Acta Diabetologica https://doi.org/10.1007/s00592-022-02014-7 ORIGINAL ARTICLE On the occasion of the centennial of insulin therapy (1922–2022), II‑Organotherapy of diabetes mellitus (1906–1923): Acomatol. Pancreina. Insulin. Alberto de Leiva‑Hidalgo1,2 · Alejandra de Leiva‑Pérez2 Received: 2 October 2022 / Accepted: 30 November 2022 © The Author(s) 2022 Abstract Aims The general objective has been the historiographical investigation of the organotherapy of diabetes mellitus between 1906 and 1923 in its scientific, social and political dimensions, with special emphasis on the most relevant contributions of researchers and institutions and on the controversies generated on the priority of the "discovery" of antidiabetic hormone. Methods We have analyzed the experimental procedures and determination of biological parameters used by researchers during the investigated period (1906–1923): pancreatic ablation techniques, induction of acinar atrophy with preservation of pancreatic islets, preparation of pancreatic extracts (PE) with antidiabetic activity, clinical chemistry procedures (glycemia, glycosuria, ketonemia, ketonuria, etc.). The field investigation has included on-site and online visits to cities, towns, build- ings, laboratories, universities, museums and research centers where the reported events took place, obtaining documents, photographic images, audiovisual recordings, as well as personal interviews complementary to the documentation consulted (primary sources, critical bibliography, reference works). The documentary archival sources have been classified according to theme, including those consulted in situ with those extracted online and digitized copies received mainly by email. Among the many archives contacted, those listed below have been most useful and have been consulted on site and on repeated vis- its: National Library of Medicine-Historical Archives (Bethesda, MD, USA); Archives, University of Toronto and Thomas Fisher Rare Books Library (Toronto, Ontario, Canada); Francis A. County Library of Medicine, Harvard University (Boston, Mass, USA); Zentralbibliothek der Humboldt-Universität (Berlin, DE), Geheimarchiv des Preuβischen Staates (Berlin, DE); Landesamt für Bürger—und Ordnungsangelegenheiten (LABO) (Berlin, DE); Arhivele Academiei Române şi Universitǎții Carol Davila (Bucharest, RO). Main results and conclusions A) The European researchers Zülzer (Z Exp Path Ther 23:307–318, 1908) and Paulescu (CR Seances Soc Biol Fil 85:558, 1921) meet the requirements of the priority rule in the discovery of the antidiabetic hormone. B) Factors of socioeconomic and political nature related with the First World War and the inter-war period delayed the process of purification of the antidiabetic hormone in Europe. C) The Canadian scientist J. Collip, University of Alberta, temporarily assimilated to the University of Toronto, and the American chemist and researcher G. Walden, with the expert collaboration of Eli Lilly & Co., were the main authors of the purification process of the antidiabetic hormone. D) The scientific evidence, reflected in the heuristics of this research, allows to assert that the basic investigation carried out by the Department of Physiology of the University of Toronto, directed by the Scottish J. Macleod, in conjunction with the clinical research undertaken by the Department of Medicine of the University of Toronto (W. Campbell, A. Fletcher, D. Graham) made it possible in record time the successful treatment of patients with what was until then a deadly disease. Keywords Pancreatic extracts · Organotherapy · Acomatol · Pancreina · Insulin · Patents Georg Ludwig Zülzer (1870–1949): Acomatol Managed by Antonio Secchi. Georg Ludwig Zülzer (also spelled Zuelzer) was born in Alberto de Leiva-Hidalgo and Alejandra de Leiva-Pérez have 1870 in Berlin, and studied in Freiburg and Berlin, where contributed equally. he defended his doctoral thesis in 1893 at the Friedrich Extended author information available on the last page of the article Wilhelm University (now Humboldt University) [1]. He 13 Vol.:(0123456789) Acta Diabetologica furthered his postgraduate training working as an assistant 1 g of the extract) a reduction in daily glycosuria from 28.6 with Raphaël Lépine and Léon Bouveret in Lyon, with Carl to 19 g was recorded [6]. H. von Noorden, Albert Neisser and Hermann Eichhorst in Frankfurt am Main and with Franz Riegel in Gießen, where Treatment of eight patients with pancreatic extracts he also met Ferdinand Blum, who would become a collabo- rator and a friend. In 1900, Zülzer started a private practice Between 1906 and 1908, Zülzer was able to treat eight as an internist in Berlin. patients with diabetes from different hospitals with his PE. They showed a certain degree of clinical improvement and Zülzer's experiences with pancreatic extracts a reduction in glycosuria and ketonuria, although often in experimental diabetes accompanied by side effects such as fever, sweating, vomit- ing, stomatitis and muscle hypertonia [7]. In 1903, influenced by Blum’s description of adrenergic dia- One special case was a 6-year-old boy with severe diabetes, betes [2], Zülzer began experimental studies with pancreatic ketosis, heavy glycosuria and malnutrition. On July 14, 1907, he and adrenal extracts, using the rabbit as an experimental received intravenously an emulsion containing 1 g of PE. Body animal. During the first years, he used for his experiments temperature rose immediately after the injection to 38.4 °C, with the laboratory of the institute headed by Professor T. W. associated vomits. On July 16, his clinical condition improved; Engelmann in Berlin. the boy gained weight, ketones disappeared and glycosuria Zülzer was the first to demonstrate that urinary glucose decreased (Fig. 1). On August 1, after the injection of 1 g of PE, excretion