouth was then begun and continued throughout the experiment in each case. The initial dose was 50 mg per kilo, which was given daily for seven to nine days. It was then increased progressively by 25 mg per kilo; 75 mg per kilo were administered for one to two days, 100 mg for two to three days, 125 mg for one to two days, 150 mg for one to two days, and a single dose of 175 mg. It will be remarked that sometimes an interval of one day had to be allowed during which no caffein was fed.

_Dog 30. Black and tan hound, male._

The dog was under observation for about eight weeks before the experiment was begun and had received a high protein diet. He was then given 50 mg caffein for nine consecutive days. On the tenth day the dose was increased to 75 mg per kilo. As no symptoms developed, this dose was increased to 100 mg per kilo, and was fed one day apart. It was then raised to 125 mg per kilo. For the first time since the drug was fed, symptoms appeared; they were noticed a few hours after feeding and persisted during the next day. Although the appetite was good, no caffein was given on this day. On the following day this dose was repeated. As the symptoms were not serious, 150 mg per kilo were given daily for the next three days, until 175 mg per kilo was reached. This dose proved fatal within six hours. Record of experiment follows:

October 9: Weight, 9 kilos, on full nitrogen diet, received daily 0.724 gram nitrogen per kilo or 87 calories per kilo, received 18 grams meat per kilo, 4 grams lard per kilo, 3 grams carbohydrates per kilo, bone dust, ad libitum.

November 3: Weight, 9.10 kilos.

November 10: Weight, 9 kilos.

November 20: Weight, 9.55 kilos.

November 29: Weight, 8.70 kilos.

December 6, 7, 8, and 9: Received subcutaneously 22 cc 2 per cent caffein. Condition good, site of injection normal.

December 10, 11, 12, 13, and 14: Received 0.4375 gram caffein by mouth equal to 0.050 gram per kilo, no symptoms, appetite and general condition good.

December 15: 11.30 a. m., received 0.6563 gram caffein by mouth, or 0.75 gram per kilo, no symptoms, appetite good, condition excellent.

December 16: 11 a. m., received 0.870 gram caffein by mouth, or 0.1 gram per kilo, weight 8.70 kilos, no symptoms.

December 17: No caffein given.

December 18: Received 0.870 gram caffein, or 0.1 gram per kilo, no symptoms.

December 20: 2.45 p. m., received 1.0875 grams caffein, or 0.125 per kilo; 4 p. m., ate food readily, seemed very uncomfortable and sick.

December 21: 9 a. m., stiffness in muscles, but no other symptoms, appetite good, no caffein given.

December 22: 11 a. m., received 1.0875 grams caffein, or 0.125 gram per kilo; 3 p. m., depressed in spirits and sick, but no other symptoms observed.

December 23: 11.30 a. m., received 1.305 grams caffein, or 0.150 gram caffein per kilo; 1.30 p. m., apparently quite sick, but no other symptoms, had good appetite.

December 24: 10 a. m., received 0.175 gram caffein per kilo; 4 p. m., when about to be fed fell over and died; no autopsy.

The total amount of caffein given dog 30 was 11.3458 grams, administered for a period of eighteen days. The average daily amount per kilo was therefore 72 mg. The feces became offensive when the amounts of caffein were increased to 75 mg per kilo. It will be observed that in this dog the appetite was uniformly good until the day of his death. Whether or not this is the cause of his resistance to caffein will be discussed later.

_Dog 32. White, male, young._

Although he was growing rapidly this dog's weight was constant, but he looked anemic. He received a high protein diet until December 3, when the rations were increased by one-third. This dog was under observation from October 26 to December 6 when the administration of caffein was begun. He then received 50 mg caffein per kilo daily for nine days consecutively without showing any effects, when the dose was increased to 75 mg per kilo, then to 100 mg per kilo. This dose was further increased to 150 mg per kilo without causing symptoms, which was repeated the next day. No symptoms having been observed after such amounts of caffein, 175 mg per kilo were fed. This dose, however, proved fatal within two hours. Record of experiment follows:

October 26: Weight, 6.90 kilos.

November 3: Weight, 6.90 kilos.

November 10: Weight, 6.90 kilos.

November 20: Weight, 6.90 kilos.

November 29: Weight, 6.55 kilos.

December 3: Put into cage, diet increased one-third.

December 6, 7, 8, 9: Weight 6.30 kilos; 12.30 p. m., received 16 cc 2 per cent caffein by subcutaneous injection in back, no symptoms of any kind noticed, site of injection normal.

December 10, 14: 0.05 gram caffein per kilo.

December 15: Received 0.4725 gram caffein by mouth, no symptoms.

December 16: Received 0.655 gram caffein, 0.100 gram per kilo.

December 17: No caffein given.

