John Macleod-Biography[128]

The 1923 Nobel Prize in Physiology or Medicine

Presentation Speech

Presentation Speech by Professor J.Sjöquist，member of the Nobel Committee for Physiology or Medicine of the Royal Caroline Institute，on December 10，1923.

Your Majesty，Your Royal Highnesses，Ladies and Gentlemen.

The Professorial Staff of the Caroline Institute has resolved to award to Dr.Frederick Grant Banting and Professor John James Richard Macleod the Nobel Prize for 1923 in Physiology or Medicine for the discovery of insulin^[1].

Although the disease which has received the name of“diabetes mellitus”has evidently been known from immemorial time-Celsus and Araeteus in their writings in the first century of our era described an illness which was characterized by an enormous secretion of urine^[2]，an unquenchable thirst and a considerable loss of flesh-it was not until the seventeenth century that the Englishman Thomas Willis made the important observation that the urine in this illness contains a sugar-like substance；and it was not until more than a hundred years later that his countryman Dobson was able to produce from such urine the kind of sugar in question.This discovery，it is true，led the study of the mysterious disease into the right paths；but nevertheless it was a long time before any real progress was made.At the time the sugar was regarded as being a substance foreign to the animal organism，which was formed only under diseased conditions.It is true that the observation by Tidemann and Gmelin in 1827，that starchy foods are under normal conditions transformed into sugar in the intestinal canal and that this is absorbed by the blood，marks an important advance；but really epochmaking was the discovery of the great French physiologist Claude Bernard in 1857 that the liver is an organ that contains a starch-like substance，glycogen^[3]，from which sugar is constantly being formed during life；in the words of Claude Bernard，the liver secretes sugar into the blood.

In connection with his investigations into the circumstances that affect the formation of sugar，Claude Bernard observed that in certain lesions of the nervous system the sugar content of the blood was increased and that the sugar passed into the urine of the animals in the experiments.For the first time，therefore，an appearance of sugar in the urine-a glycosuria^[4]，though of a transitory^[5]nature-was experimentally produced；and consequently this discovery by Claude Bernard may be characterized as the starting-point of a series of experimental researches into the causes and nature of diabetes.

Even before this，however，in the post-mortem examination of persons who had died of severe diabetes，pathologists^[6]had made the observation that the pancreas sometimes exhibits diseased changes.The attention of Claude Bernard was directed to this point，but he did not succeed in producing glycosuria by ligation of the duct which leads the secretion of the gland to the bowel or by injecting coagulating^[7]substances into it；the removal of the whole gland by operation he regarded as technically impracticable.

Hence it aroused an intense interest when in 1889 two German investigators，von Mering and Minkowski，succeeded in carrying out this operation on dogs.It was still more remarkable that the animals thus operated on，now not only excreted sugar in the urine but also became the victims of a lasting disease which in all essentials resembled the most acute form of diabetes in man，even to such an extent that the content of sugar in the blood rose above the normal and that the disease inevitably led to death with symptoms of poisoning.If a part of the gland was left behind or if a bit of it was sewn under the skin，diabetes failed to develop.

It thus became clear that the disturbance in the sugar economy of the body that appeared after the complete removal of the gland could not well be due to the failure of the pancreatic^[8]juice to pass into the bowel^[9]，but rather to the loss of some other function of the gland.

During the eighteen-eighties，above all through the investigations of the Frenchman Brown-Séquard，attention had been directed to the importance for the vital functions of certain ductless gland^[10]-like organs.Time permits me in this place only to point out that，according to the view now generally entertained，these glands exercise their effect through passing into the blood and tissue juices of certain chemically effective substances，which are called by the general name of hormones；the glands themselves，owing to the fact that they have no ducts，are called endocrine^[11]glands or glands with internal secretion.As regards the pancre-as^[12]itself，it is true that it is a secreting gland，which by means of a duct pours secretion of the gland into the intestinal canal，where that secretion has certain important functions to perform in the process of digestion；but，as Langerhans showed as long ago as 1869，the pancreas also contains anatomical formations which have no direct connection with the duct，and which，after their discoverer are called“the cell islets of Langerhans”or“insulae”.In the beginning of the eighteen-nineties Laguesse expressed the surmise that it was just these cell islets that produce the inner secretion which is so important for the combustion of sugar.

Ever since the discovery by von Mering and Minkowski of the importance of the pancreas for the sugar economy of the organism and evidently also for the development of diabetes-that is to say，for more than a third of a century-a large number of investigations in different countries have devoted a great deal of work to discovering a remedy for diabetes from the pancreatic gland.It was natural to imagine，of course，that that disease was caused by the loss of power of the pancreatic gland to produce a hormone or to produce it in sufficient quantities，and that the introduction of this hormone in the diseased organism ought to be able to exercise a favourable influence on the disease，all the more as analogous conditions were well known with regard to other organs with internal secretion，especially the thyroid gland.Many of these investigations failed，while others succeeded in actually producing extracts or juices which，when injected into the blood of diabetic dogs and even human beings，showed themselves able to bring down the increased content of sugar in the blood，to diminish or even to stop altogether the excretion of sugar into the urine，and to bring about an increase in weight.Amongst these I should like especially to mention Zuelzer，who in 1908 produced an extract which was undoubtedly effective，but which also showed injurious byeffects-consequently it could not be used to any great extent therapeutically-and also Forschbach，Scott，Murlin，Kleiner，Paulesco，and many others.

The problem was in about this position when a young assistant in physiology at the Western University in London，Ontario，Frederick G.Banting，conceived an idea that was to prove of extraordinary importance for its further development.He thought to himself that the reason for the failure to produce effective pancreatic extract，was to be sought in an antagonistic^[13]or destructive effect on the hypothetical hormone^[14]of trypsin，the protein-splitting enzyme that is produced by the secreting cells of the gland，and that there would be a greater prospect of success if these cells were destroyed by ligation of the duct of the gland and the remaining part of the gland were then used as the original material.It had previously been observed by Schulze and by Ssobolev that the ligation of the duct involved the atrophy of the acini but not of insulae.He imparted his idea to Professor Macleod of Toronto after which，together with several fellow-workers，among whom I should like especially to mention Best and Collip，he began to work under Macleod's guidance and in his laboratory in May 1921.The very first experiments in diabetic^[15]dogs were crowned with success.After the method of producing the effective extract，which at the suggestion of Sir Sharpley Schafer had been called insulin，had been improved by Collip，and after its effect on the sugar content of the blood，on the respiratory^[16]quotient^[17]，and on the capacity of the liver for forming glycogen had been established，and also the dangers which might be produced by an overdose of the remedy through an excessive reduction of the sugar content of the blood had been determined by experiments on animals under Macleod's guidance，and after it had further been proved that the trypsin in an alkaline solution really destroys the hormone，the first injection of insulin was made in a youth of fourteen years，who suffered severely from diabetes，on 23 January and the following days in 1922.The result was that the sugar content of the blood of the patient fell to the normal，the passing of sugar into the urine was reduced to a minimum，and the general state of poisoning，acidosis^[18]，which is caused by certain injurious substances which are formed in this kind of diabetes^[19]mainly through dis-turbance in the fat metabolism，often in great quantities，was checked.Since then the new remedy，the production of which does not offer any great technical difficulties，has come into use in practically all countries and with favourable results.

We must not imagine that insulin is able to cure diabetes.How could that be possible if the cause of diabetes is to be found in the fact that the cells within our organism that produce the hormone necessary for the combustion of sugar are definitively destroyed？But insulin gives us the possibility of transforming the severe form to a milder one and thereby of restoring his capacity for work and a comparative state of health to the hopeless invalid who，despite the most trying and rigorous restrictions in diet，is constantly threatened by a fatal state of poisoning.Most striking is the effect of insulin in the cases in which the state of poisoning has already passed into that of diabetic coma^[20]，against which we have hitherto been helpless and which，before the days of insulin，inevitably led to death.

It could be prophesied with a very great degree of probability that such a substance as insulin some day would be produced from the pancreatic gland，and much of the work had been done beforehand by previous investigations，several of whom very nearly reached the goal.Consequently it also has been said that its discoverer was in a preeminent^[21]degree favoured by lucky circumstances.Even if this be so，yet there would seem to be cause to remember Pasteur's words：“La chance ne favorise que l'intelligence préparée.”

The Professorial Staff of the Caroline Institute has considered the work of Banting and Macleod to be of such importance，theoretically and practically，that it has resolved to award them the great distinction of the Nobel Prize.Doctor Banting and Professor Macleod not having the opportunity of being present today，I have the honour of asking the British Minister to accept from His Majesty the King the prize，and to transfer it to the Laureates，together with the congratulations of the Professorial Staff of the Royal Caroline Institute.

