Art. XXXIII.—A. Contribution to our Knowledge of the Physiological Action of Tutin.^*

By Frank Fitchett, M.D. Edin.

A. Historical.

of attaching a boy to each of their bullock-teams, solely for the purpose of preventing their animals-feeding on this pest of the colony. Such incidents I found were of daily occurrence. I met few settlers who had not at some period had occasion from this cause to mourn the loss of sheep or bullocks—the former sometimes by the hundred, the latter by the dozen.” These remarks of Lindsay serve well to illustrate the deadly nature of the plant, and the embarrassment it offered to the pioneers of the country. They also point to the importance and great desirability of an investigation of the physiological action of the poison being made, in order that the treatment of its effects may be conducted on rational lines, and as the first step towards the discovery of an appropriate antidote.

But while sheep and cattle have been the chief victims, human beings have not proved exempt, and he was a lucky farmer who lost only his cows or his sheep. Too often one of his children succumbed to the effects of eating the berries or young shoots. Nor is this strange, for the shrub in full fruit is a very striking and attractive object. The numerous racemes of richly coloured, tempting-looking berries — at a glance not unlike black currants—could hardly be overlooked or neglected by the child or thirsty traveller ignorant of their dangerous properties.

Numerous cases of poisoning must have occurred, but the recorded deaths from this cause are not many. The following are all that occur in the literature that has been laid under contribution: 1. Thomson (4), in 1859, mentions that up till that date several children had died from eating the berries. 2. Lauder Lindsay (3) mentions the case of twelve French sailors who were poisoned by eating the berry; four of them died. 3. The Otago Colonist (5) records the death of one of two children in 1861 who had eaten the shoots. 4. The Otago Daily Times notices the death of a young man in 1862 from eating the shoots (6). 5. Easterfield and Aston (7) put on record the following cases: A girl in 1854–55, from eating the berries; a boy in 1860, from eating the berries; two cases from eating the berries—one died, the other recovered, with impaired memory.

For the purposes of this paper the writer asked of the Registrar-General a return of all cases of death from tutu poisoning that occur in the records. In his reply the Registrar-General stated that he was unable to make a return, as cases of this kind are classed in the vital statistics under the general heading “Accidental Poisoning.” He had had the statistical tables relating to inquests examined for thirty years back, and found that only four deaths occurred from eating poisonous berries, one in each of the years 1889, 1891, 1896, and 1902. It is probable that the berry in each of these cases was the tutu-berry, but in only one case—that of 1889—was it stated to be so.

Effect of the Plant on Animals.

Horses.—Statements vary as to the effect on horses. It is said that they have been known to eat freely of the plant without injury (8); and, again, that C. thymifolia is highly poisonous to them (9). They are said to refuse the young shoots, but have been known to eat the berries (3). If they escape, it is probably because they do not eat enough of the plant; that they eat freely without evil result is not credible.

Birds are regarded as immune. They certainly eat freely of the berries without, ill effect. The question of their immunity will be considered later.

Rabbits are said to be immune (10), (11), and certainly the tutu does not seem to have checked their increase. They probably do not eat the plant.

Elephants.—Sir Julius von Haast (12) records the death of an elephant from tutu-poisoning. The animal was marched inlands by its owner for a considerable distance, and on arriving at a suitable halting-place, where the vegetation was abundant, was allowed to feed. The grass had been burnt off during the previous season, and had shot up again, together with a large crop of tutu-shoots. The elephant fed for four hours, and then drank freely from a neighbouring stream. It then began to reel, fell on the ground, and died in three hours.

Sheep and Cattle.—The following extract from a letter received from the manager of a large sheep-station gives an excellent account of the effects of tutu upon sheep: “The effect on sheep is that they will stand still, trembling as if palsied, froth at the mouth, with their jaws going continually, and their teeth grinding. Suddenly they will fall over, with their limbs rigid, as if suffering from strychnine poisoning. If assisted on, to their legs they are absolutely mad, and will rush against a fence or over a precipice, and will pay no attention to man, or dog, or animals of their own kind. With animals that have eaten less of the plant, symptoms do not appear unless they are disturbed, and then the effect is shown with terrible suddenness; a bark from a dog or a sudden run for a few yards will be almost certain to start the poison to work. In cases like the above, however, the affected sheep' generally recover if left alone.” In their wild career they often injure themselves against obstacles, or rush into creeks and are drowned. More frequently they die in convulsions.

Cattle are similarly affected, but the wild delirium is even more marked, in their case. Popularly they are said to go mad; and the wild way in which they wheel round and round, gallop aimlessly about, kicking, charging, and rushing blindly against rocks and other obstacles, lends colour to the popular opinion. The wild career continues until the animal, overcome by exhaustion, falls to the ground, becomes comatose, and dies in convulsions.

In the light of this account, the symptoms displayed by one of Captain Cook's animals is interesting: “The ram was taken with fits, bordering, on madness… One night he was seized with one of these, fits and ran headlong into the sea, but soon came out again, and seemed quite easy. Presently after he was seized with another fit, and ran along the beach … and was never seen more” (13).

Animals are frequently found distended with gas, “blown” after death. This is probably due to rapid fermentation of the leaves ingested, and is similar to the condition met with in cattle after eating freely of clover. As with clover, it is more pronounced when the tutu is eaten wet.

Effect on Human Beings.

The symptoms of poisoning by the plant in the human subject include vomiting, giddiness, delirium, great excitement, stupor, coma, and convulsions.

In a fatal case reported in the Otago Colonist of the 25th October, 1861, the physician who attended the case stated in his evidence at the inquest that he found the child perfectly pale, with teeth clenched. The breathing was difficult, the lips livid, and the pupils much dilated. For about five minutes the rigidity went off, and the pupils contracted; but a relapse occurred, the teeth were set again, and the child gradually sank back, “without any symptom of convulsion or suffering.” In this case it was the

2. Botany.

The tutu plant belongs to the natural order Coriariece, a small order of very doubtful relationship possessing but a single genus, Coriaria. The genus includes some eight or ten species, and has a rather remarkable distribution, species being met with in south Europe, South America, China, Japan, north Africa, India (Himalayas), and New Zealand.

The European species, C. myrtifolia, is well known to possess toxic properties. Its leaves have been used to adulterate senna with fatal effect, and numbers of cases of death are recorded from eating the berries. In 1862 several persons were said to be poisoned by eating snails that had been fattened on its leaves and young shoots (15). The symptoms of poisoning include vomiting and convulsions, and, on the whole, closely resemble those of tutu poisoning. In 1863 Riban (15) investigated the chemistry of this species, and separated a glucoside which he named “coriamyrtin.” The physiological properties of this compound will be referred to later.

The Himalayan species, C. nepalensis, is stated to be non-toxic, but, as the same has been said both of tutu and of C. myrtifolia, the statement must be accepted with reserve. The fruit is said to be eaten with impunity.

The American species, C. thymifolia, and the New Zealand species are said to be identical, and this statement has been used to prove a former land-connection between the two countries. It is more likely, however, that the order is a very ancient one, which has died out everywhere except in those places in which it is now found (8). Moreover, the identity of the two has been questioned.

The species met with in New Zealand are given by Cheeseman (16) as three in number—(1) C. ruscifolia, (2) C. thymifolia, (3) C. angustissima. The first is known locally as the “tree-toot,” the second as the “ground-toot,” and to both the name “tutu” applies. The Maoris have no name for C. angustissima.

There seems to be some division of opinion as to whether these three really constitute separate naturally distinct species, or whether the two last are merely varieties of the first. Lauder Lindsay, who in 1868 described, though with hesitation, four species—(1) C. arborea, (2) C. tutu, (3) C. thymifolia, (4) C. angustissima—says, “If only typical species be examined the

in his extremities, a mist came over everything, and he thought that he was poisoned. The symptoms soon passed off, however, and he was none the worse.

Cattle and sheep are especially fond of the young, tender, asparagus-like shoots, but they also eat the leaves and branchlets with readiness.

3. Work of Previous Observers.

When one considers the harmful influence that this noxious plant has had upon the development of the country, it is remarkable that until recent years little effort was made to determine the nature of the poison.

In 1869 Skey (21) investigated the chemistry of C. ruscifolia. He showed that the poisonous principle is not an alkaloid, as was commonly thought, and with ether extracted from the seeds a green-coloured oil, 5 minims of which when given to a cat quickly produced the symptoms characteristic of tutu poisoning. Its highly toxic nature, together with certain peculiar chemical properties possessed by it, inclined him to the opinion that this oil was the active principle.

A year later, 1870, Hughes (10) attempted to separate an alkaloid, using the ground-shoots, and did indeed succeed in obtaining a crystalline substance, but failed to identify it. He thought that a heavy olive-coloured oily fluid which he also obtained and proved to be toxic might be the active principle and “a liquid alkaloid similar to conia.” He showed that lime destroyed the activity of the poison, and advocated its use as an antidote. In conjunction with Dr. Acheson, he conducted a series of experiments on cats and dogs, but more with the object of proving the toxicity of his extracts, and of determining the value of lime as an antidote, than with any idea of advancing our knowledge of the physiological action of the poison.