following adrenalin injection correlated with an a new peak of fever developed (39.2 °C) but glucose excretion elevation of blood glucose levels [3], and noted that the decreased and ketone bodies disappeared again. After 48–72 h simultaneous administration of a pancreatic extract (PE) pre- without available extract, the clinical and chemical parameters vented the hyperglycaemic effect of adrenalin. He measured deteriorated. Zülzer could not produce new doses; the boy was the potency of the extract by the amount needed to neutralize discharged and died some days later. the hyperglycemia caused by the administration of one mil- Zülzer presented his results at a meeting of specialists ligram of adrenalin [4]. in Internal Medicine held on 15 June 1908 in Berlin, and This spurred his interest in investigating the effects of received favorable comments in a landmark textbook on equine, bovine and porcine pancreatic extracts on glycemia endocrinology written by Professor Arthur Biedl of the and glycosuria in the pancreatectomized dog. In total, Zülzer University of Vienna [8]. devoted twelve years (from 1902 to 1914) to researching the biological effects of PE. His work resulted in seven publica- Obtaining patents for the pancreatic extract: tions and three patents [5]. Acomatol Initially, the experiments were financed by Chemische Fabrik Auf Actien (formerly E. Schering, now Bayer Health- On May 8, 1907, Chemische Fabrik auf Actien applied for a pat- Care Pharmaceuticals), that provided Zülzer with the help ent of Zülzer’s extract (Acomatol) to the German Patent Office of laboratory technicians Max Dohrn and Anton Marxer. As (Kaiserliches Patentamt). Patent number 201383 was granted an extraction medium, Zülzer used alcohol, which resulted on September 20, 1908, for a “pancreatic extract preparation in the precipitation of a large number of proteins. However, to be used to control severe hyperglycemia and diabetic coma”. he did not succeed in removing all contaminating proteins. On April 16, 1908, an application was filed to the Great Britain (To attain a higher degree of purification of the extract, it Patent Office. On March 11, 1909, Patent 8514 was granted is necessary to use fractional precipitation with increasing for the “manufacture of a pancreas preparation suitable for the concentrations of alcohol). To achieve a complete separation treatment of diabetes” On May 6, 1908, a new application was of the proteins, Zülzer heated the filtrate and added sodium filed to the United States Patent Office. USA patent 1,027,790 chloride. The last step in the extraction procedure was to was granted on May 28, 1912 (Fig. 2) [9]. vaporize the alcoholic filtrate under vacuum; the residue In 1909, J. Forschbach reproduced Zülzer’s experiments obtained in powder form contained the active substance [5]. in Minkowski’s laboratory in Breslau, with three dogs and In these experiments Zülzer macerated glandular tissue three patients. Glycosuria decreased and Forschbach noted in mortar on sand, mixed it with silica gel and pressed it that “Zülzer was the first to produce, successfully, from the on a tissue filter; he treated the liquid extract with alcohol, pancreas, a preparation that eliminates sugar excretion in precipitating contaminating proteins. The experiments were a shorter or longer period by intravenous administration”. carried out in 1905, but only published in 1908. The first one However, Forschbach ceased human experimentation after was performed in August. The dog weighed 8 kg. Diet con- observing high fever and other toxic effects in two patients sisted of 300 g of meat and 100 g of oil per day. After intra- [10]. As a consequence, Chemische Fabrik auf Actien venous administration of 5 ml of the preparation (containing decided to withdraw its investment to the research. 13 Acta Diabetologica Fig. 1  Photography of Georg L. Zülzer; unknown author and date. Historical Archive, Humboldt University, Berlin. Public domain. Case 3: A 6-year-old boy with severe diabetes, ketonuria, heavy glycosuria and malnutrition. Effect of the pancreatic extract [7] Fig. 2  G.L. Zülzer. German (1908), British (1909) and USA (1912) patents for Acomatol. Public domain 13 Acta Diabetologica Collaboration between Georg Zülzer and Camille 5- To heat the solution (80 °C), perform a dialysis, filter the content Reuter with a sterilized Berkefeld-Donde filter and dilute the final prepara- tion of the extract in 0.45 NaCl In 1911, Zülzer, who in 1908 had been appointed Chief Phy- Volumes of 15–50 ml of the pancreatic extract were slowly adminis- sician at Hasenheide Hospital in Berlin, signed an agree- tered to dogs intravenously. Blood samples (5 mL) were collected ment with the Swiss pharmaceutical company Hoffmann- at intervals. A significant number of dogs developed severe con- La Roche (now Roche) to further research on pancreatic vulsions. In 1914 when blood glucose was measured by a newly extracts. With its financial support, Zülzer was able to set up introduced micro-method, convulsions were directly related to hypoglycemia. Blood glucose decreased by 50% or even further in an experimental laboratory at Hasenheide Hospital. Camille most dogs (in one animal it went down to 17 mg/dL, before dying Reuter, chemical engineer working in Hoffmann-La Roche’s of hypoglycemia). Acute toxicity and other side effects were not research laboratory in Grenzach (Baden-Württemberg), present in these experiments. Control experiments (precipitation joined the project. of the pancreatic extract with lead acetate) gave negative results. Similar results were achieved with glandular extracts from differ- Reuter’s main tasks were to purify the extract and to ent animal sources [11, 12] boost the manufacture at large scale following a new pro- tocol. He used the hydraulic press and the Buchner funnel [*kieselguhr: diatomaceous earth]. in the process of elaborating the new pancreatic extract in Until the introduction of the first blood glucose micro-deter- collaboration with Zülzer (Fig. 3) [11]: minations in 1913 by Ivar Bang [13], previous procedures had major disadvantages, such as the need for prior separation of 1- To grind the fresh horse’s pancreas with sand after the addition of proteins from the sample and a laborious protocol that required sodium bicarbonate, mixing the product with kieselguhr [*] two days to complete blood glucose quantification [14, 15]. The 2- To apply a pressure of 350 atmospheres in a hydraulic press, col- publication of the Hagedorn-Jensen micro-method would still lecting the filtrate in a Buchner system take another five years to develop [16]. 