班廷传略

加拿大生理学家、外科医师。与C.H.贝斯特等一同从动物胰腺中提得可供临床应用的胰岛素，为临床治疗糖尿病作出贡献，因此获1923年诺贝尔生理学或医学奖。1891年11月14日生于加拿大安大略省阿利斯顿，1941年2月21日因飞机失事卒于纽芬兰。1916年毕业于多伦多大学医学院后即应征入伍，任军医上尉，曾到英国和法国前线。复员后在伦敦西方大学医学院进行研究，兼讲授解剖学与生理学。1921年回加拿大，在多伦多大学医学院任职，1923年升教授。第二次世界大战期间，从事航天医学研究。

19世纪后叶至20世纪初许多学者推测糖尿病与胰腺激素有关系，并称该激素为胰岛素，但口服动物胰脏治疗糖尿病无效。他推想，口服动物胰脏后，其中的激素可能在胃中为胰蛋白所破坏，若结扎动物胰管使产生胰蛋白的细胞萎缩而产生胰岛素的细胞不受影响，并将胰腺提取物注射应用，当可生效。1921年多伦多大学研究糖代谢的专家麦克劳德教授给他提供实验室，派贝斯特为助手。班廷和贝斯特结扎狗的胰导管6～8周后，摘出胰腺进行提取，将提取物给实验性糖尿病的狗注射，证明其有降低血糖、治疗糖尿病的作用，他们称此物为岛素。继之，擅长生物化学的J.B.科利普也参加改进提取、纯化岛素的工作，他们终于提得较纯的岛素，并将其名称改为胰岛素。1922年利用胰岛素进行第一例临床试验，获得成功。麦克劳德又改进提取方法，使胰岛素能批量生产，挽救了许多糖尿病人的生命。班廷获诺贝尔奖后，将奖金之半分给贝斯特，麦克劳德亦将奖金之半分给科利普。1923年加拿大议会授予班廷终身年金，并建立班廷研究基金，还在多伦多大学建立班廷-贝斯特医学研究所，任命他为所长。后班廷从事癌症、冠心病、硅肺等的研究。代表作有《胰腺提取物用于糖尿病的治疗》、《胰岛素、内分泌与临床》。

Frederick G.Banting-Biography^[22]

Frederick Grant Banting was born on November 14，1891，at Alliston，Ont.，Canada.He was the youngest of five children of William Thompson Banting and Margaret Grant.Educated at the Public and High Schools at Alliston，he later went to the University of Toronto to study divinity，but soon transferred to the study of medicine.In 1916 he took his M.B.degree and at once joined the Canadian Army Medical Corps，and served，during the First world War，in France.In 1918 he was wounded at the battle of Cambrai and in 1919 he was awarded the Military Cross for heroism^[23]under fire.

When the war ended in 1919，Banting returned to Canada and was for a short time a medical practitioner^[24]at London，Ontario.He studied orthopaedic^[25]medicine and was，during the year 1919-1920，Resident Surgeon at the Hospital for Sick Children，Toronto.From 1920 until 1921 he did parttime teaching in orthopaedics at the University of Western Ontario at London，Canada，besides his general practice，and from 1921 until 1922 he was Lecturer in Pharmacology^[26]at the University of Toronto.In 1922 he was awarded his M.D.degree，together with a gold medal.

Earlier，however，Banting had become deeply interested in diabetes.The work of Naunyn，Minkowski，Opie，Schafer，and others had indicated that diabetes was caused by lack of a protein hormone secreted by the islands of Langerhans in the pancreas^[27].To this hormone Schafer had given the name insulin，and it was supposed that insulin controls the metabolism of sugar，so that lack of it results in the accumulation of sugar in the blood and the excretion of the excess of sugar in the urine.Attempts to supply the missing insulin by feeding patients with fresh pancreas，or extracts of it，had failed，presumably because the protein insulin in these had been destroyed by the proteolytic enzyme of the pancreas.The problem，therefore，was how to extract insulin from the pancreas before it had been thus destroyed.

While he was considering this problem，Banting read in a medical journal an article by Moses Baron，which pointed out that，when the pancreatic duct was experimentally closed by ligatures^[28]，the cells of the pancreas which secrete trypsin degenerate，but that the islands of Langerhans remain intact.This suggested to Banting the idea that ligation of the pancreatic duct would，by destroying the cells which secrete trypsin，avoid the destruction of the insulin，so that，after sufficient time had been allowed for the degeneration of the trypsin-secreting cells，insulin might be extracted from the intact islands of Langerhans.

Determined to investigate this possibility，Banting discussed it with various people，among whom was J.J.R.Macleod，Professor of Physiology at the University of Toronto，and Macleod gave him facilities for experimental work upon it.Dr.Charles Best，then a medical student，was appointed as Banting's assistant，and together，Banting and Best started the work which was to lead to the discovery of insulin.

In 1922 Banting had been appointed Senior Demonstrator in Medicine at the University of Toronto，and in 1923 he was elected to the Banting and Best Chair of Medical Research，which had been endowed by the Legislature of the Province of Ontario.He was also appointed Honorary Consulting Physician to the Toronto General Hospital，the Hospital for Sick Children，and the Toronto Western Hospital.In the Banting and Best Institute，Banting dealt with the problems of silicosis^[29]，cancer，the mechanism of drowning and how to counteract it.During the Second World War he became greatly interested in problems connected with flying（such as blackout）.

In addition to his medical degree，Banting also obtained，in 1923，the LL.D.degree（Queens）and the D.Sc.degree（Toronto）.Prior to the award of the Nobel Prize in Physiology or Medicine for 1923，which he shared with Macleod，he received the Reeve Prize of the University of Toronto（1922）.In 1923，the Canadian Parliament granted him a Life Annuity of｜S 7，500.In 1928 Banting gave the Cameron Lecture in Edinburgh.He was appointed member of numerous medical academies and societies in his country and abroad，including the British and American Physiological Societies，and the American Pharmacological Society.He was knighted in 1934.

As a keen painter，Banting once took part of a painting expedition above the Arctic Circle，sponsored by the Government.

Banting married Marion Robertson in 1924；they had one child，William（b.1928）.This marriage ended in a divorce in 1932，and in 1937 Banting married Henrietta Ball.

When the Second World War broke out，he served as a liaison officer^[30]between the British and North American medical services and，while thus engaged，he was，in February 1941，killed in an air disaster in Newfoundland^[31].

Frederick G.Banting-Nobel Lecture

Nobel Lecture，September 15，1925

Diabetes and Insulin

Gentlemen.I very deeply appreciate the honour which you have conferred upon me in awarding the Nobel Prize for 1923 to me and Professor J.J.R.Macleod.I am fully aware of the responsibility which rests upon me to deliver an address in which certain aspects of the work on insulin may be placed before you.This I propose to do today and I regret that an earlier opportunity has not been afforded me of satisfying this obligation.

Diabetes and insulin

Since von Mering and Minkowski proved that removal of the pancreas produced severe and fatal diabetes in dogs，physiologists and clinicians have frequently endeavored to obtain from the pancreas an internal secretion which would be of value in the treatment of diabetes mellitus.Beginning with Minkowski himself，many observers tried various forms of extracts of the pancreas.Among the extractives used were water，saline，alcohol，and glycerin^[32].The extracts thus obtained were administered by mouth，subcutaneously^[33]，intravenously，or by rectum^[34]，both to experimental animals and humans suffering from diabetes.Little or no improvement was obtained and any favorable results were overshadowed by their toxic effects.In 1908，Zuelzer tried alcoholic extracts on six cases of diabetes mellitus and obtained favorable results，one case of severe diabetes becoming sugar-free.His extracts were then tried by Forschbach in Minkowski's clinic with less favorable results，and the investigation was abandoned by this group of workers.Rennie found that the islet cells existed separate from the acinar^[35]cells in certain boney fishes and in conjunction with Fraser，extracts of the principal islet cells were tried both on animals and on the human.Their results，however，were not sufficiently convincing to warrant^[36]clinical application.The problem of the extraction of the antidiabetic principle from the pancreas was then taken up for the most part by physiologists among whom were Scott，Paulesco，Kleiner，and Murlin.