Hughes's results were adversely criticized by Skey (22), who held that the temperature used in Hughes's experiments was such as must have produced many side-products by destructive distillation, and among others acetate of ammonia, the presence of which, he thought, would sufficiently account for the reactions attributed by Hughes to the presence of an alkaloid.

Twenty years elapsed before any further investigation was undertaken, and then, in 1890, W. L. Christie (11) examined the physiological action of the oil that had first been extracted by Skeey. He made a series of experiments on mammals, including one upon himself, and briefly summarised the conclusions he arrived at as follows: (1) That tutu acts on the nerve-centres after absorption into the blood; (2) that the grey matter of the motor cortex is the part chiefly affected, and that this peripheral action (sic) causes epileptiform convulsions; (3) that vomiting is chiefly due to central causes, and that by its means, and perhaps by the renal secretion, the poison is removed from the body; and, lastly, (4) that dyspncea is due to poisoning of the respiratory centres, and when death ensues it is due to asphyxia from this cause or tetanus of the respiratory muscles—both may however, I believe, occur from coma.”

The chief interest in Christie's work, however, lies in the experiment upon himself. He took, in all, 9 grains of an extract made from the leaves gathered in the spring. He calculates that each grain of extract represents 100 grains of leaf; but, in the absence of data regarding the amount of tutin in tutu-leaves at different times of the year, and of details as to the exact

method of making the extract, it is impossible to calculate what dose of tutin was taken in this case. The first dose (4 ½ grains) was taken at 2.20 on Friday afternoon, and a second dose of the same amount at 4 p.m. An hour or two later he felt sick and faint, and began to vomit. The vomiting occurred at frequent intervals, and continued for twenty-four hours. At 8 p.m. he felt slight twitches an the legs and arms; and at 10.40 p.m. the medical student who was acting as clerk of the case noted that “all the muscles seemed to get tight, and there was foaming at the mouth.” At 10.50 the pulse-rate was 102 and the breathing heavy. Twenty minutes later the pulse was still 102, but the breathing was normal, and there was profuse perspiration. At 11.24 p.m. the clerk noted that “the subject spoke in a collected manner; getting right, but drowsy.” The vomiting continued till 8 p.m. on Saturday. The following day (Sunday) he felt sick and dull, but, though shaky, managed to attend to his duties. He states that sensation, was dulled and spirits below par for seven or eight days. For a month he was not in good tone, and then for the first time he felt a sensation of “pins and needles” in his fingers and toes, and felt the floor of his bedroom woolly when he rose in the morning. He could feel accurately with his fingers, but experienced a heavy, stiff, numb sensation when, he used them, and this symptom lasted a month.

4. The Active Principle.

The first substantial advance in the investigation of the chemical properties of tutu was made in 1900, when Easterfield and Aston (23) succeeded in isolating a crystalline glucoside, to which they gave the name “tutin.” All three species of Goriaria, were experimented upon, and crystals of tutin were obtained from each. The young shoots were found to yield a greater quantity (0.03 per cent.) than either the berries or the leaves:

Preparation: In the case of C. ruscifolia, “the fresh young shoots were finely divided, the juice expressed, filtered, and evaporated to a syrup nearly neutralised by carbonate of soda and shaken up with ether. The ether on evaporation deposited crystals of tutin. These were recrystallized from alcohol until the melting-point was constant.”

Properties: Tutin is described as a colourless, odourless, intensely bitter compound, which separates from alcohol in oblique-ended prisms, and from hot concentrated solutions in water in characteristic acicular forms. It is perceptibly volatile, sublimes readily at 120° to 130° C., and melts at 208° to 209° C. (uncorrected). It contains no nitrogen, and reduces Fehling solution after inversion by acid. The compound is therefore to be considered a glucoside, but the hydrolyzed substance yields with phenylhydrazine an amorphous precipitate which is not phenylglucosazone. Examination by Zeissl's method for methoxyl groups gave negative results. Strong sulphuric acid added to a few drops of a saturated aqueous solution of tutin gives a blood-red coloration.

Solubility: 100 grams water at 10° C. dissolve 1.9 grams tutin; 100 grams either at 10° C. dissolve 1.5 grams tutin; 100 grams alcohol at 10° C. dissolve 8.2 grams tutin. It is very soluble in acetone, sparingly soluble in chloroform, and soluble in benzine and carbon-disulphide.

The optical activity has been determined by Marshall. The substance is dextrorotatory, and the specific rotatory power is + 9.25. Easterfield and Aston found that when solutions of tutin were evaporated to dryness

B. Original Work.

to 0.375 mlgm. per kilo of body-weight, 1 mlgm. of tutin being injected under the skin of a cat weighing 2.688 kilograms, and, curiously, symptoms made their appearance earlier in this case than in the last. It defaecated and began to breathe quickly within twelve minutes of the injection; salivation was noticed at fourteen minutes, and at twenty-four minutes it was panting with its mouth open, and vomited. Twitching appeared at thirty-two minutes, and the first convulsion occurred at fifty-five minutes. Convulsions were severe and frequently repeated, and it was thought that the cat would die. It had a severe convulsion at 6.5 p.m., and it was not seen again till 7.15 p.m. It then appeared rather tremulous, and was easily startled, but presented no further symptoms, and was quite well next day. It would seem that 0.375 mlgm. per kilo is very near the minimum lethal dose.

In the last three cats the constant symptoms were defaecation, rapid breathing, salivation, twitching, and general convulsions, and these generally made their appearance in the order named. The first cat did not defaecate voluntarily, and neither of the first two vomited. The vomiting in the last two occurred only once in each case. In the first convulsion in each case the tonic stage was the more pronounced, and was invariably of the opisthotonic type. Later, clonic spasms were more in evidence, and with lethal doses, when the case was making towards a termination, movement was hardly absent for a moment. With the larger doses the effect upon the mental activities of the animals was very marked. From the first they seemed dazed, and once general convulsions had set in they were oblivious of everything. With the smaller doses the cerebrum was little affected, and often after the most severe and prolonged convulsions, in the case of the cat that recovered, the animal would rise and behave as if little had happened, answering when spoken to, and even purring.

(b.) Action on Rabbits.

Rabbits are less readily affected by the poison. The largest hypodermic dose recovered from was 2 mlgm. per kilo (Exp. 5), (in a cat, 0.75 mlgm. per kilo proved fatal). A dose of 2.5 mlgm. per kilo was fatal in two hours and a half (Exp. 6), and doses larger than this killed rather rapidly (Exps. 7, 139). By oral administration a much larger dose than this is required to kill—e.g., in Exp. 8, 7.5 mlgm. per kilo proved fatal in two hours; in Exp. 9, 6 mlgm. per kilo was fatal in twelve hours; while in Exp. 10, 5 mlgm. per kilo was recovered from. In one case (Exp. 161) death preceded by typical symptoms followed the instillation of four drops of a 0.5-per-cent. solution into the conjunctival sac.

Attempts to poison rabbits with fresh tutu-leaves failed, for the animals, though deprived of all other food for several days, steadfastly refused to eat.

Symptoms.—-No important symptoms appear that have not been mentioned as occurring in cats. After a lethal dose the animal at first appears dazed, tends to assume unnatural attitudes—e.g., lies on the abdomen, with the legs projecting in front and behind—and the gait is altered. Respiration soon becomes very rapid, and there may be salivation, though it is not so marked a symptom as it is in cats. Alteration in the size of the pupil is not so noticeable. Twitching of the eyelids, lips, ears, and fore-paws occurs, and is followed by general convulsions. In the convulsion the tonic spasms are not so evident as they are in cats, but they do occur, and are of the opisthotonic type. As a rule, after the first violent convulsive movements are over, the animal continues lying on its side, and

2. Action on Birds.

It is the common opinion that birds are immune to the tutu-poison. It were not strange did a relative immunity exist, for the plump, sweet, attractive-looking berries seem to have been designed by nature for the especial purpose of inducing birds to eat; and that they do eat them freely and without injurious effect is certain. Mr. Maning, author of “Old New Zealand,” quoted by Lauder Lindsay (3), says, “Many kinds of birds live entirely on the tutu-berries when in season… The tui (Prosthemadera novce-zealandice) I have kept tame and fed for months on nothing else.”

Again, birds differ from mammals in having a higher rate of oxidation, a higher temperature, and a peculiar metabolism, which results in the excretion of urates instead of urea in the urine—features which might conceivably have an important bearing on the question of their possible immunity.

Christie (11), as the result of experiment, inclined to the general opinion that birds are immune. He injected in all 40 minims of an ethereal solution of “oil of tutu” into the “chest cavity” of a young rooster. Beyond some slight symptoms which were attributed to the ether, and a marked increase in the frequency of defaecation, no characteristic effect was noted. He observes that “there was no twitching, although the dose (40 m.) is twice as much as is necessary to convulse a cat”; and concluded that the bowels are chiefly affected in birds, and that, therefore, they are saved by rapid excretion by this channel. When the above statement is more closely examined it is found that Christie administered probably not more than 1 mlgm. of pure tutin per kilo. The symptoms were wanting because the dose was inadequate.

in continuous movement—flapping its wings and clawing at space with its feet. Respiration is spasmodic, and the forcible expulsion of air from the lungs is distinctly audible. The pupils are widely dilated—no, iris being visible—and the bird is in a state of coma, oblivious to everything, and apparently suffering no pain. In the later stages the head is brought forward, the eyes are kept closed, and the movements, which have continued without intermission from the onset, gradually become more and more feeble, and finally cease at death almost imperceptibly. The legs continue in movement longer than the wings.