3- To mix the fluid with alcohol and filter off the sediment Before the end of 1914, the laboratory at Grenzach had 4- To evaporate the filtrate at low temperature in a vacuum, add water processed over 100 kg of animal pancreas. To maintain the and neutralize the solution blood glucose lowering-effect, the pancreatic extract had to be administered at intervals of 3–4 h. In August 1914, Zül- zer’s research was interrupted since he was called to serve as a military doctor in World War I. Hoffmann-La Roche cancelled the project, concerned about the war, the potential risks of hypoglycemia and the need for close surveillance of the patients. After the war, Zülzer returned to Berlin in 1918, weakened by malaria. He became Chief of Medicine at Lankwitz Hospital, a municipal institution of the Union of Health Insurance Companies in Berlin (Verband der Krank- enkassen), with no affiliation to the university. Georg L. Zülzer claimed his priority right on the discovery of the antidiabetic hormone. Other scientists’ views on Zülzer's activities and the relevance of Acomatol In the article that described the clinical cases treated with insulin by the University of Toronto research team, pub- lished in the British Medical Journal in January 1923, the authors referred to Zülzer's previous research, stating that in 1908 he treated six diabetic patients in whom he obtained a reduction and even a disappearance of glycosu- ria and ketonuria by intravenous administration of the PE of his invention; however, due to the extreme lability of the extract and the serious side effects, Zülzer's preparation was not used in the clinical setting [17]. Fig. 3  Portrait of Camille Reuter. Unknown date and author. (Cour- On October 16th, 1923, Zülzer circulated, through tesy of V. Jörgens) the international press agent Transatlantic, a typewritten 13 Acta Diabetologica message entitled "The overcoming of diabetes. New facts Zülzer and Macleod exchanged postal correspondence in about insulin", in which he claimed his priority right on October–November 1927. the discovery of Acomatol, a preparation that was suc- In 1968, Ian Murray, Professor of Physiology at Anderson cessfully used to treat severe forms of diabetes, including College of Medicine and Head of the Department of Meta- coma, years ahead of insulin [18]. bolic Diseases, University of Glasgow, a founding member of the International Diabetes Federation, initiated an inves- (…) Without intending to deny the painstaking efforts of Dr. Banting tigation into the priority of antidiabetic hormone isolation, or to lessen his merits for the medical science I venture to say that after which he asserted his duty to vindicate the merits of the actual discovery of the preparation has been made by me a decade ago under the name of Acomatol Georg Zülzer as the first to achieve a pancreatic preparation (…) The preparation is able to reduce the content of saccharine mat- of potential therapeutic value [23]. ter in the diabetic human body is an hitherto unheard of degree, relieving the patient even from the most severe form of diabetes, the so-called coma diabeticorum Criticisms of Zülzer (…) Acomatol was completed after prolonged studies in my labo- ratories. The quantity production should just be arranged as the World War began. Insulin shows all the characteristic qualities of A. Labhart, Department of Medicine, University of Zurich, my Acomatol reviewed in detail in a 1978 publication the experiences (…) It was already in the year 1908 that I have cured the first strongly of G.L. Zülzer in experimental diabetes and stated: “Zue- diabetic persons with Acomatol lzer for 12 years doggedly pursued his idea and yet at the (…) The preparation showed then still some technical imperfections end fell short. He had the substance—the Acomatol, as which however were eliminated in 1914 he called it—in hand, yet he didn't dare to make use of it because of the misinterpretation of its effects” [24]. Fate Zülzer expressed his regret at the news that the 1923 Nobel was against him: lack of available micro-methods for blood Prize in Physiology or Medicine had been awarded to J.J.R. glucose determination, envious colleagues, difficulties in Macleod and F.G. Banting of the University of Toronto. He his academic promotion, suspension of funding, and worst claimed his priority right on the discovery of the antidia- of all, the outbreak of the Great War, ruined Zülzer’s most betic hormone in an article published in the German journal promising project. Hasenheide became a military institu- Medizinische Klinik, in which he criticized the fact that his work tion and he himself, having joined the army as a doctor, had been neglected by the German institutions [19]. ended his diabetes research. Zülzer was determined to obtain a high academic posi- tion at the University of Berlin, where his father had been J.J.R. Macleod publicly acknowledged Zülzer's Chair in the Department of Internal Medicine. During two research on pancreatic extracts research stays in Berlin in April and August 2015, we con- sulted