While these efforts were being made by the physiologists，valuable knowledge was being gained on carbohydrate metabolism.Lewis and Benedict，Folin and Wu，Schaffer and Hartman，and Ivar Bang had elaborated methods whereby the percentage of sugar in a small sample of blood might be accurately estimated.At the same time a vast amount of knowledge was accumulating on basal metabolism.Special attention was being given to the relative importance of the various foodstuffs，and emphasis was being put on dietetic treatment of diabetes.Guelpa，von Noorden，Allen，Joslin，and Woodyatt，had elaborated systems of diabetic diet.

On October 30th，1920，I was attracted by an article by Moses Baron，in which he pointed out the similarity between the degenerative changes in the acinus^[37]cells of the pancreas following experimental ligation of the duct，and the changes following blockage of the duct with gallstones^[38].Having read this article，the idea presented itself that by ligating the duct and allowing time for the degeneration of the acinus cells，a means might be provided for obtaining an extract of the islet cells free from the destroying influence of trypsin and other pancreatic enzymes.

On April 14th，1921，I began working on this idea in the Physiological Laboratory of the University of Toronto.Professor Macleod allotted me Dr.Charles Best as an associate.Our first step was to tie the pancreatic ducts in a number of dogs.At the end of seven weeks these dogs were chloroformed.The pancreas of each dog was removed and all were found to be shrivelled，fibrotic，and about one-third the original size.Histological examination showed that there were no healthy acinus cells.This material was cut into small pieces，ground with sand，and extracted with normal saline.This extract was tested on a dog rendered diabetic by the removal of the pancreas.Following the intravenous injection，the blood sugar of the depancreatized dogs was reduced to a normal or subnormal level，and the urine became sugarfree.There was a marked improvement in the general clinical condition as evidenced by the fact that the animals became stronger and more lively，the broken-down wounds healed more kindly，and the life of the animal was undoubtedly prolonged.

The beneficial results obtained from this first type of extract substantiated the view that trypsin destroyed the antidiabetic principle and suggested the idea that by getting rid of the trypsin，an active extract might be obtained.The second type of extract was made from the pancreas of dogs in which acinus cells had been exhausted of trypsin by the long-con-tinued injection of secretin.Although many of the extracts made in this manner produced marked lowering of blood sugar and improvement in the general clinical condition it was not always possible to completely exhaust the gland；consequently toxic effects frequently resulted.

The third type of extract used in this series of experiments was made from the pancreas of foetal^[39]calves of less than four months development.Laguesse had found that the pancreas of new-born contained comparatively more islet cells than the pancreas of the adult.Since other glands of internal secretion are known to contain their active principle as soon as they are differentiated in their embryological development，it occurred to me that trypsin might not be present since it is not used till after the birth of the animal.Later I found that Ibrahim had shown that trypsin is not present till seven or eight months of intrauterine^[40]development.Foetal extracts could be prepared in a much more concentrated solution than the former two varieties of extract.It produced marked lowering of blood sugar，urine became sugar free and there was marked clinical improvement.Its greatest value however was that the abundance in which it could be obtained enabled us to investigate its chemical extraction.

Up to this time saline had been used as an extractive.We now found that alcohol slightly acidified extracted the active principle，and by applying this method of extraction to the whole adult beef pancreas，active extracts comparatively free from toxic properties were obtained.

Since all large-scale production methods for the preparation of insulin today have the acid-alcohol extraction as the first step in the process，it may be well to elaborate on the methods of preparation at this stage.Insulin was prepared by the extraction of fresh glands with faintly acid alcohol.The concentration of alcohol in the original experiments varied from 40 to 60 per cent.The alcoholic solution of pancreas was filtered and the filtrate concentrated by evaporation of the alcohol and water in vacuo^[41]or in a warm air current.Lipoid material was removed by extracting the resi-due with toluene^[42]or ether^[43].The resulting product was the original whole gland extract.We were able to show that the active material contained in this extract was practically insoluble in 95%alcohol.

The extracts prepared in this way were tried on depancreatized^[44]dogs and in all cases the blood sugar was lowered.In one early case hypoglycaemic level was reached and the dog died from what we now know to be a hypo-glycaemic reaction.

It had been known that depancreatized dogs were unable to store glycogen in the liver，and that glycogen disappears in three or four days after pancreatectomy^[45].We found that by the administration of glucose and extract，the diabetic dog was enabled to store as much as 8%to 12%glycogen.Diabetic dogs seldom live more than 12 to 14 days.But with the daily administration of this whole gland extract we were able to keep a depancreatized dog alive and healthy for ten weeks.At the end of this time the dog was chloroformed and a careful autopsy^[46]failed to reveal any islet tissue.

The extract at this time was sufficiently purified to be tested on three cases of diabetes mellitus in the wards of the Toronto General Hospital.There was a marked reduction in blood sugar and the urine was rendered sugar-free.However the high protein content rendered the continuous use undesirable，due to formation of sterile^[47]abscesses.

At this stage in the investigation，February 1922，Professor Macleod abandoned his work on anoxaemia and turned his whole laboratory staff on the investigation of the physiological properties of what is now known as insulin.

Dr.Collip took up the biochemical purification of the active principle and ran the scale of fractional precipitation with 70%-95%alcohol and succeeded in obtaining a more improved end product.But unfortunately his method was not applicable to large-scale production.Dr.Best then took up the large-scale production and contributed greatly to the es-tablishment of the principles of production and purification.This work was carried out in the Connaught Laboratories under Prof.Fitzgerald who is kind enough to be here today.

It had been found that the final product obtained by the earlier methods was not sufficiently pure for prolonged clinical use，and efforts were made to secure a better product.The benzoic^[48]acid method of Maloney and Findlay which depends upon the fact that insulin is absorbed from watery solutions by benzoic acid was successfully used in Connaught Laboratories for several months.

Professor Shaffer of Washington University，St.Louis，and his collaborators，Somogyi and Doisy，introduced a method of purification which is known as the isoelectric^[49]process.This method depends upon the fact that if a watery solution of insulin is adjusted to approximately p H 5 a precipitate settles out which contains much of the potent material and relatively few impurities.Dudley has found that insulin was precipitated from water solutions by picric acid^[50]and he made use of this fact to devise a very ingenious method for the'purification of the active material.

Best and Scott who are responsible for the preparation of insulin in the Insulin Division of the Connaught Laboratories have tested all the available methods and have appropriated certain details from many of these；several new procedures which have been found advantageous have been introduced by them.The yield of insulin obtained by Best and Scott at the Connaught Laboratories，by a preliminary extraction with dilute sulphuric acid^[51]followed by alcohol，is 1，800 to 2，200 units per kg of pancreas.

The present method of preparation is as follows.The beef or pork pancreas is finely minced in a large grinder and the minced material is then treated with 5 cc of concentrated sulphuric acid，appropriately diluted^[52]，per pound of glands.The mixture is stirred for a period of three or four hours and 95 per cent alcohol is added until the concentration of alcohol is 60 to 70 per cent.Two extractions of the glands are made.The solid material is then partially removed by centrifuging the mixture and the solution is further clarified by filtering through paper.The filtrate is practically neutralized with NaOH.The clear filtrate is concentrated in vacuo to about 1／15 of its original volume.The concentrate is then heated to 50°C which results in the separation of lipoid^[53]and other materials，which are removed by filtration.Ammonium sulphate（37 g per 100 cc）is then added to the concentrate and a protein material containing all the insulin floats to the top of the liquid.The precipitate is skimmed off and dissolved in hot acid alcohol.When the precipitate has completely dissolved，10 volumes of warm alcohol are added.The solution is then neutralized with NaOH and cooled to room temperature，and kept in a refrigerator at 5°C for two days.At the end of this time the dark-coloured supernatant alcohol is decanted off.The alcohol contains practically no potency.The precipitate is dried in vacuo to remove all trace of the alcohol.It is then dissolved in acid water，in which it is readily soluble.The solution is made alkaline with NaOH to p H 7.3 to 7.5.At this alkalinity a dark-coloured precipitate settles out，and is immediately centrifuged off.This precipitate is washed once or twice with alkaline water of pH 9.0 and the washings are added to the main liquid.It is important that this process be carried out fairly quickly as insulin is destroyed in alkaline solution.The acidity is adjusted to p H 5.0 and a white precipitate readily settles out.Tricresol is added to a concentration of 0.3%in order to assist in the iso-electric precipitation and to act as a preservative.After standing one week in the ice chest，the supernatant liquid is decanted off and the resultant liquid is removed by centrifuging.The precipitate is then dissolved in a small quantity of acid water.A second iso-electric precipitation is carried out by adjusting the acidity to a pH of approximately 5.0.After standing overnight the resultant precipitate is removed by centrifuging.The precipitate，which contains the active principle in a comparatively pure form，is dissolved in acid water and the hydrogenion concentration adjusted to p H 2.5.The material is carefully tested to determine the potency and is then diluted to the desired strength of 10，20，40，or 80 units per cc.Tricresol is added to secure a concentration of 0.1 per cent.Sufficient sodium chloride^[54]is added to make the solution isotonic.The insulin solution is passed through a Mandler filter.After passing through the filter the insulin is retested carefully to determine its potency.There is practically no loss in berkefelding.The tested insulin is poured into sterile glass vials^[55]with aseptic^[56]precautions and the sterility^[57]of the final product thoroughly tested by approved methods.