December 18: Received 0.655 gram caffein daily, 0.100 gram per kilo, no symptoms.

December 20: Received 0.8188 gram caffein, 0.125 gram per kilo, no symptoms, appetite good.

December 21: Received 0.9825 gram caffein, 0.150 gram per kilo, somewhat uncomfortable, no other symptoms.

December 22: Received 0.9825 gram caffein, 0.150 gram per kilo, no symptoms except some uneasiness.

December 23: 9 a. m., no symptoms, appetite good; 11.30 a. m., received 1.146 grams caffein, 0.1759 gram per kilo; 1.30 p. m., died while making an effort to get out of cage, tonic contraction of limbs observed before death.

The amount of caffein received during the entire experimental period was 9.2223 grams, or an average per day approximately of 80 mg per kilo, and therefore 10 per cent more than dog No. 30 received. It will be observed that the appetite in dog No. 32 was likewise uniformly good, and that he received a very high protein diet which was also of a very high calorific value.

_Autopsy (dog 32)._--Stomach presented a severe inflammation of the mucosa, especially in the fundus and pyloric portions. The gastritis was more marked in pyloric portion, and the inflammatory condition extended along the whole course of small intestines, which presented numerous hemorrhagic areas, and a thick catarrhal exudate on the mucosa. The large intestine contained quite a large number of parasites, probably round worms. The liver was enlarged and the gall cyst well filled. The spleen was also considerably engorged, kidneys appeared normal, other organs all appeared normal.

_Dog 31. Black spaniel, male._

This dog had been under observation one month previous to the experiments with caffein. The usual initial dose was then administered for nine days. There were no signs of local irritation when the drug was given subcutaneously, but symptoms of toxicity were present. These disappeared, however, when the drug was administered by mouth. The dose was therefore increased to 75 mg per kilo. This, as will be seen, proved fatal within six hours. High nitrogen diet, same as No. 30.

November 3: Weight 10.250 kilos.

November 10: Weight, 10.25 kilos.

November 20: Weight, 10.30 kilos.

December 1: Put in cage.

December 6, 7, 8, 9: Weight, 10.20 kilos; received 26 cc 2 per cent caffein subcutaneously, site of injection normal.

December 6: Very restless and excited, whined when handled as though muscles were sore, appeared to be sick.

December 10-14: Condition good, received 0.51 gram caffein by mouth daily, no noteworthy symptoms, appetite continues good, somewhat restless at intervals.

December 15: 11.30 a. m., received 0.765 gram caffein per mouth (0.075 gram per kilo); 2 p. m., depressed in spirit, seemed sick and uncomfortable; 4.15 p. m., when about to feed, animal jumped up, then fell back dead.

_Autopsy (dog 31)_: Lungs congested; heart filled with blood and contained small amount of blood-stained fluid in pericardial sac. Liver deeply congested, soft and friable; gall bladder distended with bile; kidneys showed inflammation of cortex; spleen pale, normal in size and consistency; stomach practically empty, the mucosa of the pyloric portion exhibited severe gastritis, with thick catarrhal exudate. This catarrhal inflammation extended through the duodenum; remaining portion of small intestine showed mild inflammation; large intestine appeared practically normal. The total amount of caffein received by dog 31 during 10 days was 5.395 mg, or a daily average of 53.9 mg per kilo. This unusually low resistance to caffein (which was practically the only case in all the experiments on dogs presented in this research) suggests the presence of some abnormal condition. The bloody exudate in the pericardial cavity indicating pericarditis, which is likely to induce secondary changes of cardiac muscle, may be considered as a possible cause of the increased toxicity of caffein in this case.

_Dog 29. Male fox terrier, black._

This dog was kept on a low nitrogen diet for nearly five weeks before the feeding of caffein was begun. The administration of 50 mg of caffein per kilo was then carried on for eight days without showing any symptoms of toxicity. The usual increase of dose was then given--75 mg per kilo--which was followed by a manifestation of symptoms. Further increase, however, to 100 mg per kilo had no visible effect. Nevertheless it was considered advisable to suspend the feeding of caffein for one day. The same amounts were then repeated on two consecutive days. No symptoms having been observed, 125 mg per kilo were given. As symptoms of toxicity and especially loss of appetite were observed, the dog was not given any caffein the next day. Since his appetite had now improved, the experiment with larger doses was resumed. Death followed after the second dose of 150 mg per kilo. Protocol follows:

Weight, 9.90 kilos. One-third nitrogen diet. Receives 0.269 gram nitrogen per kilo (88.269 calories per kilo).

November 3: Weight, 9.85 kilos.

November 10: Weight, 9.55 kilos.

November 12: Weight, 9.40 kilos.

November 29: Weight, 9.85 kilos.