the existing documentation on Zülzer in the Histori- In a lecture at the 11th International Congress of Physiology cal Archives of Humboldt University, and in the Secret in Edinburgh in 1923 [20], J.J.R. Macleod publicly acknowl- Archives of the Prussian State (Geheimes Staatsarchiv edged Zülzer’s work in developing PE with antidiabetic activity, Preuβischer Kulturbesitz—GStA PK). The documents demonstrating in 1908 the ability of these extracts to neutralize in both archives reveal a conflicting situation between hyperglycemia induced by the administration of epinephrine Zülzer and the academics responsible for the admission as well as clinical manifestations of human diabetes mellitus. and promotion of professors at the Faculty of Medicine. Macleod also acknowledged Zülzer in his Nobel Lecture [21]: Between 1919 and 1932, the Medical Faculty voted on several occasions against his nomination as privatdozent. (…) In 1907 Zuelzer published results which must be considered, in Wolf Wilhelm Zülzer (Georg’s son, a paediatric patholo- the light of what we know now, as really demonstrating the pres- ence of the antidiabetic hormone in alcoholic extracts of pancreas gist, director of the Michigan Research Center, Detroit (…) But unfortunately, even though several diabetic patients were Children's Hospital, as well as Director of the Division of benefited by administration of the extracts, Zuelzer himself was Hematologic, Heart and Lung Diseases at the US National discouraged in continuing them because of toxic reactions in the Institutes of Health) noted in 1985: “Medical historians treated patients have written that Georg Zülzer was defeated by narrow- mindedness and uncooperativeness of his academic breth- In his book entitled Carbohydrate Metabolism and ren (…). He was a loner by temperament, as well as an Insulin [22, p. 56], Macleod stated that offender against accepted dogma” [25]. Finally, on March 19, 1932, the Prussian Minister of Zuelzer came very near to the discovery of a method for preparing insulin, as we know it today Science, Art and Education, Adolf Grimme, assigned him 13 Acta Diabetologica the Chair of Pathology and Therapy of Infectious Diseases, hypoglycemic effects; it revealed an increase in blood glu- appointment subject to the possibility of revocation [26]. cose levels by approximately 20%. A logical explanation for After Hitler's rise to power, Zülzer lost his position at this result could not be provided at that time [29]. the University, owing to his Jewish origins. Bernhard Rust, the new Minister of Health, Science and Public Education, revoked Zülzer’s teaching license on November 24, 1933 [26]. Zülzer was also expelled from Lankwitz Hospital John R Murlin (1874–1960) and all his properties were confiscated. He decided to flee the Nazi regime and in 1934 went to exile to the USA. He John Raymon Murlin and Benjamin Kramer, researchers worked in the city of New York as a clinician in private of Cornell University, initially thought that the decreased practice until his retirement to a nursing home in 1947. Zül- urinary excretion of glucose, found after the administration zer died on October 15th, 1949, as a consequence of heart of their preparations containing a dual extract of pancreas failure due to chronic myocarditis. His grave in the White and duodenal mucosa, was due to changes in renal perme- Chapel Memorial Park Cemetery, in Troy (Michigan) bears ability, rather than to a hormonal effect. Their manuscript the following inscription: “The first physician to bring dia- was published in the year 1913 [30]. betic patients out of terminal coma with his extracted pan- Years later, they noted that the disappearance of glyco- creas preparation”. suria ran parallel to a decrease of blood sugar levels and concluded that the pancreatic extract actually contained the internally secreted active principle [31]. Ernest L. Scott (1877–1966) In 1922, Murlin and coworkers observed that pancreatic extracts caused tissue toxicity with ulceration at the injection Ernest Lyman Scott, from the Department of Physiology at sites, resulting in death of some dogs [32]. Columbia University, was initiated in diabetes research in In cooperation with C. Sutter, Murlin reported the case 1911 as candidate to M. A. His first publication appeared in of a diabetic patient with ketosis treated at the Rochester 1913. Scott suggested that the external digestive enzymes of General Hospital in July 1922 with pancreatic extract admin- the pancreas had a deleterious effect on the active principle istered through a gastrointestinal catheter and by the oral of the internal secretion of the gland. It was hoped that the and subcutaneous routes. Only in this latter case could gly- presence of these enzymes could be eliminated by the atro- cosuria and ketonuria be decreased. On July 26, 1922 blood phy of the gland which followed complete ligation of the glucose level went down from 513 to 241 mg/dL [33]. ducts. Nevertheless, after various attempts in the dog, the method was abandoned as impractical [27]. In subsequent work, Scott intended to render inactive Israel Kleiner (1885–1966) these enzymes by the action of highly concentrated ethanol. For this purpose, he elaborated the following protocol: Israel S. Kleiner and S.J. Meltzer, from the Department of Medical Research at the Rockefeller Institute, investigated The glands were extracted during 72 h at 35–40 °C; then filtered and the effects of intravenous injection of a pancreas emulsion evaporated to dryness and the residue extracted with ether at room into intact and depancreatized animals. They reported that temperature. Once discarded the fraction extracted with ether, the residue was dissolved in 95% ethanol. After a new evaporation in the group of healthy animals, blood glucose concentration process, the extract was diluted