With a smaller though still lethal dose the first change is a heaviness and drowsiness, giving the bird a peculiar sleepy, stupid look. The eyes are blinked heavily, and seem to be kept open only with the greatest difficulty. This is succeeded by attempts to vomit, and, if the crop contains anything, by actual vomiting. Then follow tremulousness of the head and wings and sharp spasmodic blinking of the eyes. The head is frequently drawn back sharply, and one or other or both wings are involuntarily extended for a moment. These seizures of the head and wings recur at frequent intervals, and gradually become more severe, making it difficult for the bird to keep its feet. In the more severe seizures it is thrown back on to its tail, and sits there for a moment until the clonic movements of the extended wings have ceased. At last the bird is thrown over on to its back, and, with continuous convulsive movement, the case hastens to a termination.

With non-lethal doses little is to be observed beyond the initial drowsiness and tremulousness, and perhaps vomiting and slight convulsive movements of the wings. In none of the experiments did any of the birds recover that reached the stage of being thrown over on to the back.

In none of the birds experimented upon was Christie's observation repeated that there is increased frequency of defaecation.

3. Action on Reptiles.

The only animals of this class available for experiment were three small lizards of the species Lygosoma moco, the common lizard of New Zealand. They are very small animals, these specimens weighing 4, 5, and 7 grams, and are not very suitable for experiment.

(Exp. 33.) The first lizard (4 grams) was given a dose 5 mlgm. per kilo by hypodermic injection, and it became convulsed, and died in about two hours.

The second lizard (7 grams) (Exp. 34) received 4 mlgm. per kilo hypodermically, and died four hours and three-quarters later, after showing severe and oft-repeated convulsions. Opisthotonos was well marked, the animal bending backwards till head and tail met. There were also very definite clonic spasms of the limbs. For over an hour the animal was in almost constant movement, contorting itself and twisting in every direction.

(Exp. 35.) To the third lizard was given a dose of 3 mlgm. per kilo, but beyond exaggerated respiratory movements it displayed no symptoms, and was quite normal on the day following the injection.

Lizards are therefore affected in the same way as other animals. The lethal dose is between 3 and 4 mlgm. per kilogram.

4. Action on Amphibia.

The frogs experimented upon belong to the species Hyla aurea. This is not the native frog of New Zealand, but has been introduced from Aus-

that received 10 mlgm. per kilo, one died, and both the others showed convulsions and were very ill for more than twenty-four hours. Severe symptoms were also present with 9 mlgm. per kilo. It was noticed that frogs experimented upon in the winter were more susceptible to the poison than those used in the summer.

The relatively high lethal dose of tutin in frogs may have some connection with their mode of respiration. To a large extent this is cutaneous, and therefore a drug such as tutin, which owes its lethal power largely to its influence on the respiratory centre, might a priori be expected to be less lethal to these animals than to those that have only a lung-respiration. The fact that lizards, which have a dry skin and presumably only a slight cutaneous respiration, succumb to about one-third the lethal dose for frogs points in this direction.

Even with large doses the course of events in frogs is comparatively slow. For example, in Exp. 54, where about 60 mlgm. per kilo was given, the animal lived for an hour and a half.

5. Action on Fishes.

(b.) Comparison of Action of Tutin and Picrotoxin.

Sollmann's statement that 0.001 per cent. picrotoxin causes death in four to nine hours was tested by the improved method (see Table V, No. 85). Three fish were put into 1,000 c.c. of 0.001 picrotoxin at 11 a.m. Up to 8.30 p.m. no symptoms were seen, though the fish were under observation all day. At 8.30 they began to swim more frequently near the surface, and when seen next day one was dead, but the others were unaffected, and were returned to the trough at the end of twenty-four hours. A 0.001 per cent. of tutin (Exp. 83) showed almost similar results. Previous to this an experiment where 0.00075 per cent. and 0.0005 per cent. picrotoxin and the same strengths of tutin were tested gave doubtful results, because the amount of fluid was too small (Table V, Nos. 81, 82, 78, 79).

In other experiments picrotoxin was compared with tutin in a strength of 0.004 per cent. All the picrotoxin fish died—two at twelve hours and one at 29 ½ hours (Exp. 100); while of the tutin fish two died—at twelve hours and at eight hours and a half—and one recovered (Exp. 95). With a 0.005-per-cent. solution of picrotoxin all three fish died—viz., at 3 ½ hours, at 11 ¾ hours, and at 12 ⅔ hours (Exp. 107); while tutin of the same strength caused death to all at 22 ¼ hours and at twenty-three hours (Exp. 106): so it would appear that picrotoxin is more lethal than tutin in equal percentages.

near the surface and emitting air-bells, while at this time the other two fish showed quivering movements of the trunk. In two hours and three-quarters the small fish was dead. In three hours the larger one was dead, and the medium-sized one died in five hours. So that 0.0132 per cent. picrotoxin killed all the fish in five hours. Meanwhile the fish in the equri-molecular solution of tutin began to show symptoms at the end of three hours and three-quarters, which continued till late at night—twelve hours after the experiment was begun. The next morning two were found dead (between twelve and twenty-two hours after the experiment was begun), and the last one (medium-sized) died at twenty-four hours (Exp. 106). So the result shows that in equimolecular solutions picrotoxin is more lethal than tutin.

One must be cautious, however, in applying these results to the case of mammals. In fishes the swim-bladder regulation is disturbed both by picrotoxin and by tutin, and of the two, picrotoxin seems to have the greater influence, for air-bells are more frequently seen to be emitted by fish subjected to the action of this drug. The swim-bladder—of such paramount importance in the case of fishes—has no analogue in mammals, and it is therefore quite probable that the lethal power of these poisons may be reversed in this class of animals. Indeed, the experiments on cats (Exps. 4, 168, 169, 171) justify this statement.

(c.) Effect on Fishes of Tutin Solution that has been hydrolysed.

It has been a constant observation in the case of both lower animals and of human beings that tutu poisoning is more lethal when the stomach is empty. This suggested the possibility of the toxicity of the tutin being increased by the action of the hydrochloric acid in the stomach. Experiments on fishes were therefore undertaken to test this. To 8 or 10 c.c. of a 0.5-per-cent. solution of tutin an equal amount of 0.4 per cent. hydrochloric acid was added, and the mixed fluid kept at a temperature of 37° C. for different lengths of time in different experiments. It was then exactly neutralised with a solution of soda of corresponding strength, and made up to 1,000 c.c.

In the first experiment (Exp. 92) of this kind 7 c.c. of tutin solution was so treated (0.0035 per cent. tutin), and a control (Exp. 91) of a solution of untreated tutin in corresponding strength was used. In the hydrolysed tutin solution one fish showed symptoms at three hours and three-quarters, which continued till it died, at seven hours and a half. Another began to show symptoms at eight hours and one-third, and died between ten and twenty-two hours; while the third showed symptoms at nine hours, and died at 22 ½ hours. In the control of untreated solution one fish showed symptoms at four hours and died at four hours and a half, and the other two, of which one showed symptoms from the seventh to the tenth hour, recovered.

Another experiment of the same kind was then made, a slightly higher percentage of tutin being used—viz., 0.004 instead of 0.0035. Two testtubes were taken, each containing 8 c.c. of a 0.5 per cent. solution of tutin. To one, A (Exp. 94), 8 c.c. of 0.4 per cent. HCl were added; so that the total percentage of HCl in the fluid was 0.2. This mixture was kept at 37° C. for one hour, then neutralised and made up to 1,000 c.c. To the 8 c.c. tutin solution in the other test-tube B (Exp. 95) the same amount of HCl was added, but it was immediately neutralised, and then kept at 37° C. for one hour, and made up to 1,000 c.c. In each of these two solutions

symptoms at the end of five hours, and died in 25 ⅓ hours; a second showed symptoms at the end of six hours and a quarter, and died at twenty-eight hours; and the third showed slight symptoms at the end of eight hours, and recovered. The fluid was filtered, and three fresh fishes introduced, and of these one died within sixteen hours, one at 22 ½ hours, and one recovered (Exp. 97).

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A third experiment (Exp. 111) was made with 0.005 per cent. tutin in the same way. Symptoms appeared in two of the fish at five hours and a third; these died at eight hours and a sixth and nine hours and a sixth respectively. The third showed symptoms at six hours and two-thirds, and died at 13 5/6 hours. Usually fish live in a 0.005-per-cent. solution of tutin for an average of seventeen hours.

To summarise the results of these three experiments: Twelve fish were subjected to the influence of hydrolysed tutin (0.0035 per cent. to 0.005 per cent.). Of these, ten died at an average period of fourteen hours; the other two showed symptoms, but recovered. In the corresponding control solutions, of the twelve fish, nine died at an average period of 16.9 hours; two showed symptoms, and recovered; and one showed no symptoms. It would seem to follow that hydrolysed tutin is more toxic than tutin itself; but the rise in toxicity is not great, and in marked contrast to the action which takes place in dilute alkali at corresponding strength.