The method of estimating the potency of insulin solutions is based on the effect that insulin produces upon the blood sugar of normal animals.Rabbits serve as the test animal.They are starved for twenty-four hours before the administration of insulin.Their weight should be approximately 2kg.Insulin is distributed in strengths of 10，20，40，and 80 units per cc.The unit is one third of the amount of material required to lower the blood sugar of a 2kg rabbit which has fasted twenty-four hours from the normal level（0.118 per cent）to 0.045 per cent over a period of five hours.In a moderately severe case of diabetes，one unit causes about 2.5 grams of carbohydrate to be utilized.In earlier and milder cases，as a rule，one unit has a greater effect，accounting for three to five grams of carbohydrate.

With the improvement in the quality of insulin，the increased knowledge of its physiological action and the increased quantities at our disposal，we were now prepared for more extensive clinical investigation.In May 1922 a clinic was established in association with Dr.Gilchrist，at Christie Street Hospital for Returned Soldiers.Following this，a clinic was established in the Toronto General Hospital in association with Drs.Campbell and Fletcher，and at Toronto Hospital for Sick Children in association with Dr.Gladys Boyd.In general the routine followed in all these clinics was as follows.

After a careful history had been taken，the patient was given a complete physical examination.Special attention was directed to the finding of foci of possible infection.The teeth，tonsils，accessory sinuses^[58]，chest and digestive system were examined clinically，as well as by X-ray.Special consideration was given to biliary tract infection，constipation，and chronic appendicitis.If any source of septic absorption was located it was appropriately treated，since such conditions may lower carbohydrate tolerance.If indicated the eye grounds were examined for a possible diabetic retinitis^[59]or neuro-retinitis.

The daily routine urinalysis^[60]included the volume of the twenty-four hour specimen，the specific gravity，the reaction，and tests for albumen by heat or nitric acid.The acetone bodies were estimated by means of the Rothera and ferric chloride tests.Sugar determinations were done by means of the Benedict qualitative and quantitative solutions.In addition to the above，the blood sugars were estimated by means of the Schaffer-Hartman method and the respiratory quotients with the Douglas bag and Haldane gas-analysis apparatus.

At first the patient continued on the same diet as that previous to his admission to hospital in order to obtain some idea of the severity of his case，and to avoid complications from sudden change of diet.Coma will be discussed separately.On the second or third day he was placed upon a diet，the caloric^[61]value of which was calculated on his basal requirement.This was determined from Dubois'chart and Aub-Dubois'table.It has been estimated by Marsh，Newburgh，and Holly that the body requires two-thirds of a gram of protein per kilogram of body weight per day（1 kilo=2.2 pounds）in order to maintain nitrogenous equilibrium.The remaining calories must be supplied by carbohydrate and fats in a ratio that will prevent the production of ketone^[62]bodies.

The patient remained on this basal requirement diet at least a week.During this time，blood sugar was estimated before，and three hours after，breakfast，in order to determine the fasting level and the effect of food.The quantity of sugar excreted was estimated daily，and this amount subtrac-ted from the available carbohydrate ingested gives approximately the utilization.The available carbohydrate includes 58 per cent of the protein，10 per cent of the fat，and the total carbohydrate in the diet.It may be noted that when a patient was placed upon a diet in which the protein，fat and carbohydrates were balanced，that the amount of sugar excreted^[63]soon approached a fairly constant amount，whereas if the diet was not well-adjusted to the patient's requirements，there was wide variation in the amounts of sugar excreted.

If a patient became sugar-free and blood sugar normal on a basal requirement diet，the caloric intake was gradually increased until sugar appeared in the urine.The tolerance was thus ascertained.If a patient remained sugar-free and had a normal blood sugar when on a diet containing five hundred calories above his basal requirement he was not considered sufficiently severe for insulin treatment，since five hundred calories over and above the basal requirement are sufficient for daily activities.If，however，he was unable to me-tabolize this amount，insulin treatment was commenced.

Diabetes mellitus is due to a deficiency of the internal secretion of the pancreas.The main principle of treatment is，therefore，to correct this deficiency.If it is found that the patient is unable to keep sugar-free on a diet that is compatible with an active，useful life，sufficient insulin is administered to meet this requirement.

In severe cases insulin was administered subcutaneously three times a day，from one-half to three-quarters of an hour before meals.This was done so that the curve of hypoglycaemia produced by the insulin was superimposed on the curve of hyperglycaemia^[64]produced by the meal.In rare cases a small fourth dose was given at bed time to control nocturnal^[65]glycosuria.The less severe cases could be satisfactorily treated on a morning and evening dose or a single dose before breakfast.

When the insulin treatment was established，if sugar was present in the twenty-four hour specimen of urine，the dosage was gradually raised till the patient became sugar-free.If he was not receiving sufficient food for maintenance，diet and urinary^[66]dosage of insulin were gradually raised.If small quantities of urinary sugar persist，it was desirable to find out at what period of the day this was excreted.In order to do this，each specimen in the twenty-four hours was analysed separately.An increase in the dose previous to the appearance of glycosuria^[67]will prevent its occurrence.

In severe cases it was found preferable to give the largest dose of insulin in the morning，and reduced doses throughout the day.For example，a patient may receive fifteen units in the morning，ten units at noon，and ten units at night.If three equal doses are given there may be morning glycosuria and evening hypoglycaemia，whereas the extremes of blood sugar causing these conditions may be prevented by the above distribution.

The effect of the same dosage of extract on different individuals was found to vary considerably.Five patients，whose weights varied from forty-six to sixty-seven kilograms，each received two cubic centimetres of the same lot of insulin，and in four hours the blood sugars had decreased 0.012%，0.044%，0.128%，0.146%，and 0.018%respectively.It was found，however，that one patient would persistently give marked decreases in blood sugar after insulin，while in another the fall in blood sugar was persistently less.In our experience，the more marked decreases in blood sugar occurred in the milder cases.

The blood sugars of some of the patients were followed throughout the twenty-four hours and it was found that it was possible to gauge the dosage of insulin so as to keep the blood sugar within normal limits and still avoid the dangers of hypoglycaemia.

Coincident with the maintenance of the blood sugar at normal level the cardinal^[68]symptoms of the disease disappear.The patient loses the irritating thirst and dryness of the mouth and throat，and does not desire the large amounts of fluid with which he had previously tried to combat these symptoms.The lowered fluid intake diminishes the polyuria and from a twenty-four hour excretion of three to five litres the output falls to normal.The appetite which has been voracious^[69]is now satisfied with a normal meal，the carbohydrate of which is utilized，and the patient loses the persistent craving for food.

We found that when a patient was given too large a dose of insulin there was a marked reaction，and the hypoglycaemia which developed gave rise to symptoms which were very similar to those observed in animals.The reaction began in from one and a half to six hours after the patient received the overdose.The average time was three to four hours.The interval varied with the individual，the dosage，and the food ingested^[70].The first warning of hypo-glycaemia was an unaccountable anxiety and a feeling of impending trouble associated with restlessness.This was frequently followed by profuse perspiration.The development of this symptom was not affected by atmospheric conditions.It appeared while the patient was in a frosty outside atmosphere，or in a heated room，and was independent of physical or mental activity.At this time there was usually a very great desire for food.No particular foodstuff was desired，but bulk of any kind seemed to give satisfaction.At times the appetite is almost unappeasable^[71].

At this stage of the reaction the patient noticed a certain sensation as of clonic tremor in the muscles of the extremities.This could be controlled at first.Coordination，however，was impaired for the more delicate movements.Coincident with this there was a marked pallor of the skin with a rise in pulse rate to one hundred or one hundred and twenty beats per minute，and a dilatation of the pupils.The blood pressure during this period fell about fifteen to twenty-five millimetres of mercury，and the patient felt faint.The ability to do physical or mental work was greatly impaired.In a severe reaction there was often a considerable degree of aphasia，the patient having to grope for words.The memory for names and figures became quite faulty.

