Part 5

January 25: 3 p. m., 7 cc 2 per cent caffein (0.2 gram per kilo) solution injected into peritoneal cavity; 3.15 p. m., reflexes increased and anterior extremities paralyzed.

January 27: Rabbit recovered and is able to walk about in the room.

_Rabbit 312, maltese, female. Weight, 740 grams. Diet, oats._

March 3: 11.47 a. m., urine obtained from bladder, appearance normal, no albumen, no reduction of Fehling's solution; 11.50 a. m., 7.5 cc 2 per cent caffein (0.2 gram per kilo) injected into peritoneal cavity; 2.30 p. m. anterior extremities paralyzed, posterior extremities rigid and extended; 5 p. m. (about), rabbit died.

_Rabbit 311. Belgian hare, female. Weight, 650 grams. Diet, oats._

March 3: 11.26 a. m., urine obtained from bladder normal in appearance, albumen considerable, reduction of Fehling's solution none; 11.27 a. m., 6 cc 2 per cent caffein solution (0.2 gram per kilo) injected into peritoneal cavity; 2.30 p. m., rabbit seemed to be normal, no symptoms had developed; urine collected contained a large amount of sugar, reduction was very heavy, but no albumen was found.

March 4: 11 a. m., condition good, moves about when put on floor; gait, normal.

_Rabbit 78. Yellow and white. Weight 659 grams._

January 26: 1.30 p. m., 8.5 cc 2 per cent caffein (250 mg per kilo) injected into peritoneal cavity, under observation the rest of the afternoon, no symptoms.

January 27: 4 p. m., no symptoms developed.

_Rabbit 317. Belgian hare, female. Weight 635 grams. Diet oats._

March 15: 10.35 a. m., 8 cc 2 per cent caffein (0.252 gram per kilo) injected into peritoneal cavity; 12 noon, marked abduction of hind legs, was unable to walk after a little exertion, rabbit died between 12.30 and 12.50 p. m. _Autopsy_. Right lung hepatized and showed adhesions to costal and mediastinal pleura; liver studded with nodules of coccidiosis; spleen congested; stomach filled, mucosa normal; intestines injected; colon hemorrhagic on serosa in ventral region, near point of injection; kidneys normal.

_Rabbit 323. White, female. Weight 820 grams. Diet oats._

March 15: 10.45 a. m., 10 cc 2 per cent caffein (250 mg per kilo) injected into peritoneal cavity; 12 noon, reflexes increased, hind legs abducted but is able to walk, symptoms are mild; 1.40 p. m., tremors, weakness, and abduction of head and legs much more marked than at 12 noon.

March 16: Condition good.

March 17: Condition good, recovery apparently complete.

Since the experiments of Series A, which were intended as preliminary tests, have shown that 0.3 gram of caffein per kilo when introduced into the peritoneal cavity is rapidly absorbed and is fatal, much smaller doses were employed in subsequent trials with the drug. This is shown in series B, which may be divided into two groups. Group I, consisting of rabbits 69, 70, 92, 93, and 309, which received 0.2 gram of caffein per kilo, and Group II, Nos. 307 and 308, into which 0.15 gram of caffein per kilo was injected. Three rabbits of Group I (Nos. 92, 93, 309) died from the effects of caffein; rabbit 309 twenty minutes after injection, and rabbits Nos. 92 and 93, twenty hours and two and one-half hours, respectively, after the administration of caffein. In both of these rabbits symptoms appeared within five minutes after the injections were made. Rabbits 69 and 70, it will be noticed, survived the same amount of caffein in proportion to body weight as was given to the other members of this group. Increased peristalsis and the distribution of the dose may account for the greater resistance of rabbit No. 69. The case of rabbit No. 70 is evidently one of exceptional resistance to caffein, since both the rabbits of Group II died from the effects of a much smaller dose, namely, 0.15 gram of caffein per kilo. Moreover, macroscopical examination at the autopsy of Nos. 307 and 308 failed to show any lesions which might tend to lessen the resistance to caffein.