(d.) Action of Alkalis on Tutin.

Attention having been arrested by the fact that animals poisoned by tutin are frequently reduced to a state of coma, which may possibly contribute towards a fatal termination, the use of alkalis as a remedial agent suggested itself. Hughes had found that lime destroyed the activity of the tutu poison, and Easterfield and Aston were unable to recover tutin after a solution treated with lime had been evaporated to dryness.

The experiments were done in the same way as in the case of HCl: 10 c.c. of a 0.5-per-cent. solution of tutin was rendered alkaline with the solution

6. Action on Insects.

Insects are susceptible to the action of the poison. Weak solutions (0.1 per cent.) have no influence. A number of flies lived in perfect health

for a week on a solution of cane sugar in 0.1-per-cent. solution tutin (Exp. 115); but a 0.5-per-cent. solution quickly produced symptoms, and caused death in half an hour or so (Exp. 116).

The symptoms were very like those that characterize the action of the poison in higher animals—namely, an initial lethargy, followed by isolated tonic spasms of the wings and legs, exactly resembling the appearance met with in pigeons in the early stages of poisoning, when one or other wing is extended in rigid spasm for a moment; then marked clonic spasm of the wings recurring at intervals, and finally general convulsions, in which the insect tossed and buzzed about until exhausted. In the latest stages the flies lay on their backs, showing only occasional movements of the legs. After death, distention of the abdomen was an invariable finding, and in the case of a blowfly the exudation of a considerable drop of clear fluid from the proboscis occurred.

7. Action on Larvæ.

(Exp. 117.) The effect of the poison on maggots was tested by placing some mince that had become infected by blowflies in solutions of tutin of different strength for one hour, and then draining off the fluid. The experiment was controlled by a portion of the mince being placed in normal saline for the same period. On the day following, the maggots in the control had hatched out, and were very active; but in the mince subjected to the action of a 0.5-per-cent. solution tutin no movement was seen, and only one or two larvæ were found moving in the mince that had been soaked in tutin solutions of lesser strength.

Experiments that were made on full-grown maggots showed that confinement for forty-eight hours in tutin solutions of strengths graded up to 0.05 per cent. had apparently no influence. As immersion in a 0.05-per-cent. solution is about equivalent to a dose of 500 mlgm. per kilo, or 0.5 gram, tutin is seen to be very inactive.

8. Action on Molluscs.

(Exp. 114.) Two cockles of about the same size were placed one in sea-water and the other in a 0.5-per-cent. solution of tutin in sea-water, freshly prepared. Equal volumes of fluid were allowed. The cockle that had been placed in the tutin solution, in five minutes opened its shell and extruded its body. If touched it immediately withdrew, but came out again at once. It kept opening and closing its shell, but did not withdraw its body as the valves came together. It continued like this for twenty-four hours, responding more sluggishly to a touch as time went on, and then died. The control meanwhile had displayed no symptoms, but behaved in exactly the way the other had done when it, in turn, was placed in the tutin solution, and it also died.

9. Action on Infusoria and Amœbæ.

The action on infusoria and amœbæ was examined in the following way:—

A drop of hay-infusion, containing infusoria, amœbæ, monads, and bacteria, was placed on a slide; a drop of 0.1-per-cent. solution tutin was added, and the specimen observed for an hour. A drop of the infusion to which nothing had been added was used as a control. Another drop to which a drop of normal saline had been added was used to test the influence

of the saline in which the tutin was dissolved; and a fourth drop, to which a drop of a 0.1-per-cent. solution of quinine-sulphate had been added, was used for the sake of comparison (Exp. 118). In fifteen minutes no paramoecia were found moving in the quinine preparation, but monads were still in motion. In the three other specimens no change was observed. In thirty minutes all movement had ceased in the quinine preparation. In the tutin preparation paramoecia could still be seen moving, but their movements seemed to be somewhat irregular; amoebae, bacteria, and monads were still moving. In one hour one or two paramoocia could still be seen moving in the tutin preparation; they were by no means so numerous as at first, and rather difficult to find. Many of the monads were as active as ever, and amoebae were still throwing out pseudopods; but the preparation did not present the same appearance of general activity that it did at first. The saline preparations and the control were as active at the end of the experiment as they were at the beginning.

A further experiment (Exp. 119) was made by covering a drop of hay-infusion with a cover-glass, and then placing a drop of 0.1-per-cent. tutin solution on the edge of the cover. It was observed that such paramoecia as swam out into the drop of tutin solution remained in that drop, displayed irregular movement, and in a few minutes came to a standstill, and appeared to undergo internal disintegration, which reduced them to unrecognisable masses. The experiment was repeated with a drop of a 0.5-per-cent. solution tutin in distilled water, with similar results (Exp. 120).

It would appear, therefore, that paramoecia are injuriously affected by the poison, but not in so great a degree as they are by quinine. On amoebae, dilute solutions (about 0.05 per cent.) have very little influence. Monads are affected in a lesser degree than paramoecia.

10. Action on Bacteria.

(Exp. 121.) This was tested by placing pieces of fresh mince in test-tubes containing solutions of tutin of different strength (0.1 to 0.5 per cent.) and a piece in a test-tube containing normal saline alone. The tubes were left open to the air for twenty-four hours, and then corked and placed in an incubator at 37° C. Two days later the tubes were examined, and it was found that the tube containing saline was very offensive, but the tubes containing tutin solution were not at all offensive, and smelt rather like stomach-contents.

When subjected to microscopic examination, it was found that all the solutions contained bacteria, but in the slide prepared from the normal saline the bacteria were very much more numerous, and showed a greater variety of form and size. It would appear, therefore, that tutin has a deleterious action on certain forms of bacteria, but not on all.

That some forms of bacteria are certainly not affected by tutin is shown by the following observation: A 0.5-per-cent. solution of tutin in normal saline which had been allowed to stand for six months in the laboratory was examined microscopically, and found to contain fairly numerous large motile bacilli resembling B. subtilis. A green growth that had developed and formed a layer at the bottom of the bottle was found to consist of small rounded yeast-like cells tinged with chlorophyl.

11. Action on Yeast.

The action of tutin on the fermentation of glucose by yeast was studied in a few experiments (Exps. 122, 123, 124). It was found that fermentation

proceeded vigorously in the presence of even 0.4 per cent. tutin. Some slight differences in the rate of fermentation that were noted were probably due to differences in the amount of yeast added. (For details of experiments, see protocols, Exps. 122, 123, 124.)

12. Germination of Seeds.

The action of tutin on the germination of seeds was tested by soaking mustard-seeds in solutions (in normal saline) of various strengths (0.1, 0.2, 0.3, 0.4, and 0.5 per cent.) for twenty-four hours (Exp. 125). The seeds were then sown on pieces of felt in separate tin boxes, appropriately labelled, and kept moist with water. An equal number of seeds was sown on each piece of felt, and the experiment was controlled by seeds that had been soaked for the same length of time in normal saline. The day following, all the seeds had germinated, and they were therefore replaced in their respective tutin solutions for a further period of twenty-four hours, and then sown again. During the next week the progress made by the seedlings was observed, and the effect of the tutin on their development noted.

It was found that the seeds progressed more or less in order of the weakness of the solutions in which they had been soaked: thus, the growth of those that had been soaked in normal saline, in 0.1 per cent., and in 0.2 per cent. tutin was the most vigorous, while those that had been soaked in 0.3, 0.4, and 0.5 per cent made slow progress, and all the seedlings in these lots did not continue to grow. In a fortnight, however, little difference was to be noted in any of the seedlings that had developed at all, one or two of the 0.5-per-cent. seedlings that were growing being as well developed as those that had been soaked in saline.

This result may possibly be due to the effect previously noted on bacteria certain forms of bacteria being necessary to the growth of plants. From experiments on invertebrates, bacteria, &c., it may be inferred that tutin is toxic to any given form of life in proportion to its complexity of organization. Wherever a nervous system is sufficiently developed, the toxic action is greatest; but that it has some injurious influence on primitive protoplasm may also be seen from its effects on paramoecia, bacteria, &c.

13. Action of Tutin on Tissues.

were not used, in order to allow of ready interchange of O₂ and CO₂. The results are shown in Table VIII (Exps. 126 to 134 inclusive).

(Exp. 134.) The rate of progress of a small fragment of cork along the surface of the gullet was noted before and after applying tutin (0.3 per cent. in saline). Average rate before = 50.4″ for 1 cm. travelled; average rate after = 34.8″ for 1 cm. travelled. The method presented many difficulties, as it is impossible to keep the fragment moving on the same line each time, and the mucus secreted clogs its movements.

From these results it is impossible to draw a definite conclusion, for although in some specimens taken from sea-water animals the ciliary action ceased on the application of tutin, in other specimens from the frog's gullet tutin had little or no action.

(c.) Action of Tutin on Striped Muscle and Nerve-terminations.