The onset of hypoglycaemic symptoms depends not only on the extent，but also on the rapidity of fall in blood sugar.The level at which symptoms occur is slightly higher in the diabetic with marked hyperglycaemia^[72]than in a patient whose blood sugar is normal.When the blood sugar is suddenly reduced from a high level premonitory symptoms^[73]may occur with a blood sugar between the normal levels of 0.100%and 0.080%，while the more marked symptoms of prostration^[74]，perspiration^[75]，and in coordination develop between 0.080%and 0.042%.As a patient becomes accustomed to a normal blood sugar the threshold of these reactions becomes lower.One patient who formerly had premonitory symptoms of hypoglycaemia at 0.096%now has no reaction at 0.076%，but symptoms commence between this level and 0.062%.

The ingestion of carbohydrate，in the form of orange juice（four to eight ounces），or of glucose^[76]，relieves these symptoms in from one-quarter to one-half hour.If the reaction is severe，or if coma or convulsions occur，epinephrine or intravenous glucose should be given.The former acts in from three to ten minutes，but in order that the symptoms should not recur，glucose must be given by mouth as soon as the patient has sufficiently recovered.The patients were warned that when these reactions occurred they were to obtain carbohydrate immediately.

“Fats only burn in the fire of carbohydrate.”The ability of the severe diabetic^[77]to burn glucose is markedly impaired，therefore the excess of fat is incompletely oxidized，giving rise to ketone bodies.These appear in the blood and urine as acetone，diacetic and hetaoxybutyric acids.Insulin causes increased carbohydrate metabolism，and consequently fats are completely burned.This is substantiated by the fact that acetone^[78]and sugar disappear from the urine almost simultaneously following adequate amounts of insulin.When insulin is discontinued in these cases，acetone bodies and sugar reappear in the urine.

Since the Rothera test is exceedingly delicate（sensitive to 1 part of aceto-acetic acid in 30，000），patients on a high fat diet may be sugar-free and still show traces of acetone bodies.A comparison with the ferric^[79]-chloride^[80]test（which is sensitive to only 1 part in 7，000）is，therefore，desirable.The persistence of ketone bodies in amounts which can be determined by the ferric-chloride test necessitates either an increase in the carbohydrate or a decrease in fat of the diet.

When the production of acetone bodies is more rapid than the excretion they accumulate in the blood，giving rise to air hunger，drowsiness^[81]，and coma.The need of insulin is then imperative.After its administration，the utilization of carbohydrate by the body gives complete combustion of the fats.When a patient was admitted to hospital in coma the blood-sugar tests and a urinalysis were done as soon as possible.（The urine was obtained by catheterization^[82]if necessary.）While these tests were being carried out，the large bowel was evacuated^[83]with copious enemata.If the blood sugar was high and acetone present in large amounts in the urine，from thirty to fifty units of insulin were given subcutaneously.Blood and urinary sugar were frequently estimated because of the danger of hypoglycaemia.To prevent this，from thirty to fifty grams of glucose in ten per cent solution were given intravenously.If the patient was profoundly comatose，the insulin was administered intravenously with the glucose.

The patient usually regained consciousness in from three to six hours.From this time on，fluids and glucose were administered by mouth if retained.The patient was urged to take at least two hundred cubic centimeters of fluid per hour.In from eight to ten hours，the ketone bodies were markedly reduced.On the following day protein was given every four hours as the white of one egg in two hundred cubic centimetres of orange juice.In two to three days，when ketone bodies had disappeared from the urine，fat was cautiously added，and the patient was slowly raised to a basal requirement diet.He was then treated as an ordinary diabetic.During the period of coma the patient was kept warm and toxic materials eliminated from the bowel by purgation^[84]and repeated enemata.A large amount of fluid was given to dilute the toxic bodies and promote their elimination.This was administered intravenously，subcutaneously，or per rectum.If signs of circulatory failure developed these were treated by appropriate stimulation.

Striking results were obtained with the above procedure.However，it was found that the longer the period of untreated coma the more grave was the prognosis^[85]and the slower the recovery if it occurred.Cases complicated by severe infection，gangrene^[86]，pneumonia^[87]，or intestinal intoxication^[88]may recover from acidosis^[89]and coma^[90]，but succumb to the complication.

Marked lipaemia was present in three cases.This disappeared in the course of a week to ten days after the patient was placed on insulin and on a diet in which the fat was restricted.The urine of one patient became acetone-free while lipaemia persisted.

The severe diabetic，whose ability to burn carbohydrate is markedly impaired，has a persistently low respiratory quotient，from 0.7 to 0.8，which is but little raised by the ingestion^[91]of glucose：when glucose and insulin are given together，the respiratory quotient is markedly increased，showing that carbohydrate is being metabolized.The highest values have been obtained when pure glucose was used with insulin.Less extensive rises have been secured when the patient，while on a mixed diet，received insulin.

All the patients gained in weight on the additional calories.There was an increase in sexual vigour and there was a greater ability to do mental and physical work.Nearly all of the patients have returned to their former employment，and while still under supervision，they administer their own insulin and arrange their own diets with satisfactory results.

All diabetics who have not an adequate knowledge of the dietetic treatment of their disease should be admitted to hospital in order that they may receive instruction in the preparation of their calculated and weighed diet-that they may learn the qualitative tests for sugar and acetone in the urine-that their carbohydrate tolerance may be accurately determined；and that the use of insulin，if required，may be safely instituted.Mild cases，especially if over fifty years of age，can be controlled by diet.Cases that cannot be adequately controlled by dietetic treatment alone should be given sufficient insulin to enable them to attain to a diet on which they may“carry on”.

One of the commonest complications of diabetes，especially in untreated patients over fifty，is gangrene.It is often associated with varying degrees of sclerosis^[92]of the leg arteries，which makes it extremely difficult to obtain healing.This may be accomplished by the use of insulin，but when permanent impairment has occurred it is advisable to amputate.Amputation is also advisable when an infection is so severe that the life of the patient is in jeopardy^[93].Treatment of these cases is difficult because，due to the infection，there is a marked variation in the daily production of insulin by their own pancreas.But with careful treatment they can be rendered free from acetone and sugar，and their general condition improved.Operation is then performed preferably under nitrous oxide^[94]and oxygen anaesthetic^[95].If the blood sugar is maintained normal，and acidosis is prevented，the wound heals kindly，provided that the amputation has been high enough to assure a good blood supply.For varying periods after the operation，the patient remains on insulin treatment.In nearly all cases at the end of three or four weeks，mild hypoglycaemic reactions indicate an overdose of insulin.It is then necessary to increase the diet or decrease the insulin.In some cases the tolerance improves sufficiently to warrant the discontinuance of insulin.

Diabetic patients requiring major operations，such as appendectomy^[96]，cholecystectomy^[97]，and tonsillecto-my^[98]，or removal of teeth，are first rendered sugar-and acetone-free，unless the severity of symptoms demand immediate attention.Patients formerly considered bad surgical risks，if given proper dietetic treatment with insulin may be protected from the acidosis^[99]，hyperglycaemia^[100]，and glycosuria^[101]which otherwise usually result from the anaesthetic.In the diabetic，infections such as boils and carbuncles，and also intercurrent infections such as bronchitis，influenza，and fevers are favorably influenced by the normal blood sugar and increased metabolism which the administration of insulin permits.In the diabetic with tuberculosis，insulin allows the administration of proper nourishment to combat the tubercle infection.

During the past year and a half I have not been in active practice but have remained associated with the clinics.I have also kept in personal touch with the first fifteen patients who received insulin treatment.These patients were all extreme-ly severe diabetics for whom diet had done its best.Of these fifteen patients，seven were children under fifteen years.It has been possible through the intelligent co-operation of the parents to continue a proper balance between diet and insulin dosage，and to maintain six of the seven children sugarfree.None of these have had to return to hospital，and all have gained in tolerance，and require from one-half to one-third less insulin than when they first began treatment.They have all gained in height and weight，and for the most part have developed into healthy normal children.The one child whose diet and insulin has not been properly controlled has been back in hospital repeatedly and is steadily losing in tolerance.Of the remaining eight cases there were four women and three men whose ages ranged from twenty-five to thirty-five years.The weight of the women varied from seventy-four to seventy-nine pounds.Two of the women，although they have gained to normal or overweight and now have no symptoms of disease，have not shown any increase in tolerance，due，perhaps，to the fact that they have not kept sugar-free.All the others，both men and women，have been able to reduce their dose of insulin from two-thirds to one-fifth of the original requirement.The one remaining case was admitted for amputation.She had had diabetes for six years，and at the time of admission，her blood sugar was 0.350%，and large amounts of acetone and sugar were being excreted^[102]in the urine.She was rendered sugar-and acetone-free by means of insulin before the operation was performed.Amputation was done at the middle third of the thigh.The stump was entirely healed in three weeks.Within six weeks of her operation，insulin was discontinued and her diet was increased without the return of diabetic symptoms.It is now three years since her operation and she is sugarfree on a liberal diet without insulin.