That a dose of 0.15 gram per kilo is therefore in all probability the minimum fatal dose for the rabbit when injected into the peritoneal cavity appears from the results of the experiments in series C, in which smaller doses, 0.125 gram of caffein per kilo caused mild symptoms only, while 0.1 gram per kilo rarely induced any symptoms. It may be remarked that the rabbits of series C were fed carrots while rabbits Nos. 307 and 308 received oats. Their resistance to caffein may be different, but, as was pointed out in the earlier part of this investigation, diet does not seem to influence the toxicity of the single dose of caffein. Doses of 150 and of 100 to 125 mg per kilo, when injected into the peritoneal cavity, may be considered, respectively, as the minimum fatal and minimum toxic doses for the gray rabbit. Analysis of the experiments in series D shows much greater resistance to caffein than in the other rabbits which received it intraperitoneally. Thus, after the administration of 0.2 gram per kilo to each of five rabbits, no effect was observed in two cases (Nos. 310, 311), while in two others (Nos. 74, 75) symptoms developed, but they survived. Only one rabbit, No. 312, died from the effects of this dose; the autopsy showed the presence of degeneration of the liver and petechial hemorrhages on the cortex of the kidneys in the case of No. 310, which was probably the cause of death rather than the caffein.

Two decigrams of caffein can not be considered, therefore, the fatal dose for rabbits. This is further corroborated by the results obtained in experiments with larger doses. Rabbit 78, which received 257 mg per kilo, failed to show any symptoms. The same amount in proportion to body weight in No. 323 caused mild symptoms only, while the rapid death of rabbit No. 317 after the same dose of caffein may be explained by the lesion found at autopsy, thus affording additional evidence that disease may decrease the resistance to caffein. It will be observed that all the members of this series were young rabbits and, as will be shown later, young animals of other species are likewise more resistant to caffein than adult animals. Similar results were obtained by von Anrep, who observed that atropin is less toxic in young than in full-grown animals.

Observations were also made on the diuretic effect of caffein when injected into the peritoneal cavity. The results shown in the following table indicates the stimulating effect on renal secretion whether the diet consisted of oats or of carrots. The urine of some rabbits contained moderate amounts of sugar after from 0.2 to 0.15 gram of caffein per kilo was given; albumen was observed in one case, but in none of the others. In rabbit No. 311 albumin was found before the injection of caffein, but none in the urine which was collected three hours after caffein was injected.

_Effect of caffein on renal secretion._

+--------+---------+-------+--------------------+--------- No. | Weight.| Caffein | Urine.| Time. | Diet. | |per kilo.| | | -----+--------+---------+-------+--------------------+--------- |_Grams._| _Gram._ | _cc._ | | 307 | 1,320 | 0.150 | 100 | 4.5 hours | Oats. 308 | 1,305 | .150 | 180 | do. | Do. 295 | 1,205 | .100 | 100 | 2 hours 20 minutes | Carrots. 293 | 1,605 | .100 | 90 | 3 hours | Do. 292 | 1,595 | .100 | 105 | 4 hours | Do. -----+--------+---------+-------+--------------------+---------

NOTE.--The amount of urine secreted in three hours by control rabbits, on a carrot diet, varied between 35 and 50 cc, the average weight of the animals being a little above 1,600 grams. The secretion of urine on an oat diet was much less for an equal period of time.

TABLE 3.--_Intraperitoneal injections._

SERIES A.

----+--------+-------+-------------+--------------+------------------- | |Caffein| Time of | Duration | No.| Weight.| per | appearance | of life. | Remarks. | | kilo. | of symptoms.| | ----+--------+-------+-------------+--------------+------------------- |_Grams._|_Gram._| | | 71| 1,659 | 0.3 |85 minutes |24 hours |Gray. 61| 2,143 | .3 | |At the end of |Black. | | | | injection | 72| 1,402 | .3 |8 minutes |1.25 hour |Gray and white. ----+--------+-------+-------------+--------------+-------------------

SERIES B, GROUP I.

----+--------+-------+-----------+--------------+--------------------- 70| 1,487 | 0.2 | |10 days |Gray and white. 93| 1,492 | .2 |5 minutes |2.5 hours |Maltese; given second | | | | | dose after 3 days, | | | | | died 2.5 hours later. 69| 1,492 | .2 |About |Survived |White. | | | 5 minutes | | 92| 1,388 | .2 |5 minutes |24 hours |Yellow. 309| 1,500 | .2 | |20 minutes |Belgian; oats. ----+--------+-------+-----------+--------------+---------------------

SERIES B, GROUP II.

----+--------+-------+-------------+--------------+------------------- 308| 1,350 | 0.15 |3 hours and |About 2.5 days|Belgian; oats. | | | 40 minutes| | 307| 1,320 | .15 |1 hour |24 hours | Do. | | | 24 minutes| | ----+--------+-------+-------------+--------------+-------------------

SERIES C.