December 6: Weight, 9.90 kilos; 11.35 a. m., received 25 cc 2 per cent caffein solution by subcutaneous injection in back; 4 p. m., no symptoms, appetite good.

December 7-9: Received 25 cc caffein 2 per cent solution--subcutaneous injection, no symptoms, area of injection inflamed and swollen.

December 10, 13: Site of injection showed increased inflammation, received 0.495 gram caffein (50 mg per kilo) in 30 grams meat daily without showing any symptoms.

December 14: 12 noon, received 0.7425 gram caffein by mouth (0.075 per kilo); 2.30 p. m., restless and uneasy.

December 15: 11.30 a. m., received 0.7425 gram caffein by mouth; 2 p. m., depressed in spirits, although continues to have good appetite.

December 16: Weight, 9.50 kilos; 3.15 p. m., received 0.9509 gram caffein by mouth; 4.50 p. m., no symptoms.

December 17: Animal rested.

December 18: Received 0.950 gram caffein by mouth, no symptoms.

December 19: Received 0.9509 gram caffein by mouth, no symptoms.

December 20: 2.45 p. m., received 1.1875 grams caffein (0.125 gram per kilo); 4 p. m., restless and quite sick; ate only a little food.

December 21: 9 a. m., still uncomfortable, allowed to rest, no caffein given, gradually recovered appetite.

December 22: 11 a. m., received 1.875 grams caffein; 3 p. m., seemed sick, but showed no other symptoms, appetite fair.

December 23: 9 a. m., showed no symptoms from the day before, ate food gradually, seemed sick; 11.30 a. m., received 1.425 grams caffein (0.150 gram per kilo); 1.30 p. m., looked and behaved as if very sick, no other symptoms; 3.45 p. m., in attempting to get out of box fell over on back, had convulsions, whined, dyspnoea, died within 30 seconds.

_Autopsy_: Stomach exhibited mild inflammation; small intestine inflamed and hemorrhagic areas on mucosa; liver engorged and friable; spleen normal; kidneys slightly congested; other organs appeared normal. The total amount of caffein fed to Dog 29 was 12.135 grams, which was given in 18 days. The average daily amount per kilo was therefore 67.68 mg.

_Dog 28. Male fox terrier._

Low nitrogen diet was begun about four weeks before the feeding of caffein; 50 mg of caffein was then fed for seven consecutive days. Partial loss of appetite was observed after the first dose. As the experiment progressed the desire for food steadily diminished, and the feces became fetid. Symptoms of intoxication manifested themselves early in the experiment, and vomiting occurred after the fourth dose. The dog was then put on a diet exclusively of meat. After an intermission of 10 days 109 mg caffein per kilo were given. Since there were no symptoms, the following day the amount was increased to 125 mg per kilo. This dose proved fatal within 16 to 20 hours.

This dog was stout and strong, weight 12.25 kilos, received daily 0.269 gram nitrogen per kilo (88.269 calories per kilo).

November 3: Weight, 11.75 kilos.

November 10: Weight, 11.95 kilos.

November 20: Weight, 11.20 kilos. All through this period had been kept in a cold, poorly ventilated room, put in a warm room, with bedding and good ventilation.

November 29: Weight, 11.95 kilos.

December 1: Put in a cage; weight, 11.95 kilos.

December 6: Weight, 11.95 kilos; 11.45 a. m., received 0.050 gram caffein per kilo; then received 30 cc 2 per cent caffein (0.6 gram) in practically one subcutaneous injection; 4.30 p. m., ate only part of food.

December 7: 10.25 a. m., received 30 cc 2 per cent caffein by subcutaneous injection (0.6 gram, or 50 mg, per kilo); 1.45 p. m., seemed sensitive to touch, no desire for food, depressed in spirit.

December 8: 11.40 a. m., received 30 cc 2 per cent caffein by subcutaneous injection (50 mg per kilo); 1 p. m., depressed in spirit, hind legs seemed somewhat stiff, no desire for regular food, site of injection inflamed.

December 9: 10.50 a. m., received 30 cc 2 per cent caffein by subcutaneous injection (50 mg per kilo); 2.30 p. m., had vomited, no desire for regular food.

December 10: Inflammation of site of injection, and swelling very pronounced; 2 p. m., received 0.5975 gram caffein, or 50 mg per kilo, with 30 grams of meat, refused regular food.

December 11, 12: Received 0.5975 gram caffein by mouth, no symptoms except refusal of regular food, feces fetid.

December 13-22: Put on meat diet exclusively, high temperature, no caffein, weight 10 kilos, appetite good, feces fetid.

December 22: 12 a. m., weight 11 kilos, received 1.2 grams caffein by mouth (0.109 gram per kilo); 4 p. m., no symptoms.