It may be of interest to mention a few cases in greater detail to further illustrate the improvement in carbohydrate tolerance following insulin treatment.

Case 1：male，aged 29 years，had suffered from chronic appendicitis.The urine of the patient in December，1916，was sugar-free.About the middle of March，1917，he sud-denly developed polyuria^[103]，polyphagia^[104]，and polydipsia^[105]，and lost fourteen pounds in weight in a fortnight.There was marked weakness.Urinary sugar was discovered to be as high as eight per cent at this time.On April 4th，the patient was placed on Allen treatment，and slowly regained a tolerance of about two hundred grams available carbohydrate.He returned to his army duties in September 1917，and was able to carry on uninterruptedly until March，1919.His tolerance had decreased during this time to about one hundred and fifty grams.Following discharge from the army in March，1919，the course of the patient was slowly downhill until October，1921，when a particularly severe form of influenza shattered his tolerance.Up to this time the patient was maintained practically sugar-free，but following the attack of influenza，his tolerance fell to about sixty-six grams of available carbohydrate.He began to lose weight rapidly.Thirst，hunger，and polyuria returned.His strength dimin-ished and，owing to mental and physical lassitude^[106]，he found it impossible to continue his work.Glycosuria became persistent and acetone bodies made their appearance，and steadily increased.A distinct odour of acetone was at times distinguishable in the patient's breath.

On February 11th，1922，this patient was taken to the Physiology Department of the University of Toronto，and the respiratory^[107]quotient was found to be 0.74，and unchanged by the ingestion of thirty grams of pure glucose.Then 5 cc of insulin were given subcutaneously，and within two hours the patient's respiratory quotient had risen to 0.90.The urine was sugar-free and he had shaken off his mental and physical torpor.Following this experiment，the patient did not again receive insulin until May 15th as the product was being further improved.Since the latter date，the patient has been constantly on insulin.

During the first six months of insulin treatment it was impossible to maintain him sugar-free，although he received about 120 units per day.However，he gained in weight and his clinical condition improved.About January，1923，with the improvement in the quality of insulin，the patient became sugar-free and has remained sugar-free with the exception of one or two occasions.During the first nine months he required no reduction in the dose of insulin，but since that time，on the average of every two months，he has had a series of hypoglycaemic reactions which necessitated the reduction of the dose.One exception to this occurred in June，1924，at which time appendectomy^[108]was performed following a mild attack of appendicitis.An increased dose was required to maintain him sugar-free during this period.At the present time he requires but 20 units of insulin，or onesixth of his original requirement.His diet has been practically constant during the whole period of observation.All symptoms attributable to diabetes have long since disappeared.He has gained twenty-five pounds in weight and apart from the necessity of taking insulin and controlling his diet he leads an active normal life.

This case is a striking example of the fact that it is only in cases who are maintained sugar-free over long periods of time that an improvement in tolerance is obtained with a consequent reduction in the dose of insulin.

Case 2：female，age 15 years.In the autumn of 1918，the patient had polydipsia and polyuria，and complained of weakness.During the winter she suffered from pains in the legs and back，and from insomnia^[109].In March，1919，these symptoms became more severe.The appetite became excessive and there was some pruritus.The weight by this time had fallen from seventy-five pounds to sixty-two pounds.Glycosuria was discovered and she was placed under the care of Dr.F.M.Allen，to whom we are very much indebted for complete record of the case from April，1919，till August，1922.During this period the diet was controlled so as to maintain the urine free from sugar.Despite this careful dietetic regime the patient's condition became progressively worse.

When she came under my care on August 16th，1922，the examination showed：patient emaciated^[110]；skin dry；slight edema of ankles；hair brittle and thin；abdomen prominent；marked weakness.The patient was brought on a stretcher and weighed forty-five pounds.Nothing of note in the respiratory^[111]，cardiovascular^[112]，digestive，or nervous system.

At this time she was receiving a diet of protein 50g，fat 71g，carbohydrate 20g（919 calories）.Insulin treatment was started immediately.At this early stage，the unit of insulin had not been worked out，and it is therefore difficult to accurately estimate the dosage she received.The diet was increased daily so that，at the end of two weeks，she was receiving protein 63g，fat 208g，carbohydrate 97g（2512 calories）.This diet was continued up to January 1st，1923.Insulin was given 15 to 30 minutes before the morning and evening meals.A sufficient amount was given to maintain the urine free of sugar.Each specimen of urine was examined and the dose was increased slightly if traces of sugar appeared.When hypoglycaemia occurred，orange juice or glucose candy was given.Between August 16th and January 1st，the urine was sugar-free，except on ten occasions when traces of sugar appeared，and on two other occasions when less than 2g was excreted.Acetone was absent from the urine.

On this treatment the patient gained rapidly in strength，and was soon able to take vigorous exercise.Her weight increased from 45 to 105 pounds in the first six months.The diet included such foodstuffs as cereals，bread，potato，rice，corn，tapioca^[113]，corn starch，and even honey.

At present（June 1925）she is in the best of health，and to use her own words“never felt better in all my life”.She has grown four inches and weighs 134 pounds.Her present diet which is only approximate because she has dispensed with the weighing of food，is 125g carbohydrate，50g protein，50g fat.This diet is practically the same as that of December，1922.The insulin required to maintain her sugarfree has been reduced about one-third.

Dr.Gladys Boyd，who is now in charge of the diabetics at the Hospital for Sick Children，Toronto，has been able to follow a number of cases of children under insulin treatment.She has estimated the insulin requirement per 10g of carbohydrate in a number of cases，and in general her results show a decided increase in tolerance in all cases in which glycosuria^[114]and hypergly-caemia are adequately controlled.To illustrate-Case 1，which required 6.9 units per 10g carbohydrate in March，1923，only required 2.6 units in January，1924.Case 2，which required 7.8 units per 10g in January，1925，in June 1925 required only 2.8 units.Case 3，which required 6.5 units per 10g in April，1922，required only 3.7 units in January，1925.

From a review of the work，Dr.Boyd has found that all the patients had had hyperglycaemia or even glycosuria at times，but if such occurrences were only transitory^[115]and infrequent，improvement in tolerance occurred.Even short periods of rest to the pancreas by means of balanced diet and insulin resulted in improvement in tolerance.Two of our earliest cases，Fanny Z.and Elsie N.are the only exceptions to this rule.Fanny is to all appearances in the best of health with a blood sugar of 0.3%to 0.4%.She has been admitted in coma four times.During her stay in hospital she improves but does as she chooses on discharge.Her tolerance is becoming less all the time.Elsie keeps in touch with us but is looked after by another physician.He purposely allows her to have glycosuria at night.She is fine physically，but requires much more insulin than formerly.

Dr.Boyd has also found that in those cases who can handle sufficient food without insulin，although the disease has been kept under control there has not been such striking increase in tolerance.

The best evidence that there is regeneration^[116]of the pancreas^[117]with insulin treatment is provided by Drs.Boyd and Robinson.The following is the case reported by them.

Clinical history：B.N.，white，male，aged 9 years.Family history：Father and one maternal uncle have diabetes.Diabetes diagnosed in this child when he was two years old.He was placed on a suitable Allen diet，which was strictly adhered to，and for a time did well except for recurrent attacks of dysentery，which lowered his tolerance.Failure to gain in stature or weight in any way commensurate with his age was noted and the general condition became worse each year until he was more or less a chronic invalid with increasingly frequent attacks of acidosis^[118]during the last year before starting insulin.

He was admitted to the Hospital for Sick Children，Toronto，the end of December，1922.At this time he was an emaciated dwarf，more or less drowsy and unhappy.His weight was thirty pounds，and his height thirty-nine inches.His tolerance to carbohydrate had decreased until he was unable to utilize 15g of such food.Insulin treatment was started at once and his diet increased to a diet suitable for a boy of his age.Sufficient insulin was given to keep him sugar-free and his blood sugar normal.He was discharged on an adequate diet plus insulin.Progress，both in general condition and in improvement of pancreatic^[119]function，was steady.His tolerance to carbohydrate trebled in the year，as shown either by the fact that 30 units of insulin controlled the disease as adequately as go units a year before，or，stated in another way，without insulin he could now handle 54g carbohydrate instead of 15.From a chronic invalid in 1922 he became“the leader of the gang”，in 1923.He was killed by fracturing his skull when sleigh^[120]riding.He lived for about three hours after receiving the injury and an immediate post-mortem examination was made.The pancreas was removed within thirty minutes of death.

