
Art. XXXII.—Some Experiments on Tutin and Tutu Poisoning.
[Read before the Otago Institute, 7th October, 1913.]
The following is a short description of some work on tutu and tutin poisoning that has occupied me at various periods since the publication of the joint paper on tutin by Dr. Fitchett and myself in the “Quarterly Journal of Experimental Physiology” (vol. 2, p. 335, 1909). In the beginning of that paper we gave the history of the previous work done on the subject, and a practically similar account is also given by Dr. Fitchett in a paper published in these Transactions (vol. 41, p. 286, 1909).
Since the publication of these papers, a paper entitled “On the Toxicology of the Tutu-plant” has been published by W. W. Ford in the “Journal of Pharmacology and Therapeutics” (vol. 2, 1910). Dr. Ford had received some tutin from Mr. Aston, Wellington, and had experimented in very

much the same way and with practically the same results as Dr. Fitchett and myself. He found, as we did, that tutin is extremely toxic, the lethal dose to guinea-pigs and rabbits being a few milligrams per kilogram body-weight, and that it is not readily destroyed—e.g., it withstands treatment with acids, and does not lose its properties on standing. Like ourselves, he failed to produce immunity to its action. A large part of his work was done on the fate of tutin in the tissues, and his most striking assertion is that the tutin can be localized in the brain. From our experiments Fitchett and myself had come to the conclusion that tutin attacks the nerve-centres, but Ford goes further and says he is able to prove its existence in the brain as a “detoxified” body which reduces Fehling's solution after being hydrolyzed with HCl. I have repeated his experiments, following his method so far as I can gather it from his paper, but I have not succeeded in getting a positive result. The same problem had presented itself to me before I saw Ford's paper, and I had already done some of the experiments he describes—e.g., I also found that brain-tissue from a recently killed animal does not diminish the toxic power of tutin (Exp. 226).
Recent Experiments on the Fate of Tutin in the Body.
Our previous work on this point had led to no positive conclusions. We found no evidence for the existence of tutin in the urine or in the milk of rabbits. We believed we had some evidence of accumulation of the drug, as successive sublethal doses proved fatal (see Exp. 13 in Fitchett's paper), but we suggested that the facts might be explained in another way—viz., that the vital mechanisms became more and more susceptible to the action of the drug.
In considering the possible fate of a poison in the body several possibilities present themselves: (a) The poison may remain unchanged and become uniformly distributed throughout the tissues; (b) it may remain unchanged, but some tissues may have more and some less; (c) it may be changed into some other substance which may be uniformly or irregularly distributed. Besides these possibilities, the poison may be excreted more or less rapidly in changed or unchanged form; in the case of tutin this is unlikely, at least so far as the unchanged poison is concerned (see previous papers). Concerning the other possibilities, all that one can say at present is that experiments show that (a) is unlikely—e.g., Exps. 288 and 240 show that the extract of relatively large amounts of muscle has no toxic effect. This fact is of considerable practical importance to sheep-farmers, for I believe it would be quite safe to feed the dogs on the meat of sheep accidentally poisoned by tutu.
Besides the muscles, I have examined the liver, blood, and brain of animals after administration of large doses—larger relatively than is likely to occur in stock spontaneously eating the tutu-plant—but I have not been able to detect any evidence of its presence in any tissue except blood and brain, and in those instances only in traces.
In examining the tissues for presence of tutin two methods presented themselves—that used by Ford, in which the tissue is examined for a reducing substance after hydrolyzing; and, secondly, the biological method, in which the toxicity of extracts of the tissue is examined. As already stated, I was unable to obtain Dr. Ford's results, and accordingly made use of the second method to a greater extent. The tissue was usually dried, care being taken that the reaction was acid; the dried material was ex-