This was tested by determining the strength of stimulus just sufficient to cause contraction of a nerve-muscle preparation before and after the application of tutin. The preparation (gastrocnemius - sciatic) was made from the frog. An induction-coil with one Daniell cell and mercury-key was used. The muscle in each case was placed first in normal saline (0.75 per cent.) and the strength of the minimal stimulus determined. The tutin solution was then substituted for the saline, and the excitability tested from time to time.

Four experiments were made (Exps. 135 to 138 inclusive). In three of these the one muscle was placed in 0.025, 0.05, and in 0.5 per cent. tutin solution, while the other muscle was placed in normal saline. In another experiment the muscle of one preparation was placed in 0.5-per-cent. solution tutin, and the nerve of the other in 0.5-per-cent. tutin. (Exp. 136).

14. Action on the Different Systems.

given hypodermically. But in ordinary cases of poisoning by the tutu plant there may be irritation of the gastric mucosa as well. Christie's assertion (11) that tutin is excreted by the stomach, and so causes vomiting by local irritation, is unsupported. No tutin could be extracted from the gastric and small-intestine contents of a rabbit which had been killed by intravenous injections of large doses of tutin during a blood-pressure experiment.

Stomach.—In cases of herbivora poisoned by tutu, accumulation of gas in the stomach is a marked feature. This is probably due to the rapid fermentation of the leaves ingested, though possibly it may be due, in part, to air swallowed during the convulsions.

To test the fermentability of tutu-leaves, a mash of minced leaves with a little water was placed in an incubator at 40° C., and examined after the lapse of an hour. On stirring the mixture it was seen to be permeated with bubbles of gas, but it was not determined how far this was due to the expansion of imprisoned air.

In the experiments with the pure substance, distension by gas was not observed. Pure tutin and artificial gastric juice when incubated in a fermentation-tube gave no appearance of gas-development.

The action of hydrochloric acid in hydrolysing tutin has already been referred to under “Fishes.” It was found that the toxicity was slightly increased.

Intestines.—The action on peristaltic movement was not investigated experimentally, but from observations made on animals killed by tutin, where the abdomen was opened immediately, it was clear that there was no diminution of peristaltic movement. That it was even increased was inferred from the frequency with which defaecation occurred. The stools were often loose, and were expelled with some violence.

(b.) Haemopoietic System.

In one experiment, where a rabbit received a fatal dose of tutin and died in one hour, blood-films were taken before the injection of tutin and just before death (Exp. 139). The only point of difference seemed to be that the leucocytes appeared fewer in number after tutin. They seemed to be quite normal in regard to granules and staining-power.

(c.) Circulatory and Respiratory Systems.

Previous observers have noticed that the tutu poison has no depressing influence upon the circulation. Christie (11) found that the heart continued beating after the respiration had ceased. In one of his experiments (on a cat) he opened the thorax immediately on the cessation of all movement, and observed the heart to beat for twenty-two minutes after the last respiratory gasp. In his own case, where he took 9 grains of an extract made from the leaves, the pulse-rate rose to 102, and this observation led him to conclude that the action of the poison was to accelerate the beat of the heart. In attempting to bleed poisoned animals by opening veins and by slitting the ears he found that the blood did not run readily, and concluded that the arterioles were contracted. He noticed, also, what had been observed previously in cases of accidental poisoning—that at first the respirations were increased in frequency and in force, and that later they became feeble and irregular, and finally ceased before the heart stopped beating.

Breathing of the Cheyne-Stokes type occurred in some experiments, the climax of the period of respiratory activity corresponding to the highest point of the Traube-Hering curve, which appears simultaneously.

(d.) Urinary System.

Micturition was a marked feature in cats poisoned by tutin. It occurred voluntarily several times before convulsions occurred, and during the convulsions (and this is true also of rabbits and guinea-pigs) involuntary discharges of urine occurred. In the majority of cases the bladder was found distended after death, even where a free discharge of urine had taken place during the convulsions. This points to an increased secretion of urine. Attempts were made to estimate the rate of flow in anaesthetised animals (Exps. 144, 145), but without satisfactory results.

This increased secretion pointed to the possibility of tutin being eliminated in the urine, and so an attempt was made to recover tutin from the urine of poisoned animals. The urine was collected by incising the bladder after death. It was evaporated to dryness and exhausted with ether, but no crystals of tutin could be detected on evaporating off the ether. The residue from the ethereal solution was in one case dissolved in saline and injected under the skin of a frog (Exp. 151), but with negative results. The urine was in several cases examined for abnormal constituents, but none was found.

(e.) Genital System.

Many of the animals experimented upon were pregnant females in different stages of pregnancy, but in no case was abortion observed, although the animal was under the action of the poison long enough for it to occur.

(f.) Nervous System.

The symptoms that appear in animals poisoned by tutin point clearly to the central nervous system as the part of the body that is specially affected by the action of the poison. The two cardinal symptoms are convulsions and a dulling or blunting of the mental faculties that in the early stage makes the animal appear dazed and stupid, and in the later stages passes into actual coma.

Convulsions.—The convulsions are obviously of central origin. They bear a close resemblance to an ordinary epileptic fit, which is generally held to originate in the cortex. Whether the tutin convulsions originate in the cortex or not, it is certain that the nerve-cells in the basal ganglia, pons, medulla, and cord are profoundly affected. This is shown by the action on the vomiting centre, respiratory centre, and cells of the cord. Among the first symptoms to appear are twitching of the ears, blinking of the eyes, and movements of the lips. These are followed by jerking of the head and movements of the fore and hind limbs. These movements may be attributed to irritation of the cells of either the upper or the lower motor neurones. In order to determine more accurately the site of action the following experiment was made:—

(Exp. 152.) The right cerebral hemisphere was exposed in a cat and a large dose of tutin injected hypodermically. When general convulsive movements had developed, the right cerebral hemisphere was removed. Now, had the convulsions been cortical in origin, one would expect them to have ceased on the left side of the body; but they continued as before,

From the examination of sections of the cerebrum, medulla, and cord there is evidence of great congestion of these parts in tutin poisoning. The central nervous system was examined in several cases after death, and congestion of the membranes was found in all. In one case the grey matter of the cord when seen on section was distinctly reddish. Pieces of cord, medulla, and cerebrum were fixed in 8 per cent, formol, and sections were made and stained by Nissl's method, and also by Muir's eosin and methyline-blue method. On microscopic examination the Nissl granules showed no obvious change, but there was very evident congestion, shown especially well by Muir's method. The capillaries and small vessels seemed more numerous than normal, because they were rendered visible by being crowded with red blood-corpuscles. At several places there were collections of corpuscles, pointing to small extravasations of blood. These changes were present in the grey matter of the cord, medulla, and cerebral cortex. The appearance at once suggested that more or less permanent damage would have resulted had the animal lived. It has been observed in cases of tutu poisoning that sometimes the victim does not completely recover, a permanent mental alienation remaining as an after-effect.

Lauder Lindsay (3), in one case of poisoning he records, in which convulsions were a prominent symptom, notes that the subject never completely recovered, “there remaining to this day a peculiar form of nervous irritability not observable prior to this toot-poisoning.” The present writer has knowledge of another case, although, unfortunately, no authentic details are available, where two children were poisoned by tutu. They were both very seriously ill, and one died. The other recovered, but incompletely, mental enfeeblement and a squint remaining as after-effects. Sequelæ of these kinds may possibly be explained as resulting from permanent damage done to the nervous tissue by the extreme congestion and the small hæmorrhages which occur in the cerebral cortex as well as through-out the whole grey matter of the central nervous system.

Sympathetic Nervous System.—This was not specially examined by experimental methods, but that the nerve-cells here were also affected was shown by the viscid character of the saliva secreted by cats, by the dilatation of the pupil that occurred during convulsions, and by the erection of the hairs of the tail.

In short, it is probable that tutin affects every kind of nerve-cell; the fact that the medulla is seen to be specially affected being due to the sensitiveness of the cells in that region.

Effect on the Pupil and Conjunctiva.—When dropped into the eye of a rabbit, tutin solutions cause no local irritation and no change in the size of the pupil (Exps. 160, 161). The same holds good for the cat (Exp. 162) and for the excised eye of the frog (Exp. 163). During convulsions in the cat and in pigeons dilatation is well marked.

The action of coriamyrtin was studied in the same way, because it is said by Riban (15) to cause contraction of the pupil in rabbits when applied locally. It was found to cause contraction in the excised eye-ball of the frog, but the results of applying it to the eye of a rabbit and of a cat were negative (Exps. 164, 165).

(g.) Action of Tutin on General Nutrition.

It is not an uncommon opinion among farmers that animals that eat the plant in moderation thrive well on land where tutu abounds. To test whether tutin had any injurious influence on general nutrition, a young

15. Fate Of Tutin In The Body.

It is impossible to trace in the body the fate of a substance which, like tutin, contains only C, H. and O; but that the tutin is not rapidly destroyed or eliminated was shown by the following experiment (Exp. 13): A guinea-pig was given by mouth on alternate days a dose of tutin which was near the minimum lethal dose; thus, 1-5 mlgm. per kilo was given on the 17th, on the 19th, and on the 22nd February. No symptoms appeared, so the animal was able either to oxidize or to excrete this amount (1.5 mlgm. per kilo) in two days. On the 24th it received 2 mlgm. per kilo, and a similar dose was given on the 26th. On the 27th it was found dead. From this it appears that a guinea-pig is unable to dispose of 2 mlgm. per kilo within two days. Enough tutin must have been still present in the body on the afternoon of the 26th, when the second dose of 2 mlgm. per kilo was given, to raise the total amount present to the lethal dose.