in saline solution and ready for levels became equal to those seen before glucose admin- injection istration at 90 min of the infusion of isotonic glucose. In Scott also prepared watery extracts. After handling the alcoholic untreated pancreatectomized animals, however, glucose extracts as above, the preparation was evaporated to dryness under levels 90 min after glucose administration were more than a vacuum and extracted in absolute alcohol at 38 °C. After pouring off, the residue was extracted with water rendered acid by acetic two times higher than the original value. In a third group acid. Then the preparation was filtered and evaporated. Before of animals, the addition of PE allowed for a very close to injecting, the ethanol was eliminated and saline solution added normal response. These experiments suggested that internal pancreatic secretion contributed to the rapid disappearance In general, the intravenous injections of the PE to pancre- of circulating glucose [34]. atectomized dogs temporarily decreased the sugar excretion. In 1919, Kleiner published a set of experiments con- The interpretation suggested by Scott at this point was that ducted between 1915 and 1919 supporting the existence of the PE could reduce the output of sugar by a toxic action, internal pancreas secretion and showing beneficial effects rather than therapeutic [28]. for the treatment of experimental diabetes. Intravenous Scott performed additional experiments with cats. Surprisingly, the administration of PE did not show 13 Acta Diabetologica administration of pancreatic emulsion achieved a highly Paulescu invented an original technique of experimental significant blood glucose reduction in most of the 16 dogs hypophysectomy, entering the cranial vault laterally, lift- with diabetes after pancreatectomy. Submaxillary gland ing the temporal lobe which allowed optimal visualization emulsions (control group) did not change blood glucose of the gland and complete ablation. This procedure was levels. No relevant toxic effects occurred. Unfortunately, successful in twenty-four experimental cases [45, 46]. The Kleiner left the Rockefeller Institute and abandoned this adoption by Harvey Cushing of this new surgical approach area of research [35]. resulted in much lower mortality rate in human surgery In 2010, Jeffrey M. Friedman carried out a statistical in comparison with the previous transpharyngeal method, analysis of the raw data presented by I.S. Kleiner. The study facilitating the “carefold avoidance of the total removal reaffirmed the validity of Kleiner's conclusions, confirm- of the anterior lobe” [47, 48]. Lewis Redford, Surgical ing the existence of an endocrine pancreatic secretion with Fellow working with Cushing, made the following obser- antidiabetic action. Unfiltered aqueous pancreatic extract vation: “The best work has been done in Romania by the generated from fresh adult pancreas, diluted in physiologi- physiologists Nicolae Paulescu, who has developed what cal saline and slowly administered intravenously was effec- seemed to be an ideal surgical approach to the pituitary of tive in inducing a temporary decrease in blood glucose to dogs” [49, p. 208]. The inspiration of Paulescu enabled normal values and elimination of glycosuria in the dog with Cushing to carry out extensive animal studies to evaluate post-pancreatectomy diabetes [36]. the functions of the pituitary gland [50]. Paulescu investigated the glycogenic function of the liver, heart and muscle, in fasting and postprandial stages Nicolae C. Paulescu: The discovery [51]. Years later, he persued additional research on the of pancreina metabolism of glycogen in diabetes induced by pancreas ablation [52] and by floridzine [53]. In collaboration with Paulescu, outstanding endocrine researcher Lancereaux, he developed a four-volume Traité de Méde- cine (3,870 pages). The first volume was printed in 1903, Nicolae Constantin Paulescu was born in Bucharest on and the three additional ones in 1908, 1912 and 1930, October 30, 1869. He went to Paris in 1888 for his medical respectively [54]. In different chapters, this comprehensive education. In 1891 he obtained a medical post as an extern text covered many aspects of the biology of the pancreatic at the Hôtel-Dieu. In 1897 he defended his doctoral thesis gland (embryology, anatomy, histology, physiology, clini- “Recherches sur la structure de la Rate” and was appointed cal assessment, pancreatic disorders from various causes Deputy Surgeon at the Notre Dame du Perpétuel-Sécours and the influence of carbohydrates, lipids and proteins on Hospital. Paulescu entered the Faculty of Science, obtaining glycogen metabolism [37]. the degrees in Biological Chemistry and General Physiol- ogy, a doctorate in Natural Sciences [37] and, finally, Doctor Tecnique and metabolic effects of pancreatectomy from the University of Paris, the Sorbonne (1901) [38]. Dur- ing these years he was supervised by Étienne Lancereaux Paulescu was obliged to close the laboratory in 1916, and Albert Dastre, with whom he initiated, already in when Bucharest was occupied by Austrian and German 1889, investigations with the aim to isolate the antidiabetic troops. All documents he wrote between 1916 and 1919 pancreatic substance [37]. He was introduced in surgical were published in the following years. One of these docu- procedures for experimental research and investigated the ments was the three volumes of the Traité de Physiologie physiology of the thyroid [39] and adrenal glands [40], the Médicale [55], a textbook where he described on pages significance of albuminuria [41] and the treatment of arterial 313–314 of volume II an original procedure of pancreas aneurysms [42, 43]. ablation he carried out in the experimental laboratory Paulescu was offered the post of professor and Chair before August 1916. of