----+--------+-------+-------------+--------------+------------------- 223| 1,165 | 0.125|2 hours |Survived |Belgian; carrots. 293| 1,605 | .1 | | do. | Do. 295| 1,205 | .1 | | do. | Do. 292| 1,595 | .1 | | do. | Do. 298| 1,205 | .125|1.5 hours | do. | Do. ----+--------+-------+-------------+--------------+-------------------

SERIES D.

----+--------+-------+-------------+--------------+------------------- 310| 880 | 0.2 |2 days(1) |About |Belgian; oats. | | | | 2.5 days(1)| 311| 650 | .2 | |Survived | Do. 312| 740 | .2 |40 minutes |4.5 hours |Maltese; oats. 78| 659 | .257|15 minutes |Survived |Yellow and white; | | | | | oats. 75| 842 | .2 | do. | do. |Gray and white. 74| 692 | .2 | do. | do. | 317| 635 | .252|1 hour |About 2 hours |Belgian; oats. | | | 25 minutes| | 323| 820 | .25 |1 hour |Survived |White; oats. | | | 15 minutes| | ----+--------+-------+-------------+--------------+-------------------

(1) Not due to caffein.

INTRAMUSCULAR INJECTION.

Well-fed rabbits, which received a diet exclusively of oats, were used for these experiments. The injections were made into the lumbar or into the gluteal muscles.

SERIES A.

In this series the caffein was injected into the gluteal muscles.

_Rabbit 284. Brown and white, female. Weight, 1,100 grams._

December 14: 2 p. m., 11 cc 2 per cent caffein injected into the gluteal muscles (0.2 gram per kilo), under observation until 5 p. m., had frequent convulsions; at 5 p. m. in a comatose condition. Rabbit was found dead the next morning.

_Rabbit 286, white and black, female. Weight, 1,315 grams._

December 15: 2.30 p. m., 13 cc 2 per cent caffein injected into the gluteal muscles (0.1977 gram per kilo), tremors and increased reflexes observed during the next two hours, but no other symptoms.

December 17: Rabbit alive.

_Rabbit 285, yellow and white, female. Weight, 1,385 grams._

December 14: 10.15 a. m., 14 cc 2 per cent caffein injected into the gluteal muscles (0.2 gram per kilo), general tremors, but no convulsions observed. Rabbit survived.

December 17: Rabbit still alive.

_Rabbit 287. Belgian hare, female. Weight, 1,140 grams._

December 15: 2.15 p. m., 11 cc of 2 per cent caffein injected into the gluteal muscles; 2.30 p. m., tonic contractions of posterior limbs. Paralysis and death at 2.40 p. m.

SERIES B.

In series B the caffein was injected into the lumbar muscles.

_Rabbit 307. Belgian hare, female. Weight, 1,175 grams._

February 16: 11.05 a. m., 8 cc 2 per cent caffein injected (0.136 gram per kilo) into the lumbar muscles; under observation until 4 p. m., no symptoms; 4 p. m., allowed to walk on the floor; after walking a short distance loss of coordination and paralysis of posterior extremities; 5.20 p. m., found dead.

_Rabbit 306. Belgian hare, female. Weight, 1,860 grams._

February 16: 11 a. m., 12.5 cc 2 per cent caffein injected into the lumbar muscles; 12 noon, no symptoms; 2 p. m., walked about 10 feet, exhaustion and paralysis; 3 p. m. found dead.

_Rabbit 181. Belgian hare. Weight, 1,230 grams._ (Was experimented on some time previously.)

February 16: 10.55 a. m., 8 cc 2 per cent caffein injected into the lumbar muscles; (0.130 gram per kilo); 12 noon, no symptoms; 2 p. m., no symptoms; 3 p. m., put on the floor, walked about 10 feet and was exhausted, posterior extremities paralyzed; 4 p. m., found dead.

SERIES C.

In the fall of the same year additional experiments were carried out with doses ranging from 100 to 200 milligrams of caffein per kilo, which were injected into the lumbar muscles. The results are given in the following abbreviated protocols:

_Rabbit 425. Belgian hare. Weight 1,520 grams._

September 27: 10.30 a. m., 7.5 cc 2 per cent caffein injected into the lumbar muscles; 2 p. m., reflexes increased.

September 28: Rabbit normal.

October 5: Weight, 1,620 grams; 2.50 p. m., 10 cc 2 per cent caffein injected into lumbar muscles; 3.05 p. m., reflexes increased.

October 13: Weight, 1,520 grams; 10.30 a. m., 10 cc 2 per cent caffein (131 mg per kilo) injected; 11 a. m., no symptoms; 11.30 a. m., reflexes much increased.

October 14: Alive, no symptoms.