16. Immunity Or Tolerance.

Several experiments were made with the object of determining whether tolerance of the effects of the poison can be established.

A pigeon (Exp. 15) that had been subjected to gradually increasing doses by mouth over a period of ten days withstood 16 mlgm. per kilo. The minimum lethal dose in pigeons by oral administration is about 10.25 mlgm. per kilo. A second pigeon (Exp. 17), treated in the same way for over three weeks, succumbed to 12 mlgm. per kilo. A guinea-pig (Exp. 167), which was being treated in the same way, and had received several small doses, was killed by a dose of 7.5 mlgm. per kilo given by mistake, so that no great degree of tolerance had been established. A rabbit, already referred to above when considering the effects on general metabolism, received small doses, gradually increasing to 7 mlgm. per kilo, over a period of two months. A dose of 8 mlgm. per kilo then caused severe symptoms; so that it would appear that a slight degree of tolerance had developed, for the lethal dose, by oral administration, in rabbits is about 7 mlgm. per kilo. Five days later this rabbit succumbed to a dose of 11.6 mlgm. per kilo per os.

The general conclusion reached was that tolerance can be established only in a very slight degree, if at all.

(Exp. 172.) Twelve mlgm. per kilo proved fatal to a frog. It displayed the symptoms seen in tutin poisoning. The time of death could not be noted, so no strict comparison can be made with tutin. The minimum lethal dose of tutin in frogs is between 10 and 11 mlgm.

On the whole, the impression left on one's mind is that there is comparatively little difference in the toxic power of the two substances; but the mental effects seemed more marked in the case of the cat poisoned by tutin.

18. Action Of Remedies.

Although not strictly within the scope of the title, of this paper, notes of some experiments made to show the influence of remedial measures will be included here. These experiments were made from time to time while the physiological action of tutin was being examined, and before the experiments on the action of alkalis on tutin had been undertaken. They are necessarily, therefore, incomplete; but some points have been investigated and some observations made which it is hoped may prove of value in the treatment of cases of poisoning.

In tutu poisoning various remedies have been suggested from time to time, and the rationale of some of these is difficult to understand. With shepherds, bleeding is a favourite method of treatment. It is usually done by slashing the ears or tail, or by incising the roof of the mouth. It is said to be of special advantage in young sheep, but in older sheep it is regarded by some as being dangerous, and as tending rather to hasten the end than to promote recovery. Carbonate of ammonia is also used, a lump about the size of a walnut being dissolved in water and poured down the animal's throat.

In 1870 Hughes advocated the use of lime as an antidote. He was led to do this from the observation he had made that lime destroyed the activity of the poison. Cases in human beings have been treated with lime, and, it as said, successfully.

The Maoris depended largely upon partial asphyxiation as a means of treatment. This was effected either by holding the patient under water till he was nearly drowned, and repeating the immersion as soon as he showed signs of returning life, or by suspending him head downwards over the smoke of a fire. Another method (31) was to bury the patient in the ground up to the neck, apparently with the object of restraining the convulsive movements.

Professor Marshall (32), in a report made to the Agricultural Department of the New Zealand Government, recommends bleeding and the intravenous injection of chloral-hydrate. In connection with the use of chloral-hydrate it may be noted that Crichton Brown (34) states that he was able, by the administration of chloral-hydrate, to prevent death in a rabbit which had received five times the minimum lethal dose of picrotoxin.

It will be noticed that in a rabbit (Exp. 174) which received a lethal dose (3 mlgm. per kilo hypodermically) death was prevented by 0.6 gram of chloral-hydrate. It should be stated that the chloral was given first by the rectum, and the tutin administered hypodermically as soon as the anæsthetic effect of the chloral was established, so that every chance was given to the action of the chloral. In two rabbits where 4 mlgm. of tutin per kilo was given (Exps. 173, 175) death occurred. The one received 1 gram of chloral per rectum in one dose, and the tutin hypodermically immediately afterwards; it died in six hours, in tutin convulsions. The

diminished by the absorption of the CO₂, which no doubt forms part of the mixture of gases present, so the distension that so frequently occurs in these cases would be diminished.

The comatose condition which forms part of the symptoms of tutin poisoning suggested the possibility of some form of acid poisoning being present, and, if this be so, the injection of alkalis should prove of benefit-Sodium-carbonate was tried intravenously in one of the blood-pressure-experiments, but no apparent effect was observed. In another case sodium-carbonate was injected into the rectum of a rabbit that seemed likely to die of tutin poisoning, but here also it was ineffective. So that for the present one can only say that alkali would render unabsorbed tutin non-toxic.

19. General Summary.

1. Investigations made on the action of the pure principle tutin (C₁₇H₂₀O₇) confirmed the results of previous observers that it is in itself sufficient to account for the main bulk, if not the whole, of the symptoms of poisoning by the tutu plant.

2. These symptoms, as they occur in cats, have been fully described, and the differences which appear in other animals noted.

3. Tutin, or its metabolic products, acts mainly on nerve-cells, producing first increased excitability and then exhaustion. It specially affects the cells of the respiratory centre, causing increased rate and depth of respiration.

4. Death may occur during the phase of increased excitability (asphyxia during convulsions) or in the phase of exhaustion. Various reflex acts—vomiting, defæcation, micturition—may occur during the stage of increased excitability. A comatose condition, possibly due to exhaustion of the cells of the cerebral cortex, is a marked feature in proportion to the strength of the dose. It deepens as death approaches. Small hæmorrhages into and congestion of the grey matter of the brain and cord are marked features in fatal cases. The Nissl granules seem unchanged when death occurs in a short time.

5. In strong solutions tutin has a slight deleterious action on tissues less highly specialised than nerve tissue—e.g., ciliated epithelium and muscle. It retards the growth of some forms of bacteria, and injuriously affects paramœcia and other low forms of life in relatively strong solutions.

6. The symptoms of poisoning by tutin are in a general way similar in widely different forms of life (flies, pigeons, cats, &c.), and can all be referred to an action on nerve-cells.

7. The minimum lethal dose in milligrams per kilo of body-weight for different classes of animals is as follows:—

8. The effects on the various systems can all be referred to the influence on their nerve mechanisms—e.g.: Alimentary system, salivation, vomiting (on hypodermic injection). The circulatory system is not injuriously affected

by tutin. Cardiac inhibition does not occur, and the heart beats forcibly up to the time of death. Respiration is quickened and deepened. The pupil is not affected by local application, but dilates during the tutin, fit. General metabolism is not affected.

9. From experiments on birds and rabbits, some slight “degree of tolerance seems to be acquired. The natural relative immunity of birds is discussed.

10. Accumulation of the drug, or of its effects, may occur. Thus a guinea-pig was found to be unable to dispose of 2 mlgm. per kilo per os administered every second day.

11. The toxic action of tutin was compared with that of other members of the picrotoxin group. It was found to be, more toxic, than the sample of picrotoxin employed. The action of coriamyrtin was found to be very similar to that of tutin.

12. Attempts were made to antagonize the action, of tutin with chloral- hydrate and other drugs, with a slight degree of success.

Attention is drawn to the powerful action of weak alkalis on tutin. The toxic power of tutin is completely destroyed by 0.2 per cent. sodium-hydrate acting upon it for five minutes at 37° C., and possibly the action of weaker alkalis—e.g., lime and magnesia—would be equally destructive.

The suggestion is made that weak alkali should be used to destroy the tutin in the stomach in the case of stock poisoned by eating the tutu plant.

I may state here that the bulk of the work was done in the physiological laboratory of the Otago University, and I gladly take the opportunity of acknowledging my great indebtedness to Professor Malcolm, both for this privilege and for the essential aid he has afforded me by advice and criticism. I wish also to acknowledge my obligation to Mr. Aston for supplies of tutin and coriamyrtin, and for several references; to Dr. Hocken, for the use of his invaluable library; to Professor Benham, for the identification of specimens; to Mr. Deans, of the Acclimatisation Society, for supplies of trout-fry; and to Nurse Stronach, of the Infectious Diseases Hospital, Lake Logan, for supplies of minnows.

Protocols.
Exp. 1.

Temperature in rectum, 102° Fahr:

P.M.—Rigor mortis extremely well marked; no sign of fluid at point of injection; blood very dark and fluid.

Abdomen: Right horn of uterus contains one fœtus nearly full size, which looks as if it had died in spasm; one hind leg twisted over the other; right paw behind right ear; claws extruded.

Gall-bladder contained bile. No obvious abnormalities.

Thorax: Great veins and right side of heart distended with blood; left side contained some dark blood; lungs showed small hæmorrhages, as also did the thymus.

Brain and cord: The membranes seemed somewhat injected; grey matter of cord appeared distinctly pinkish to the naked eye.