From this clinical history one might expect the pancreas to show marked degeneration.However，on section there was little sign of degeneration，but on the other hand there was strong evidence to support the view of active regeneration both of acinar^[121]and islet tissue.These regenerative changes were more marked in the periphery^[122]and smaller lobules^[123]of the pancreas than in the central area.

The acinar cells were found to he actively proliferating^[124]in cords and clusters forming small lobules in some areas，and were in close association with newly formed func-tioning ducts.

The islets were greatly increased in number，particularly in the periphery，there being about four times as many per field as in the central area.These cells were large but might be overlooked with an ordinary stain.However，they could be identified as islet cells by Bowie's special granule^[125]-stain.This stain also demonstrated that these cells were almost entirely beta cells and were probably concerned in the increased carbohydrate tolerance.On the other hand，those islets in the central areas showed an increased number of cells all in an active state of nutrition，but closely packed together.The special stain showed a normal ratio of alpha and beta cells.

These sections were studied by Bensley，Opie，Allen，and others，who concurred in the opinion of Drs.Boyd and Robinson.

Dr.F.M.Allen，Morristown N.J.，after using insulin for three years states as his belief，“That there has been improvement of tolerance in some cases beyond what was possi-ble without insulin”.“This observation is trustworthy only in cases where prolonged strict control of symptoms by diet was previously employed.On the other hand，the marked increase of tolerance is limited to a minority of cases and has not proved to be continuous in any of them.In other words the improvement always stops short of a cure.There is certainly no decline of tolerance with the passage of time，provided the case is kept under proper control.”

This summary is the belief of the most conservative of the outstanding clinicians^[126]in the United States engaged in diabetic work on a large scale.

Dr.E.P.Joslin，Boston，Mass.，who has one of the largest diabetic clinics in the world，has also found that，“The diabetic who is able to reduce his insulin is the diabetic who is absolutely faithful to diet and restricts gain in weight to a moderate degree.”

Joslin and his associates have carefully analysed the gain in weight and height of their thirty-two diabetic children under fifteen years of age.Their conclusions are：

（1）The gain in weight of the diabetic child treated with insulin resembles that of the normal child，but the diabetic child is still under weight for his age，though often not for his height.

（2）The increase in height of the diabetic child treated with insulin，though occasionally normal，is usually below that of the normal child.So far he has not grown tall like the normal child，either at the expense of growing thin or while being well nourished.

Of the 130 children treated with insulin，120 are still living，while of the 164 who did not receive insulin，there are 152 dead.Of the 120 still living，40%have either not increased or have actually decreased their insulin.Dr.Joslin believes that if the 60%who have had to increase their insulin had received similar treatment，they too would have been able to reduce their insulin.

Sixteen children under ten years of age who have taken insulin under Dr.Joslin's care for an average of two years，are all alive，and now their duration of life is more than three times the duration of life of diabetic children of similar age treated by Dr.Joslin prior to 1915.

Regardless of the severity of the disease，it has been found that by carefully adjusting the diet and the dose of insulin，all patients may be maintained sugar-free.Since this is possible，it is to be strongly advocated，because we have abundant evidence for the belief that there is regeneration of the islet cells of the pancreas when the strain thrown upon them by a high blood sugar is relieved.The increase in tolerance is evidenced by the decreasing-dosage of artificially administered insulin.In fact，in some moderately severe cases，the tolerance has increased sufficiently that they no longer require insulin.

Diabetes mellitus may be considered fundamentally as a disordered metabolism，primarily of carbohydrates，and secondarily of protein and fat.It is indisputably proven that for normal metabolism of carbohydrate in the body，adequate amounts of insulin are essential.It follows，therefore，that the treatment consists in giving just sufficient insulin to make up for the deficiency in the patient's pancreas.

Insulin enables the severe diabetic to burn carbohydrate，as shown by the rise in the respiratory quotient following the administration of glucose and insulin.It permits glucose to be stored as glycogen in the liver for future use.The burning of carbohydrate enables the complete oxidation of fats，and acidosis disappears.The normality of blood sugar relieves the depressing thirst，and consequently there is a diminished intake and output of fluid.Since the tissue cells are properly nourished by the increased diet，there is no longer the constant calling for food，hence hunger pain of the severe diabetic is replaced by normal appetite.On the increased caloric intake，the patients gain rapidly in strength and weight.With the relief of the symptoms of his disease，and with the increased strength and vigor resulting from the increased diet，the pessimistic，melancholy^[127]diabetic becomes optimistic and cheerful.

Insulin is not a cure for diabetes；it is a treatment.It enables the diabetic to bum sufficient carbohydrates，so that proteins and fats may be added to the diet in sufficient quantities to provide energy for the economic burdens of life.

John Macleod-Biography^[128]

John James Richard Macleod was born on September 6，1876 at Cluny，near Dunkeld，Perthshire，Scotland.He was the son of the Rev.Robert Macleod.When later the family moved to Aberdeen，Macleod went to the Grammar School there and later entered the Marischal College of the University of Aberdeen to study medicine.

In 1898 he took his medical degree with honours and was awarded the Anderson Travelling Fellowship，which enabled him to work for a year at the Institute for Physiology at the University of Leipzig.

In 1899 he was appointed Demonstrator of Physiology at the London Hospital Medical School under Professor Leonard Hill and in 1902 he was appointed Lecturer in Biochemistry at the same College.In that year he was awarded the McKinnon Research Studentship of the Royal Society，which he held until 1903，when he was appointed Professor of Physiology at the Western Reserve University at Cleveland，Ohio，U.S.A.

During his tenure of this post he was occupied by various war duties and acted，for part of the winter session of 1916，as Professor of Physiology at McGill University，Montreal.

In 1918 he was elected Professor of Physiology at the U-niversity of Toronto，Canada.Here he was Director of the Physiological Laboratory and Associate Dean of the Faculty of Medicine.

In 1928 he was appointed Regius Professor of Physiology at the University of Aberdeen，a post which he held，together with that of Consultant Physiologist to the Rowett Institute for Animal Nutrition，in spite of failing health，until his early death.

Macleod's name will always be associated with his work on carbohydrate metabolism and especially with his collaboration with Frederick Banting and Charles Best in the discovery of insulin.For this work on the discovery of insulin，in 1921，Banting and Macleod were jointly awarded the Nobel Prize for Physiology or Medicine for 1923.

Macleod had，before this discovery，been interested in carbohydrate metabolism and especially in diabetes since 1905 and he had published some 37 papers on carbohydrate metabolism and 12 papers on experimentally produced glycosuria.Previously he had followed the earlier great work of von Mering and Minkowski，which has been published in 1889，and although he believed that the pancreas was the organ involved，he had not been able to prove exactly what part it played.Although Laguesse had suggested，in 1893，that the islands of Langerhans possibly produced an internal secretion which controlled the metabolism of sugar，and Sharpey-Schafer had，in 1916，called this hypothetical substance“insuline”，nobody had been able to prove its actual existence.Others had made extracts of the pancreas，some of which had proved to be active in affecting the metabolism of sugar，but none of these products had been found reliable，until Banting and Best，jointly with Macleod，could announce their great discovery in February 1922.The process of manufacturing the pancreatic extract which could be used for the treatment of human patients was patented；the financial proceeds of the patent were given to the British Medical Research Council for the Encouragement of Research，the discoverers receiving no payment at all.Subsequently，the active principle of these earlier pancreatic extracts，insulin，was isolated in pure form by John Jacob Abel in 1926，and eventually it became available as a manufactured product.

Earlier，in 1908，Macleod had done experimental work on the possible part played by the central nervous system in the causation of hyperglycaemia^[129]and in 1932 he returned to this subject，basing his work on the experiments done by Claude Bernard on puncture diabetes，and Macleod then concluded，from experiments done on rabbits，that stimulation of gluconeogenesis^[130]in the liver occurred by way of the parasympathetic^[131]nervous system.

Macleod also did much work in fields other than carbohydrate metabolism.His first paper，published in 1899，when he was working at the London Hospital，had been on the phosphorus content of muscle and he also worked on air sickness，electric shock，purine^[132]bases，the chemistry of the tubercle bacillus and the carbamates^[133].