_Rabbit 426. Belgian hare, female. Weight, 1,425 grams._

September 27: 7 cc 2 per cent caffein injected into the lumbar muscles at 10.30 a. m.; 2 p. m., reflexes increased.

September 28: Rabbit normal.

October 5: Weight, 1,425 grams; 2.55 p. m., 9 cc 2 per cent caffein injected into lumbar muscles; 3.05 p. m., reflexes increased.

October 13: Weight, 1,405 grams; 10.30 a. m., 10 cc 2 per cent caffein (142 mg per kilo) injected; 11 a. m., no symptoms; 11.30 a. m., reflexes increased.

October 14: Rabbit alive, no symptoms.

_Rabbit 427. Belgian hare, female. Weight, 1,780 grams._

September 27: 9 cc 2 per cent caffein injected into the lumbar muscles; 2 p. m., reflexes increased.

September 28: Rabbit normal.

October 5: Weight, 1,850 grams; 3 p. m., 11.5 cc 2 per cent caffein injected into lumbar muscles; 3.10 p. m., reflexes increased.

October 13: Weight, 1,830 grams; 10.40 a. m., 14 cc 2 per cent caffein (153 mg per kilo) injected into lumbar muscles; 11 a. m., no symptoms; 11.30 a. m., reflexes increased.

October 14: Rabbit alive, no symptoms.

_Rabbit 453. Belgian hare, male. Weight, 1,160 grams._

October 12: 3.45 p. m., 11.5 cc 2 per cent caffein in aqueous solution injected into lumbar muscles; 4.15 p. m., reflexes increased; 4.30 p. m., paralyzed.

October 13: 9 a. m., found dead. _Autopsy_: Gastric mucosa hemorrhagic; liver darkened; other organs normal.

_Rabbit 455. Belgian hare, gray, female. Weight, 1,185 grams._

October 12: 3.30 p. m., 11.5 cc 2 per cent caffein injected into the lumbar muscles; 4 p. m., reflexes increased.

October 13: Rabbit weighed 1,070 grams, no symptom of caffein poisoning, reflexes normal; 10.30 a. m., 10 cc 2 per cent caffein injected into the lumbar muscles; 11.30 a. m., jumped off the table, had attack of convulsions and died. _Autopsy_: Findings same as in No. 453.

_Rabbit 428. Belgian hare, gray, male. Weight, 1,650 grams._

October 5: 4 p. m., 14.8 cc 2 per cent caffein (0.18 gram per kilo) injected into the lumbar muscles.

October 6: Found dead.

_Rabbit 429. Belgian hare, male. Weight, 1,340 grams._

October 5: 4 p. m., 13.5 cc 2 per cent caffein (0.2 gram per kilo) injected into lumbar muscles.

October 8: Rabbit found dead.

SERIES D.

Further experiments making injections into both the lumbar and the gluteal muscles, were made in this series.

_Rabbit 577. Gray male. Weight, 1,380 grams._

February 14: 3 p. m. 14 cc 2 per cent caffein injected into the gluteal muscles of the right side; 3.10 p. m., restless, jumped off the table and walked about, reflexes increased; 3.45 p. m., passed 30 cc clear, straw-colored urine; 4.45 p. m., allowed to walk about, ran across the room, about 20 feet, looked tired, stretched himself out on the floor, then raised himself and walked about showing no disturbance of gait.

February 15: 9 a. m., found dead.

_Rabbit 578. Gray, female. Weight, 1,670 grams._

February 14: 3.05 p. m., 18 cc 2 per cent caffein solution injected into the gluteal muscles of the right side; 3.15 reflexes increased, but not restless; 5 p. m., allowed to walk about, no symptoms observed.

February 15: Found dead.

_Rabbit 579. White and gray, male. Weight, 1,490 grams._

February 14: 3.15 p. m., 15 cc 2 per cent caffein solution injected into the gluteal muscles of the right side; put in cage; 3.30 p. m., reflexes increased; 5 p. m., taken out of cage and allowed to walk across the room, no special symptoms noticed.

February 23: Still alive.

_Rabbit 580. Gray male. Weight, 1,510 grams._

February 14: 3.35 p. m., 15 cc 2 per cent caffein solution injected into lumbar muscles.

February 23: Still alive, in good condition.

_Rabbit 581. Gray female. Weight, 1,680 grams._

February 14: 3.45 p. m., 17 cc 2 per cent caffein solution injected into the lumbar muscles of the right side; 4 p. m., reflexes increased; 4.15 p. m., jumped off the table and had wild convulsions, became very restless, walked about the laboratory; 4.25 p. m., had convulsions occasionally; 4.30 p. m., extremities extended and quite rigid; 4.35 p. m., convulsions and death.