Microscopic: The cord and medulla were hardened in 8 per cent, formol, and carried through into paraffin, and sections cut. The sections were stained by Nissl's method with toluidin blue. On examination the granules were found to be present and normal in appearance. A marked feature was the congestion of the grey matter. Capillaries can be seen close up to the nerve-cells quite full of corpuscles. At other places the collections of corpuscles suggest that minute hæmorrhages had occurred. The cord, medulla, and cerebrum show these appearances, but the congestion of the medulla is the most marked.

Exp. 2.

Exp. 3.

Exp. 4.

Exp. 5.

Exp. 6.

Exp. 7.

Exp. 8.

Exp. 9.

Exp. 10.

Exp. 11, 12.

Two guinea-pigs, A and B. Weight of A, 624 grains (Exp. 11); weight of B, 684 grams (Exp. 12). To A, gave 2 mlgm. per kilo body-weight; to B, gave 3, mlgm. per kilo body-weight.

Result.—A killed in 70 minutes by 2 mlgm. per kilo; B in 35 minutes by 3 mlgm. per kilo.

Exp. 13.

Guinea-pig. Weight, 751 grams.

Result.—Was able to eliminate 1.5 mlgm. per kilo given, every second day, but unable to eliminate 2 mlgm. per kilo in two days.

In this experiment the influence of the prolonged administration of tutin on the weight of the animal was tested by keeping a second guinea-pig under similar conditions as a control. The weights of the two animals as taken during the course of the experiment were as follows:—

Result.—Control gained 38 grams; tutin animal gained 16 gram.

Exp. 14.

P.M. (3.30).—Rigor well marked; abdomen opened; some grass observed in stomach and intestines, but no great accumulation of gas.

Exp. 15.

P.M.—Crop noted to be empty; nothing abnormal detected.

Result.—16 mlgm. per kilo non-lethal; 20 mlgm. per kilo killed in one hour.

Exp. 16

P.M.—Crop opened; found empty, except for a small amount of grumous fluid containing small yellow particles. Sour smell, and distinct acid reaction. No abnormality observed.

Result.—Death in 16 minutes from 17 mlgm. per kilo.

Exp. 17.

Result—No tolerance developed. Death from a dose of 12 mlgm. per kilo.

Exp. 18.

Result.—Recovery from 15 mlgm. per kilo.

Exp. 19.

P.M.—Crop half-full. No abnormality detected.

Result.—Death in 9 minutes from a dose of about 16 mlgm. per kilo.

Exp. 20.

P.M.—Crop full of half-digested food. No abnormalities observed.

Result.—Death in 5 minutes from a dose of 16 mlgm. per kilo.

Exp. 21.

P.M.—Crop empty. No abnormality observed.

Result.—Killed in 35 minutes by a dose of 15 mlgm. per kilo.

Exp. 22.

P.M.—Crop half-full. No abnormality observed.

Result.—Death in 72 minutes from 15 mlgm, per kilo. Life evidently prolonged by the chloral.

Exp. 23.

Result.—Death in 19 minutes from 13 mlgm. per kilo.

Exp. 24.

Result.—Death in 136 minutes from a dose of 12 mlgm. per-kilo.

Exp. 25.

Result—Death in 46 minutes from a dose of 12 mlgm. per kilo.

Exp. 26.

Result.—Recovery from a dose of 9 mlgm. per kilo.

Exp. 27.

Result.—Recovery from a dose of 9.5 mlgm. per kilo.

Exp. 28.

Result.—Death in 45 minutes from a dose of 10 mlgm. per kilo.

Exp. 29.

Result.—Recovered from a dose of 10 mlgm. per kilo.

Exp. 30.

Exps. 31, 32. (See text.)
Exp. 33.

Exp. 34.

Exp. 35.

Exps. 36, 37, 38.

No sign of convulsion noticed in these frogs. The two that died seemed extremely thin and emaciated. Frog (1) looked very much thinner on the 17th than, on the two days before. The weather was cold on the 16th. On the 17th the temperature was 12° C. in the morning, but this does not explain why frog (3); with 3 mlgm. per kilo, seems unaffected. Loss of weight might be due to drying; but the air has not been dry. The weather is cold and wet.

Exps. 39, 40, 41.

Frogs (1) and (3) were apparently normal.

In the two experiments above, the number of minims in 1 c.c. was taken as 15; in the subsequent experiments as 17. The doses per kilo body-weight above are therefore smaller than as stated.

Exps. 42, 43.

Exp. 44.

Exp. 45.

Exp. 46.

Exp. 47.

Exp. 48.

Exps. 49, 50, 51.
Three frogs. Weights: (1),23 grams (Exp. 49); (2) 39 grama (Exp.50); (3), 30 grams
Exp.50

Exp. 52.
Frog. Weight, 40 grams.

Exp. 53.
Pigeon. Weight, 335 grams.

Exp. 54.
Frog. Weight, 20 grams.

Exp. 55.
Frog (female). Weight of frog, 38 grams; ovaries, 57 grams.

Exp. 56.
Frog (small).

Exp. 57.
Frog (small).

Exp. 58.
Frog (small). Weight, 12-5 grams.

Exp. 59

Exps. 60 to 76.

These experiments were very like the above (see Table III).

Table, of Experiments on Fishes.

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Control to the above fishes tinder the same conditions (200 c.c. fluid) showed symptoms of asphyxiation in 4 hours, and the fishes in tutin and picrotoxin died practically in order of their weights.

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Table of Experiments on Fishes—continued.

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Flinds 94 (A), 95 (B), and 96 (C) filtered and tried over again. (1.), (m.), (s.) = large, medium, small.

Exp. 108.
Three minnows (large, medium, and small).

Exp. 114.

Exp. 115.

Exp. 116.

Exp. 117.

Exp. 118.

A drop of hay-infusion, containing paramœcia, amœbae, monads, and, bacteria, was placed on each of four slides, labelled A, B, C, D.

Exp. 119.

Experiment repeated, using a 0.5-per-cent. solution tutin in distilled water, with similar results.

Exp. 120.

This experiment was essentially similar to Exp. 119.

Exp. 121.

Exp. 12.2.

Four fermentation-tubes were filled—(1) with glucose solution and tutin (0.25 per cent, of the latter); (2) glucose solution, with 0.1 per cent, tutin; (3) glucose solution alone; (4) water. A few drops of a yeast emulsion were added to each.

Exp. 123.

Exp. 124

Four fermentation-tubes filled with the following:—A: Water (16 c.c.), 25 per cent. glucose (4 c.c.); B: Water (20 c.c.); C: Water (8 c.c.), plus 0.5 per cent, tutin solution (8 c.c.) plus 25 per cent, glucose (4 c.c.) (equals 0.2 per cent, tutin in mixed fluid); D: 0.5 per cent, tutin (16 c.c.), plus glucose (4 c.c.) (equals 0.4 per cent, tutin in mixed fluid). A few drops of freshly procured emulsion of brewers' yeast added to each; D received rather more than the others.

This shows that the amount of yeast added exerts a greater influence on the amount of fermentation that occurs than does the presence of tutin.

Exp. 125.

Five test-tubes were taken, containing a 0.1-per-cent., a 0.2-per-cent., a 0.3-per-cent., a 0.4-per-cent., and. a 0.5-per-cent. solution tutin in normal saline; and one test-tube containing normal saline alone. Equal volumes of fluid in each test-tube.

Exp. 126.

A few bars were taken from the gill of a cockle, and mounted in sea-water, and observed under the low power.

Exp. 127.

Normal saline and afterwards sea-water were then perfused under cover-glass, but recovery did not take place.

Exp. 128.

A solution of 1/1000 KOH in sea-water perfused under cover-glass, and ciliary movement immediately resuscitated.

Exp. 129

As this piece of gill was rather large, a fresh specimen was taken from a mussel.

Exp. 130.

Exp. 131.

Two or three gill-bars from a small rock-oyster were taken and mounted in seawater under a cover-glass. Two preparations were made, and one used as a control. (Low power.)

Exp. 132.

A fresh preparation was made from the gill of a cockle, and examined with the low power without a cover-glass. A control was used.

Exp. 133.

Three preparations of ciliated epithelium from the gullet of a frog. No. 1 was mounted in normal saline without a cover-glass, and used as a control; No. 2 was mounted in a drop of a 0.1-per-cent. solution tutin, without a cover-glass: No. 3 was mounted in a drop of a 0.5-per-cent. solution tutin, without a coverglass. Three microscopes were used, and the specimens examined under the low power, with the draw-tube out.

Exp. 134.

The gullet of a frog was exposed, and the rate of progress along it (by ciliary action) of a small fragment of cork was observed. A centimeter scale was arranged to lie parallel with the gullet, and the time the cork took to travel 1 cm. noted as follows:—

Exp. 135.

Two frogs' nerve-muscle preparations made. Muscles placed in watch-glasses with normal saline. Nerve laid on frog-plate, and kept moist with saline, and stimulated with break shocks. A (m saline), minimal stimulus found to be with coil at 17 cm. B (in saline), minimal stimulus at 21.5 cm. B was then placed in a watch-glass cortaming a 0.025-per-cent. solution tutin in normal saline at 5.54 p.m.

Exp. 136.

In this experiment, the nerve of the one preparation (A) was placed in tutin solution (0.5 per cent.), and the muscle of the other (B) in the same. Before placing in tutin solution, A contracts at 28 cm., and B at 34 cm.