In addition he wrote 11 books and monographs，among which were his Recent Advances in Physiology（with Sir Leonard Hill）（1905）；Physiology and Biochemistry of Modern Medicine，which had reached its 9th edition in 1941；Diabetes：its Pathological Physiology（1925）；Carbohydrate Metabolism and Insulin（1926）；and his Vanuxem lectures，published in 1928 as the Fuel of Life.

In 1919 Macleod was elected a Fellow of the Royal Society of Canada，in 1923 of the Royal Society，London，in 1930 of the Royal College of Physicians，London，and in 1932 of the Royal Society of Edinburgh.During 1921-1923 he was President of the American Physiological Society，and during 1925-1926 of the Royal Canadian Institute.He held honorary doctorates of the Universities of Toronto，Cambridge，Aberdeen and Pennsylvania，the Western Reserve University and the Jefferson Medical College.He was an honorary fellow of the Accademia Medica，Rome，and also a corresponding member of the Medical and Surgical Society，Bologna，the SocietáMedica Chirurgica，Rome，and the Deutsche Akademie der Naturforscher Leopoldina，Halle，and Foreign Associate Fellow of the College of Physicians，Philadelphia^[134].

Macleod was a very successful teacher and director of research.His lucid lectures were delivered in an attractive manner and his pupils and research associates found him a sympathetic and stimulating worker，who demanded exact work and the humility that was a feature of his character.He would not tolerate careless work.He was much interested in the development of medical education and especially in the introduction of scientific methods of investigation into clinical work.

Outside the laboratory he was keenly interested in golf and gardening and the arts，especially painting.A sensitive，loyal and affectionate^[135]man of engaging personality，his serene^[136]spirit met with courage and optimism the painful and crippling disabilities which troubled the final years of his busy life.

Macleod was married to Mary McWalter.He died on March 16，1935.

【注释】

[1]insulin n.胰岛素

[2]urine n.尿

[3]glycogen n.肝糖，糖原质

[4]glycosuria n.糖尿

[5]transitory adj.短时间的

[6]pathologist n.病理学者

[7]coagulate v.凝结adj.凝结的

[8]pancreatic adj.胰腺的

[9]bowel n.肠

[10]ductless gland n.无管腺

[11]endocrine n.内分泌

[12]pancreas n.［解］胰腺

[13]antagonistic adj.反对的，敌对的

[14]hormone n.荷尔蒙，激素

[15]diabetic adj.［医］糖尿病的n.糖尿病患者

[16]respiratory adj.呼吸的

[17]quotient n.商，份额

[18]acidosis n.酸液过多症，酸毒症

[19]diabetes n.［医］糖尿病，多尿症

[20]coma n.昏迷

[21]preeminent adj.卓越的

[22]注：发现胰岛素

[23]heroism n.英雄品质，英勇，勇敢的事迹，豪侠的行为，英雄主义

[24]practitioner n.从业者，开业者

[25]orthopaedic adj.［医］整形外科的

[26]pharmacology n.药理学

[27]pancreas n.［解］胰腺

[28]ligature n.绷带

[29]silicosis n.［医］硅肺病

[30]liaison officer n.［军］联络官

[31]Newfoundland n.纽芬兰（岛）（加拿大一省）

[32]glycerin n.甘油，丙三醇

[33]subcutaneously adv.皮下地

[34]rectum n.直肠

[35]acinar adj.［解］腺泡的

[36]warrant n.授权，正当理由，根据，证明，凭证，委任状，批准，许可证vt.保证，辩解，担保，批准，使有正当理由

[37]acinus n.小核果，浆果，葡萄状腺

[38]gallstone n.［医］胆石

[39]foetal adj.似胎儿的，胎儿的

[40]intrauterine adj.子宫内的

[41]vacuo［拉］真空

[42]toluene n.［化］甲苯（染料或火药的原料）

[43]ether n.天空醚，大气，苍天，［物］以太

[44]depancreatize v.［医］截除胰腺，切除胰脏

[45]pancreatectomy n.［医］胰切除术

[46]autopsy n.（为查明死因而做的）尸体解剖，验尸

[47]sterile adj.贫脊的，不育的，不结果的，消过毒的，毫无结果的

[48]benzoic adj.安息香的

[49]isoelectric adj.［物］等电位的，零电位差的

[50]picric acid n.〈化〉苦味酸

[51]sulphuric acid硫酸

[52]diluted adj.无力的，冲淡的

[53]lipoid n.［化］类脂adj.类脂的

[54]chloride n.［化］氯化物

[55]vial n.小瓶vt.装入小瓶

[56]aseptic n.防腐剂adj.无菌的

[57]sterility n.不毛，不育，中性，无结果，无菌状态，思想贫乏

[58]sinus n.窦，穴，湾，凹处

[59]retinitis n.［医］视网膜炎

[60]urinalysis n.［医］尿分析，验尿

[61]caloric n.热量adj.热量的

[62]ketone n.［化］酮

[63]excrete vt.排泄，分泌

[64]hyperglycaemia n.多糖症，高血糖症

[65]nocturnal adj.夜的，夜曲的

[66]urinary adj.尿的，泌尿器的

[67]glycosuria n.糖尿

[68]cardinal adj.主要的，最重要的

[69]voracious adj.狼吞虎咽的，贪婪的

[70]ingest vt.摄取，咽下，吸收

[71]unappeasable adj.无法平息的，不能满足的

[72]hyperglycaemia n.多糖症，高血糖症

[73]premonitory symptoms n.地震征兆

[74]prostration n.俯卧

[75]perspiration n.排汗

[76]glucose n.葡萄糖

[77]diabetic adj.［医］糖尿病的n.糖尿病患者

[78]acetone n.［化］丙酮

[79]ferric adj.铁的，含铁的，［化］（正）铁的，三价铁的

[80]chloride n.［化］氯化物

[81]drowsiness n.睡意

[82]catheterization n.导管插入（术）

[83]evacuated adj.撤退者的

[84]purgation n.净化，洗涤，清洗，洗罪

[85]prognosis n.预后

[86]gangrene n.坏疽，腐败堕落的

[87]pneumonia n.［医］肺炎

[88]intoxication n.陶醉

[89]acidosis n.酸液过多症，酸毒症

[90]coma n.昏迷

[91]ingestion n.摄取

[92]sclerosis n.［医］硬化症，硬化，硬结

[93]jeopardy n.危险，危险

[94]nitrous oxide n.［化］一氧化二氮，笑气（=laughing gas）

[95]anaesthetic n.麻醉剂adj.麻醉的

[96]appendectomy n.阑尾切除术

[97]cholecystectomy n.［医］胆囊切除术

[98]tonsillectomy n.［医］扁桃腺切除术

[99]acidosis n.酸液过多症，酸毒症

[100]hyperglycaemia n.多糖症，高血糖症

[101]glycosuria n.糖尿

[102]excrete vt.排泄，分泌

[103]polyuria n.［医］多尿（症）

[104]polyphagia n.多食症，杂食性

[105]polydipsia n.［医］烦渴

[106]lassitude n.疲乏

[107]respiratory adj.呼吸的

[108]appendectomy n.阑尾切除术

[109]insomnia n.失眠，失眠症

[110]emaciated adj.瘦弱的，衰弱的

[111]respiratory adj.呼吸的

[112]cardiovascular adj.心脏血管的

[113]tapioca n.（食用）木薯粉，［植］木薯

[114]glycosuria n.糖尿

[115]transitory adj.短时间的

[116]regeneration n.再生，重建

[117]pancreas n.［解］胰腺

[118]acidosis n.酸液过多症，酸毒症

[119]pancreatic adj.胰腺的

[120]sleigh n.雪橇v.乘雪橇，用雪橇运输

[121]acinar adj.［解］腺泡的

[122]periphery n.外围

[123]lobule n.小裂片，小叶

[124]proliferate v.增生扩散

[125]granule n.小粒，颗粒，细粒

[126]clinician n.［医］临床医生，临床教师

[127]melancholy n.忧郁adj.忧郁的

[128]注：发现胰岛素

[129]hyperglycaemia n.多糖症，高血糖症

[130]gluconeogenesis n.［生化］糖质新生

[131]parasympathetic n.副交感神经，副交感神经系统

[132]purine n.［生化］嘌呤，咖啡碱

[133]carbamate n.［化］氨基甲酸盐

[134]Philadelphia n.费城（美国宾西法尼亚州东南部港市）

[135]affectionate adj.亲爱的，挚爱的

[136]serene adj.平静的