_Rabbit 582. Gray male. Weight, 1,870 grams._

February 14: 4.15 p. m., 18 cc 2 per cent caffein solution injected into the lumbar muscles of right side; 5 p. m., reflexes increased; walked about in the room, then rested; 5.15 p. m., had short spasm when handled.

February 23: Alive; good condition.

The data presented in these experiments show that the toxicity of caffein when injected into the muscles of the lumbar regions is the same as when injected into the gluteal muscles. The rabbits of series A received approximately 0.2 gram caffein per kilo and two died as a result of this treatment. The other two survived but symptoms of caffein intoxication were observed.

In series B smaller doses proved fatal, from which it would appear that caffein is more toxic when injected into the lumbar muscles. Further observations, however, failed to corroborate the results obtained in this series. Thus, in series C, 130 to 150 mg of caffein per kilo injected into the lumbar muscles produced mild symptoms only. Experiments with larger doses showed that 0.180 gram caffein per kilo may cause death. It will be noticed, on the other hand, that rabbit No. 455 survived a dose of 0.2 gram per kilo. New experiments were therefore carried out in which the same amounts of caffein in proportion to the weight of the animals were injected into the lumbar muscles as into the gluteal muscles. As shown in the experiments of series D, one rabbit (No. 581) died shortly after caffein was injected into the lumbar muscles; two recovered. Two of the three which received injections into the gluteal muscles were found dead the next day; one recovered. Post-mortem examination failed to indicate the presence of any abnormalities. The rate of absorption of caffein from the gluteal and from the lumbar muscles seems to be, therefore, the same, or not to differ very much. The observations of Auer and Meltzer(7) are of interest in this connection. According to their investigations adrenalin is more rapidly absorbed from the lumbar than from the gluteal muscles. This is in all probability due to the greater delicacy of the test they employed (since they judged the rate of absorption by the effect of adrenalin on blood pressure) as well as to the much greater activity of the substance.

TABLE 4.--_Intramuscular injections._

SERIES A.

----+--------+-------+----------+-----------+----------+-------------- | |Caffein| | | | No.| Weight.| per | Symptoms | Duration | Site of | Remarks. | | kilo | after-- | of life. |injection.| ----+--------+-------+----------+-----------+----------+-------------- |_Grams._|_Gram._| | | | 284| 1,100 | 0.200 | 3 hours |Less than | Gluteal |White and | | | | 20 hours | | brown female. 286| 1,315 | .1977| 2 hours |Survived | do. |White and | | | | | | black female. 285| 1,385 | .200 | Present | do. | do. |Yellow and | | | | | | white female. 287| 1,140 | .210 |15 minutes|25 minutes | do. |Gray female. ----+--------+-------+----------+-----------+----------+--------------

SERIES B.

----+--------+-------+----------+-----------+----------+------------- 307| 1,175 | 0.136 | 5 hours |6 hours, | Lumbar |Gray female. | | | | 20 minutes| | 306| 1,860 | .134 | 3 hours |4 hours | do. | Do. 181| 1,230 | .130 | 4 hours |5 hours | do. |Gray. ----+--------+-------+----------+-----------+----------+-------------

SERIES C.

----+--------+-------+----------+-----------+----------+------------- 425| 1,520 | 0.131 | 1 hour |Survived | Lumbar |Gray. 426| 1,405 | .142 |30 minutes| do. | do. |Gray female. 427| 1,830 | .153 |50 minutes| do. | do. | Do. 453| 1,160 | .200 |30 minutes|Less than | do. |Gray male. | | | | 20 hours | | 455| 1,185 | .200 | do. |Survived | do. |Gray female. 428| 1,650 | .180 | |Less than | do. |Gray male. | | | | 20 hours | | 429| 1,340 | .200 | | do. | do. | Do. ----+--------+-------+----------+-----------+----------+-------------

SERIES D. ----+--------+-------+----------+-----------+----------+------------- 577| 1,380 | 0.200 |10 minutes|Less than | Gluteal |Gray male. | | | | 18 hours | | 578| 1,670 | .210 | do. | do. | do. | Do. 579| 1,490 | .200 |15 minutes|Survived | do. |White and | | | | | | gray male. 580| 1,510 | .200 | | do. | Lumbar |Gray male. 581| 1,680 | .200 |15 minutes|50 minutes | do. | Do. 582| 1,870 | .192 |45 minutes|Survived | do. | Do. ----+--------+-------+----------+-----------+----------+-------------