Medicine, at the University of Freiburg (Switzerland). Instead, he decided to return to Romania in 1900, where he “Une condition expérimentale “sine qua non” est que cette abla- was appointed Assistant Professor of Physiology and then tion soit tout a fait complète. Pour remplir ce postulat, nous avons imaginé un procédé particulier –qui nous a donné entière satisfac- promoted to Professor of Physiology in 1905 [44]. tion (…). During the first fifteen years in Bucharest, he organized L’opération dure environ une demi-heure, à savoir: 5 minutes, pour the laboratory of Experimental Physiology, made important enlever le lobe du foie; 15 minutes, pour extirper le pancréas; 10 developments in endocrine physiology and experimental sur- minutes, jusqu’à la fin du pansement. gery and worked as clinician in the Hospital Saint Vincent de Paul. 13 Acta Diabetologica Pour pouvoir opérer dans des bonnes conditions, il faut choisir des De même, on reprend 25 cc et 10 cc de sang carotidien, pour doser la chiens jeunes, qui pèsent de 8 à 12 kg, car ils ont tissu conjonctif glycose et l’urée, immédiatement après l’injection, puis une heure peu résistant et des lobules pancréatiques très friables, qui peuvent plus tard, et ainsi de suite. être facilement déchiquetés. De plus, chez ces animaux, l’extrémité La séparation de la glycose du sang se fait par l’alcool à 96º. Son splénique de la glande n’est pas trop profondément située et on peut dosage dans le sang et dans l’urine, s’effectue par le procédé indi- l’enlever, sans grande peine”. qué plus haut (T.I., p. 99). La séparation de l’urée du sang se fait de la même façon que celle English translation: de la glycose. Son dosage s’effectue à l’aide de l’hypobromite de soude”. A "sine qua non" experimental condition is that this ablation is complete. To fulfil this postulate, we have devised a particular English translation: procedure which has given us complete satisfaction (...). The operation lasts about half an hour, namely: 5 minutes to remove Afterwards, taking meticulous aseptic precautions, this gland is the liver lobe; 15 minutes to remove the pancreas; 10 minutes until chopped in a Latapie grinder, sterilised in the oven. Then, ten times the end of the dressing. its weight of sterilised distilled water is added to the preparation, To be able to operate in good conditions, it is necessary to choose and after shaking it several times, it is placed in the icebox. young dogs, weighing between 8 and 12 kg, because they have After 24 hours, this diluted hash is filtered through a double sterilised weak connective tissue and very friable pancreatic lobules, which tarlatan compress and 7 per 1,000 of NaCl is added to the filtrarum. can be easily shredded. In addition, in these animals, the splenic Thus prepared, the extract is introduced into a sterilised Mohr's extremity of the gland is not too deeply located and can be removed burette, connected by a rubber tube to a cannula, and it is pushed without much difficulty. into an external jugular vein by the force of gravity, with an average speed of 100 cc for 15 to 20 minutes (...). Paulescu demonstrated the requisite of complete ablation The attenuation of the diabetic syndrome begins immediately after to induce experimental diabetes: “Total extirpation of the the injection (...). pancreas is immediately followed by an intense and very But before the injection, 24 cc of blood are taken from the carotid grave diabetes…partial ablation of the pancreas does not artery to measure the glucose and 10 cc of blood to measure the urea. bring about diabetes if the remaining fragment is greater Similarly, 25 cc and 10 cc of blood from the carotid artery are taken than a tenth of the gland”. He also observed that ligature of to measure glucose and urea immediately after the injection, and the pancreatic ducts did not induce glycosuria, neither the again one hour later, and so on. surgical removal of the duodenal region of the gland [55]. Glycose is separated from the blood by using 96º alcohol. Its determi- nation in blood and urine is carried out by the procedure indicated above (T.I., p. 99). Development of the pancreatic extract. Intravenous The separation of urea from blood is done in the same way as for glycose. Its determination is carried out with the help of sodium administration to the experimental animal hypobromite. In pages 321–327 of the Traité de Physiologie Médicale, Paulescu transferred the fresh gland under aseptic condi- Paulescu described the procedure to develop the PE, fol- tions to a Latapie mortar for its fragmentation, adding ten lowed by four experiments performed in the interval from volumes of sterilized water, followed by agitation and refrig- November 12 to December 29, 1916 [55]. erated storage. Twenty-four hours later, the mixture was fil- tered through a double sterilized tartalan compress, adding a “Puis, en prenant des précautions minutieuses d’aseptique, on hache cette glande dans un broyeur Latapie, stérilisé au four. Ensuite, on solution of sodium chloride (0,7%). Then the aqueous extract ajoute, à ce hachis, dix fois son poids d’eau distillée stérilisée, et was administered to the pancreatectomized dog via a sterile après l’avoir agité à plusieurs reprises, on le place à la glacière. Mohr burette, cannulated into a central or peripheral vein at Au bout de 24 heures, on filtre ce hachis dilué, à travers une double a rate of 100 mL in 15–20 min. The attenuation of the dia- compresse de tarlatane stérili-sée, et on ajoute, au filtrarum, 7 pour betic symptoms appeared immediately after the intravenous 1.000 de NaCl. injection. Ainsi préparé, l’extrait est introduit dans une burette Mohr stérilisée, reliée, par un tube en caoutchouc, à une canule, et il est poussée dans una veine jugulaire externe par la force de la gravi-té, avec une Pancreina vitesse