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Protocols.
[Only those experiments are described here that are not fully given in the text. For future reference the experiments are numbered in continuity with those of Dr. Fitchett's paper.]
199. Rabbit, used for blood-pressure experiment, received 10 c.c. of 0·5 per cent, tutin injected intravenously in two doses. Animal killed three-quarters of an hour after the first and half an hour after the last dose. Vessels washed out with saline. Blood and washings were precipitated with alcohol, but not examined till some four months later, then extracted repeatedly with alcohol. The alcohol was driven off, and a watery solution of the residue made up to 10 c.c.; 2 c.c. of this was injected into a rabbit without obvious effect, but 1½ c.c. killed a 21 grm. frog in twenty-seven hours, with symptoms of nervous disturbance, such as twitching of hind limbs–muscular weakness with irritable reflexes.
225. 30 c.c. of fresh defibrinated rabbit-blood incubated five hours at 40° C. with 2·62 c.c. of 0·5 per cent. tutin was administered by stomachtube to a rabbit weighing 1·31 kilo (=dose of 10 mlgrm. per kilo). Severe tutin symptoms resulted, with recovery of animal. The dose here was slightly above the usual fatal one.
226. Mashed brain of recently killed rabbit + 5 c.c. of 0·5 per cent, tutin, incubated overnight at 40° C. Extracted with alcohol, and watery extract of residue made. A dose of this extract proved fatal to a frog, while a similar dose of an extract of rabbit-brain without the addition of tutin did not kill a control frog.
229. Cat, pregnant, weight 3·7 kilo, gave 3·7 c.c. of 0·5 per cent, tutin solution (five years old). Dose, 5 mlgrm. per kilo; actual amount, 0·0185 grm. Twitchings began in four minutes. To allow of complete absorption of the dose, she was kept under A.C.E. anaesthesia for about twenty minutes. The convulsions were largely modified in severity by the anaesthesia. On removal of the anaesthesia they became very severe. Animal was killed by bleeding forty minutes after dose given. Uterus contained three foetuses. Brain and upper part of spinal cord. weighed 27·5 grm.; brains of foetuses, 7·5 grm.; blood obtained, 56 grm.
Cat's brain was mashed in mortar, extracted repeatedly with boiling water, filtered, evaporated to 27·5 grm. (= original weight). Tested with Fehling before and after hydrolysis with HC1, but got no reduction. Some
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of the fluid was concentrated and injected into a frog; it caused death. A similar quantity treated with alkali, neutralized, and injected into another frog also caused death. Neither frog showed tutin symptoms.
Foetal brains treated together in similar way and filtrate concentrated to small bulk: whole fitrate hydrolyzed with HCI gave slight but distinct reduction. No test made before hydrolysis because of small bulk of fluid, and result would need to be controlled by using normal foetal brains.
Blood.—An. extract of the blood of this animal gave reduction with Fehling without hydrolyzing. To distinguish whether tutin was present, the blood was evaporated to dryness on filter paper, and extracted with hot ether; the ethereal extract was extracted with water after driving off the ether. This extract gave no reduction before or after treatment with HCl.
237. Young cat, weight about 850 grm., received orally the alcoholic extract of dried blood of rabbit 234 (dosed with 10 mlgrm. tutin). As described in the text above, the extract represented about 33 c.c. of blood. At 12 noon, dose administered; 12.3 p.m., very distinct twitching of the ears; 12.14 p.m., twitching continues, licking its lips and swallowing frequently; 12.22 p.m., drowsy appearance, deliberate winking, and exaggerated breathing; 1.3 p.m., no symptoms apparent except drowsiness. When seen again at 3.18 p.m. it appeared normal.
(If the tutin in rabbit 234 had been uniformly distributed, the 33 c.c. of blood would have contained 0·164 mlgrm: this to a cat weighing 800 grm. = 0·2 mlgrm. per kilo. But a control experiment on a sister cat showed that 0·21 mlgrm. per kilo given orally in milk produced no effect. We may conclude, therefore, that the blood of No. 234 contained more than its due proportion of the poison.)
238. Young cat, weight 760 grm., received an extract of the muscles of rabbit 234 (dosed with 10 mlgrm. tutin). The dried muscle weighed 35 grm., representing, therefore, over 100 grm. body-flesh. The greater part of this extract was successfully administered to the cat, and beyond urination and some quickening of respiration no obvious effect followed.
240. Rabbit, weight 1,350 grm., received 10 mlgrm. tutin intravenously. Twenty-two minutes afterwards, when practically dead, the blood was collected, and the liver, muscles, brain, and cord excised and treated in the usual way for extraction of tutin. The extract of muscle, representing 100 grm. to 200 grm. of flesh produced no symptoms on oral administration to a cat weighing 812 grm. The extract of liver, representing 30 grm. fresh substance, tested on another young cat produced no symptoms beyond urination and defaecation, which might be attributed to the bile salts and other normal extractives.
244. Young cat, weight 812 grm., received hypodermically 15 mlgrm. tutin. Symptoms became severe in ten minutes, and lasted an hour, at end of which time the animal was killed by bleeding. The temperature fell steadily all the time (37° C. to 32° C. at death). A test-tubeful of blood of this animal yielded very little serum, which did not reduce Fehling either before or after hydrolyzing, while the blood of a normal cat killed by chloroforming at the same time gave the usual proportion of serum, and this serum reduced Fehling easily before, and still more so after, hydrolyzing with HCl.
My sincere thanks are due to Mr. Aston, of the Agricultural Department, Wellington, for supplying me with pure crystallized tutin.