Exp. 137.

One muscle in saline (B) and one in a 0.05-per-cent, solution tutin (A). Nerves exposed to air, and kept moist. Stimulated as before. Before tutin applied. A contracts at 30.5 cm., and B at 25.5 cm.

Exp. 138.

Same as previous experiment. Before tutin (interrupted current), A contracts at 36 cm., and B at 32 cm.; single shocks, A contracts at 27 cm., and B at 25.5 cm.

Exp. 139.
Rabbit (young). Weight, 804 grams.

Blood-films were taken, dried in the air, and fixed with Jenner's stain; and next morning at—

P.M.—Stomach not distended; contained only food; blood-film taken from the left ventricle only; heart had ceased beating; lungs not congested; bladder full.

Result.—10 mlgm. per kilo caused death m less than one hour.

Exps. 140-151.

Experiments 140 to 151 inclusive referred to the blood-pressure work, details of which are omitted.

Exp. 152.

P.M.—The chest was opened at once, and slight heart-movements found to be still present. The bladder, notwithstanding the large quantities of urine ejected during the experiment, was full. The cerebral hemispheres were found to be entirely removed; the corpora quadrigemina were intact; the cord was found to be divided within the dura mater at the level of the 5th dorsal vertebra; the cord was crushed through rather than cut through (scissors, not too sharp, having been used to divide it), but its continuity was completely destroyed.

Exp. 153.

Exp. 154.

A frog (Hyla aurea), pithed in the ordinary way three hours previously, was suspended by the head. At intervals of a few minutes its, feet (as far as the ankle) were dipped into dilute sulphuric acid, of a strength of 1 in 1,000. After each dipping the feet were carefully washed in fresh water. The time was taken by a seconds-clock, and the result was as follows:—

6.3 minims of a 0.1-per-cent, solution tutin(11 mlgm. per kilo) was then injected, under the skin of the back, and after an interval of 30 minutes the feet were again dipped in the acid:—

Here a further dose of 10 minims was injected, and after an interval of 15 minutes the feet were dipped as before:—

Each, time the feet were withdrawn together, and, it was thought, more actively than before the tutin was injected.

Exp. 155.

The experiment was repeated with a second frog, pithed in the same way four hours previously. The result was as follows:—

10 minims of a 0.1-per-cent. solution tutin was then injected under the skin of the back, and after an interval of half an hour the reaction-time again tested. The time when movement first took place only was noted.

At 22 seconds there was a slight twitch, but no further movement took place, although 70 seconds were counted. Pinching or pricking a foot met with an immediate response, but the acid had no influence. Five minutes later no reflex movement could be elicited by any means, and the heart could no longer be seen beating through the chest-wall.

Frog A was then tested again, and found to respond as actively as before.

Exp. 156.

A third frog, pithed one hour previously, was treated in the same way.

2 minims of a 0.5-per-cent. solution was then injected under the skin of the back, and 10 minutes were allowed to elapse before the feet were dipped again.

1 minim of a 0.5-per-cent. solution tutin was then injected into the heart.

Exp. 157.

5 minims of a 0.1-per-cent. solution tutin (10 mlgm. per kilo) was then injected under the skin of the back, and six minutes later the test reapplied.

Exp. 158.

The experiment was repeated a fifth time. In this case the frog had been pithed in the usual way one hour previously, but it appeared to retain some power of voluntary movement, and was so restless that it was suspected that the cerebrum had not been completely destroyed. Extensive general movement followed each application of the acid.

1 minim of a 0.5 per-cent. solution tutin was then injected (about 12 mlgm. per kilo) under the skin of the back, and 15 minutes later the feet were again dipped into the acid; but before this was done a typical tutin convulsion occurred.

On examination the optic lobes were found to be intact.

Exp. 159.

The experiment was repeated with a sixth frog. This was beheaded a little in front of the anterior border of the tympanic membrane, so as to sever all in front of the top of the medulla.

1 minim of a 0.5-per-cent. solution was then injected under the skin of the back, and 8 minutes later the test reapplied.

As a result of letting the frog fall on the table, a typical tutin spasm, with croaking, occurred here. Seemed to require severe irritation to induce a spasm. A spasm occurred on striking sharply with a glass rod.

250 seconds were counted, but feet not withdrawn. Slight twitches occurred, however, at 58″, 71″, 86″, 94″, and 120″. 100 seconds more, but no effect.

Although the acid had ceased to have any influence, it was still possible to elicit reflex action on painful stimulation—e.g., pinching.

Ten minutes later reflexes could not be elicited in any way. The thorax was then opened, and the heart found to be still beating. The auricles were distended. Twitching occurred as the cord was being pithed.

Exp. 160.

A few drops of a solution of tutin were introduced into the right eye of a young rabbit. Within a few minutes there seemed to be a very slight dilatation. In an hour and a quarter the right pupil was thought to be slightly the larger by an observer ignorant of which eye had been subjected to the test, but the difference was so very slight, if existing at all, that the result was regarded as doubtful.

Exp. 161.

Four small drops of a 0.5-per-cent. solution tutin were introduced into the left conjunctival sac of a rabbit. It was observed for 15 minutes, and no change in the size of the pupil was noted. No hyperæmia of the conjunctiva resulted, nor did the conjunctiva become less sensitive.

This rabbit died twenty-four hours later, after exhibiting symptoms of tutin poisoning.

Exp. 162.

Exp. 164.

Exp. 165.

Exp. 166.
Rabbit. Weight, 777 grams.

Exp. 167.
Guinea-pig. Weight, 627 grams.

P.M.—Stomach distended with gas; intestines contain mixed gas and fluid contents; peritoneum much injected; appearance suggestive of general peritonitis.

Exp. 168.
Cat. Weight, 2.686 kilograms.

Exp. 169.

Exp. 170.
Cat. Weight, 2.699 kilograms.

This cat previously received 1 mlgm. tutin, and recovered; will now receive an equimolecular solution of picrotoxin, 887/336 × 1 mlgm. picrotoxin = 2.66 mlgm. picrotoxin = 18 minims of a 0.25-per-cent. solution picrotoxin.

Exp. 171.
Cat. Weight, 3 kilograms.

P.M.—Three hours afterwards. Membranes of cord and brain markedly congested; intestines pale; heart, right side enlarged, left side contracted; hæmorrhagic patches in lung; urinary bladder empty; gall-bladder moderately full; uterus contained three fœtuses; pieces of cord and brain fixed in 8 per cent. formal sections, made by paraffin method.

Exp. 173.
Rabbit. Weight, 1.264 kilograms.

Exps. 176, 177, 178, 179.

Four frogs. Weights: A, 27 grams (Exp. 176); B, 37-5 grams (Exp. 177); C, 28 grams (Exp. 178); D, 34.5 grams (Exp. 179).

P.M.

6.0. To each frog was given 0.012 gram chloral-hydrate, hypodermically. It was intended to give 0.008 gram chloral-hydrate to frogs C and D, but by mistake they received the same dose (0.012 gram) as A and B.

6.5. B received 15.3 minims of a 0.05-per-cent. solution tutin (12 mlgm. per kilo), hypodermically; C received 10.4 minims of a 0.05-per-cent. solution tutin (11 mlgm. per kilo), hypodermically; D received 14 minims of a 0.05-per-cent. solution tutin (12 mlgm. per kilo), hypodermically.

Next day. A is normal, B is dying, C is dead, D is dying.

Exp. 180.

P.M. Rabbit. Weight, 1.4 kilograms.

5.55. Gave 1.5 grams urethane, by stomach-tube.

6.20. Apparently under, very sleepy and limp. Gave 4 mlgm. tutin per kilo, hypodermically.

7.30. Symptoms first observed; they were not present at 7.15 p.m.

8.0. Twitching of ears and head, and starts of fore part of body; no salivation; was handled a little roughly in trying to see if salivation present, and went into a typical fit; it lay on one side, and fit succeeded fit in rapid succession; movements chiefly clonic; no tonic phase could be detected.

8.30. As fits still continue, and animal seemed doomed, tried about 1 c. c. of a 10-percent. sodium-carbonate, per rectum. While giving injection noticed involuntary urination.

8.45. Sodium-carbonate had produced no obvious effect, so tried chloroform; administered it as carefully as possible, with a Skinner's mask; breathing was shallow and rapid; under influence of chloroform animal became quiet, movements of limbs ceased, head moved slowly backwards, and breathing ceased suddenly without the least warning; the heart could not be felt beating, and artificial respiration had no effect.

Exp. 181.

Feb. 4. Rabbit was chloroformed, stomach-tube passed; 0.85 c.c. paraldehyde (1.2 c.c. P.M. per kilo), dissolved in 10 c.c. water, given.

5.0. Noticed that reflexes had returned.

6.15. Asleep.

Feb. 5. Recovered.

Exp. 182.

P.M. Rabbit. Weight, 1.551 kilograms.

4.48. Gave 1/200 grain hypodermic tabloid of hyoscine hydrobrom, hypodermically.

5.15. Apparently no effect, so gave a second dose of 1/200 grain, per os.

6.5. No effect.

Next day. Normal.

References To Literature