moyenne de 100 cc pour 15 à 20 minutes (…). L’attenuation du syndrôme diabétique commence immédiatement Paulescu presented the results of the administration of his aprés l’injection (…). pancreatic extract in three scientific meetings of the Roma- Mais, avant l’injection, on prend, de la carotide, 24 cc de sang, pour nian Society of Biology (branch of the European Society of doser la glycose, et 10 cc de sang pour doser l’urée. Biology) in Cluj (April 21), Iaşi (May 19) and Bucharest (June 23). As a consequence, four short articles covering nine experiments were published in the issue of Comptes 13 Acta Diabetologica Fig. 4  Left Nicolae Paulescu published four short articles covering administration of pancreina on the glycemic profile in the pancreatec- nine experiments in CR Société de Biologie, showing the results of tomized dog; c serial study of the variations in glycemia and gluco- the administration of his pancreatic extract to dogs with experimen- suria over 24 h induced by the injection of pancreina; d evaluation of tal diabetes. Right The four graphs are from his long original article, the intensity of the glycemic response in the animal with experimen- published in August 1921. They illustrate: a the development of dia- tal diabetes depending on the amount of pancreatic extract adminis- betes after complete pancreatic ablation; b the effect of parenteral tered [60]. (Courtesy of Constantin Ionescu-Tirgoviste) rendus des Séances de la Société de Biologie et de ses fil- vein generated transient hypoglycemia and reduction of ials (Paris), dated on July 23, 1921. (Fig. 4). Main effects urea levels in blood and urine [59]. observed were: On August 31, 1921, Paulescu published a large and 1. The total ablation of the pancreas in the dog determined comprehensive article, already accepted in June 1921 by increased content of glucose, urea and ketone bodies in Archives Internationales de Physiologie (Fig. 5) [60]. blood and urine. The injection of the pancreatic extract In the document, a summary of experiments performed induced a temporary suppression of hyperglycemia, dis- along 20 years of research, demonstrated the hypoglycemic appearance of glycosuria, and decreased concentration effect of the aqueous extract, in association to the reduc- of urea and ketone bodies in blood and urine. Similar tion of glycosuria and ketonuria in pancreatectomized dogs. effects were observed when the extract was injected in a The manuscript displays the technique to achieve a sterile branch of the portal vein (mesenteric or splenic venula) extract, the methods used to estimate blood glucose (proce- or in the jugular vein [56]. dure of Pflüger), glycosuria (as Claude Bernard), ketones (as 2. The effect on blood sugar started soon after the injec- Deniges), urea (sodium hypobromite), the clinical manifesta- tion, reached the maximum levels in about two hours tions observed in the animals, informed autopsies, registry and disappeared in approximately twelve hours. A paral- of body temperature, exact date and timing of each step and lel decrease of glucose excretion was associated to the duration of each experiment. In the article he included new reduction of the hyperglycemia [57]. information focused in control experiments. The adminis- 3. The quantitative changes were relative to the amount of tration of normal saline and splenic extract did not reduce gland being administered. With a third of the pancreas, a the hyperglycemia of pancreatectomized dogs. The admin- slight reduction was observed; with two thirds, the effect istration of the pancreatic extract, that he named pancreina, was markedly intense [58] generated fever as the main side effect. Paulescu induced a 4. In the non-depancreatized, non-diabetic dog with intact bout of fever (up to 38.9 °C) to the animals by the intraspinal pancreas, the administration of the extract in the yugular injection of a solution of sodium nucleate; both hyperglyce- mia and glycosuria remained unchanged [37, 59, 60]. 13 Acta Diabetologica Fig. 5  Main results and conclusions of the experiments carried out by response observed in control experiments (other organs than the pan- NC Paulescu demonstrating the effects of pancreina on the metabo- creas, saline and sodium nucleinate) [60] lism of carbohydrates, proteins and ketone bodies and the absence of Fig. 6  Nicolae Paulescu: Registration of the pancreina patent. Romanian Ministry of Industry (April 10, 1922) 13 Acta Diabetologica The patent of pancreina (1922) due to the persistence of toxic effects, particularly, fever [37, 61, 63]. On April 10, 1922, Paulescu successfully processed the reg- istration to the Romanian Ministry of Industry of the patent Paulescu’s letters to Frederick Banting of the pancreatic extract that he called pancreina (Fig. 6) and to the President of the Nobel Commission [61, p. 83–84]. in 1923 To purify pancreina, Paulescu subjected the aqueous glandular triturate to a double pad filtration of sterile hydro- On February 5, 1923, Paulescu wrote to Frederick Bant- philic gauze. Then, the addition of 1% hydrochloric acid ing, enclosing his 1921 publications on the treatment of generated a protein precipitate that was filtered again before experimental diabetes with pancreina. He never got a neutralizing with caustic soda. The final stage of the process response from the Canadian investigator. On November 6, consisted of sterile filtration through a Berzelius leaf and 1923, Paulescu wrote to the President of the Nobel Com- evaporation by means of heat at a temperature not exceed- mission, enclosing a copy of his publication in the August ing 50 °C [62]. 