tracted with hot alcohol, and the alcoholic extract concentrated, neutralized, and taken up with water to form a clear liquid in some cases, in others an emulsion.
Blood.—Since the practice of bleeding is universally recognized as valuable in the treatment of stock poisoned by tutu, my attention was specially drawn to this tissue. In bleeding “tutued” sheep it is a common practice to slit a vein near the eye—the blood flows down on the face, and much of it is said to be swallowed. This led me to try whether fresh blood has any influence on the toxicity of tutin. Exp. 225 shows that it has little, if any, effect. The other experiments recorded here had as their object the discovery of tutin in the blood; and the results show that shortly after the administration of large doses of tutin the blood contains somo poison—either tutin itself in traces or some other toxic body arising from it or resulting from its action. The experiments in support of this are the following:–
Exp. No. 234: Rabbit, weight 2,010 grm., received 10 mlgrm. tutin intravenously. Respiration immediately became quickened; seven minutes after injection the animal began to show muscular symptoms, and at eight minutes a typical convulsion broke out. Twenty minutes after the in-jection, and when nearly dead, it was killed by bleeding; the blood was defibrinated, and within twenty minutes 10 c.c. of it was injected in two portions into the ear-vein of a rabbit (No. 235) weighing 1,350 grm. The animal became dazed and sleepy-looking; it sat with its eyeballs rolled up under the half-closed lids, and the respiration-rate was markedly increased. It also licked its lips repeatedly as if salivating, but within an hour it seemed to have completely recovered. These symptoms point to the presence of some tutin or an allied toxic body in the blood. If all the tutin were present in the blood as such the dose would have been 1 mlgrm., and the animal would have shown much more decided symptoms. The residue of the blood of the first rabbit was evaporated to dryness; it then weighed 7·2 grm., and therefore represented about 35 c.c. of blood = one-third of the total blood of the animal. The dried residue was treated with alcohol, and the alcoholic extract taken up with water as already described. The whole quantity was given by mouth to a kitten (No. 237). It developed some twitching of the ears, licking of its lips, and swallowing movements, &c., but recovered completely. The symptoms here are suggestive of small amounts of tutin in the blood; and on calculating the percentage present, if the tutin were uniformly distributed throughout the body, one may safely conclude that the blood had more than its due share of the poison, while, as shown above, the blood did not contain all the poison.
Exp. No. 232: Rabbit, weight 1,588 grm., received hypodermically 1·5 c.c. of 0·5 per cent. tutin = 7·5 mlgrm. The animal had severe symptoms, and was killed half an hour after the dose was administered. The brain and cord weighed 11 grm. The blood and washings of blood-vessels yielded 9·8 grm. dried material; it was calculated, therefore, that the amount of blood obtained = 43 c.c. = more than half the total blood.
Both tissues were acidified with HC1 and evaporated to dryness, then both were extracted with absolute alcohol and the extract dissolved in water after removing the alcohol.
The whole of the extract of brain and cord, measuring 3 c.c., was injected into the peritoneum of a rabbit weighing 673 grm., but no symptoms of any kind followed.

The watery extract of blood obtained in the same way also measured 3 c.c. Half of it was injected intravenously into a small rabbit (weight, 671 grm.). Before the injection was finished it jerked its head backwards, and when released fell over on its back, clawed the air in a convulsive manner, and was dead in a few seconds.
The remaining half of the extract was given hypodermically to another small rabbit. During the injection the animal writhed as if in pain, later on the latter half of the body became paralysed, the respiration became very feeble, but in about half an hour it had completely recovered.
Exp. No. 233: The blood of a normal rabbit, measuring about 30 grm., was treated in an exactly similar way to that of No. 232, and the final watery extract injected into the small rabbit that had survived (vide supra). No symptoms followed, and there is little doubt but that the blood in this case (232) contained some toxic body, although the symptoms produced were not those of tutin.
Other experiments bearing on this part of the subject are detailed in the protocols of experiments 199, 229, and 244; but I consider the results less conclusive here, because frogs frequently die when injected with foreign material, and the blood, at least in cats, may give reduction without being hydrolyzed, while in Exp. 244 the blood of a tutinized cat failed to give the reduction test.
These results indicate that the beneficial effects of bleeding can hardly be ascribed to the removal of the poison in the shed blood, for the tutin absorbed from ingested tutu is not likely to be present in such high concentration as in these experiments, and yet the blood in these cases had comparatively feeble toxic power although the quantities used were relatively large—certainly larger relatively than would occur in bleeding as usually practised on sheep.
Brain.—From Dr. Ford's statements one might be led to believe that practically all of a moderate dose of tutin becomes concentrated in the brain of the animal. As already stated, I was unable to confirm this by the chemical test, and the brological test, with one exception, also gave negative results. Thus in the experiment described above (No. 232) the hydrolyzed brain of the rabbit produced no effect.
In Exp. 229 the extract of brain gave no reduction after hydrolysis, but some reduction was obtained with the extract of foetal brains. This latter point requires further investigation; a reducing body may be normally present. The result of injection of the hydrolyzed brain extract into a frog is inconclusive, because the control frog, in which the extract was treated with KOH to destroy the suspected tutin, also died.
In Exp. 240 a rabbit weighing 1,350 grm. received 10 mlgrm. tutin intravenously. The brain, with the spinal cord, weighing 10·3 grm., was extracted with alcohol, and the extract was dissolved in water. One-third of this solution was injected into a cat without effect. The material used here was not hydrolyzed, and the absence of symptoms corresponds to Ford's results. Another portion of the extract of brain was tested for reduction after hydrolysis, without result.
Exp. 244 similarly gave a negative result by the reduction method. The only experiment which gave some indication that the brain may contain a toxic body was No. 236. In this case a young cat, weighing 760 grm., received the emulsified brain of rabbit 234, which had been poisoned with 10 mlgrm. of tutin. After about an hour it showed some twitching of the ears, licking of its lips, swallowing movements, deliberate

winking, and exaggerated respiratory movements; within two hours all these symptoms had disappeared, and at no time were they very pronounced. The animal was fasting, and it may be that the tutin compound, with the tissues, was dissociated in the stomach. This is the only experiment that lends some support to Ford's results.
Liver.:—One experiment (No. 240) was done on tutin in the liver, with negative results.
Experiments on the Relative Toxicity of two Varieties of Tutu.
There is a general consensus of opinion among sheep-farmers that the “fern” tutu (Coriaria angustissima) is more deadly than the ordinary “tree” tutu (Coriaria ruscifolia). Easterfield and Aston found that all varieties of the plant contain tutin, and estimated the percentage in young shoots of C. ruscifolia at 0·03, but no systematic quantitative estimation of the amount of tutin in the different varieties has yet been made.
The experiments which are described here were undertaken with the view of comparing the toxicity of the varieties mentioned above, but I wish to emphasize that this does not settle the practical question as to which is the most deadly to stock, for the palatability and the ease with which the plant may yield its poison are probably more important than slight differences in the percentage of tutin present.
Exp. 201: A quantity (464 grm.) of C. ruscifolia leaves, stalks, and fruit was gathered in the end of February. It was minced, covered with water, allowed to steep for fourteen hours, and then pressed through flannel and made up to 1,000 c.c. Of this fluid 50 c.c. per kilo administered by stomach-tube to a rabbit (201) caused death with typical tutin symptoms in three hours and three-quarters. From our former experiments, Fitchett and I found that death resulted in rabbits from a dose of 6 to 8 mlgrm. tutin orally administered; but there is, of course,. the great probability that in order to kill the animal more than that amount would be required when the tutin is given mixed with the other extractives of the plant. The experiment shows, however, that at least 0·03 per cent. tutin was present (0·007 grm. in 50 c.c. = 0·14 grm. in 464 grm. of tutu = 0·03 per cent.).
In March of the same year samples of two varieties of tutu were obtained from the slopes of Swampy Hill, near Dunedin. The one variety was certainly C. ruscifolia, and from the descriptions given I believe the other to have been C. angustissima. Each sample was extracted once with water as described above, and a few days later a second extract was made in the same way, and the toxicity of the extracts determined on rabbits by oral administration.
The results were as follows:-
Coriaria ruscifolia (800 grm.).
First extract measured 1,800 c.c. (No. 212) 16 c.c. per kilo: no effect. (No. 213) 40 c.c. per kilo: distinct symptoms; recovered. (No. 214) 45 c.c. per kilo: death in one hour and a half. Assuming that 45 c.c. of the extract contains 0·008 grm., this gives 0·32 grm. tutin in the first extract.
Second extract measured 1,150 c.c. (No. 210) 40 c.c. per kilo: gave slight symptoms. (No. 215) 50 c.c. per kilo: caused death in three to four hours. Assuming that 50 c.c. of this extract contains 0·008 grm., the second extract contained 0·184 grm., or a total of 0·504 grm. tutu in 800 grm. tutu = 0·063 per cent.
A third extract of the same material was made. It measured 1,000 c.c., and of this 60 c.c. per kilo gave no symptoms.

Coriaria angustissima (400 grm.).
First extract measured 862 c.c. (No. 216) 38 c.c. per kilo: very severe symptoms; recovered. Assuming from previous experiments that the dose here was 7·5 mlgrm., the extract may be said to have contained 0·17 grm. tutin.
Second extract measured 862 grm (No. 211) 40 c.c. per kilo: slight symptoms; recovered. (No. 218) 50 c.c. per kilo: more marked symptoms.
The symptoms observed here corresponded to those produced by a dose of about 5 mlgrm. pure tutin, and on that basis the extract would contain 0·107 grm. tutin. The total in 400 grm. leaf would therefore be 0·277 grm., or a percentage of 0·069; but owing to pressure of other work the lethal doses of the extracts of this variety were not so exactly determined as for C. ruscifolia.
These experiments, so far as they go, indicate that there is little difference between the actual amounts of tutin present in the two varieties.
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

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.