1921 issue of Archives Internationales de Physiologie. He Paulescu also carried out multiple experiments to opti- bitterly protested the Nobel award to Banting and Macleod mize the administration of pancreina. To this end, he com- and demanded justice, insisting on the priority of his pub- pared intravenous, subcutaneous, oral, and intrarectal routes, lications on the discovery of the antidiabetic hormone and concluding that only parenteral administration (intravenous that the Toronto team had violated his intellectual property and subcutaneous) was effective. The limited clinical expe- rights (Fig. 7). rience achieved with a scarce number of “thin” and “fat” diabetic subjects determined the interruption of the trial, Fig. 7  Left Letter from Nicolae Paulescu to Frederick Banting (Feb- the award of the Nobel Prize in Physiology or Medicine to Frederick ruary 5, 1923). University of Toronto Archive. Right Letter of protest Banting and John Macleod (NC Paulescu personal papers; courtesy of from Nicolae Paulescu to the Chairman of the Nobel Commission for Dan Angelescu) 13 Acta Diabetologica demonstrated that the pancreas contains an antidiabetic (…) Please, allow me to protest against the fact that this [Nobel] dis- tinction has been awarded to people who do not deserve it. Indeed, substance which has been given the name of insulin” [67]. the discovery of these physiological and therapeutic effects belongs Sir Edward Albert Sharpey-Schäfer briefly described the entirely to me research activities of N.C. Paulescu [68]: (…) In these articles, they do nothing but repeat what I had already said, well in advance, about the decrease in hyperglycemia and (…) NC Paulescu experiments commenced in 1916 but interrupted glycosuria, blood and urinary urea, acetonemia and acetonuria, by the German invasion [sic*] of Romania were published in 1921. under the influence of intravenous injections of pancreatic extract, They established that—in dogs rendered diabetic by extirpation in a diabetic animal of the pancreas and showing all the signs of severe diabetes-, (1) NC Paulescu, November 6, 1923 increase of sugar in the blood and its appearance in the urine; (2) increase of acetone bodies in the blood and their appearance in The response of the Nobel Commission was to send the the urine; (3) increase urea in blood and urine—these signs can be made to disappear by intravenous injection of pancreatic extract. pamphlet entitled "The Nobel Prize Winners of 1923" with The extract he used was made from fresh pancreas with ice-cold the award presentation speech by SJ Sjöquist, highlighting distilled, “ stérile autant que possible”. It was introduced gradually the contents of Banting and Best's first article of February into a vein of the diabetic dog, and its effect in a short time was to 1922 [26]. bring down the blood sugar level to normal or less, and to greatly reduce or abolish the glycosuria In 1930. Paulescu wrote: “I previously believed that the (…) In later experiments attempts were made by Paulescu to isolate scientist works safely because of the conviction that the the active substance (termed by him pancreina) by submitting the date of publication protects him from any injustice. Unfor- aqueous extract to the action of acids and alkalis, of alcohol and tunately, today I am forced to admit that I was completely heat. He found that boiling destroys the activity, and that an extract, wrong” [64]. from which most of the proteins have been precipitated by alcohol or by neutralizing the acidified extract by soda, contains the active International experts publicly expressed their substance. He further found that no effect was obtained by oral solidarity with the vindication of Paulescu's administration scientific merits and recognized his priority E.A. Sharpey-Schäfer, 1926 in the discovery of the antidiabetic hormone (1923–1934) [* Note: the invasion of Romania by the Austrian army, assisted by the German military forces]. The Spitalul medical journal published Paulescu's claim In 1926, J.J.R. Macleod recognized Paulescu's scientific addressed to the Biological Society of Bucharest (affiliate merits and his contribution to the discovery of pancreatic of the Parisian), expanding on the chronology, results of hormone, praising the experimental results published in his experiments and related publications between 1916 and the Comptes Rendus de la Societé de Biologie in April 1921 [26]. 1921, eleven months before the first article by Banting In a letter dated November 5, 1921, Ernest L. Scott recog- and Best [22; p.66]: nized the importance of Paulescu's findings, consistent with (…) Paulescu’s researches were communicated at a meeting of the his experiments at Columbia University in 1921, which sug- Reunion Roumaine de Biologie in the spring of 1921, when he gested the existence of a pancreatic hormone that regulates described the effects produced by intravenous injections of sterile carbohydrate metabolism [26, 65]. pancreatic extracts on the percentage of sugar, of acetone bodies, John R. Murlin wrote in 1922 that results published by and of urea in the blood and urine of depancreatized dogs. There can be no doubt that all three substances became markedly reduced Paulescu in 1921 showed that intravenous administration in amount, in blood and urine, as a result of the injections. The of a sterile pancreatic extract to pancreatectomized dogs results were the same whether the injection was made into a branch reduced and even temporarily eliminated hyperglycemia of the portal vein or into a yugular vein and excessive production of urea and ketone bodies [26, 32]. (…) The effects were noticeable in one hour following the injection, Alfredo Sordelli, President of the Academy of Exact, attain their maximum in two hours, and passed off in twelve hours. They varied with the amount of gland present in the injected extract Physical and Natural Sciences of the Harvey Society, Acad- Paulescu also observed that the blood sugar, as well as the blood emy of Sciences (New York) and of the Société de Biologie urea, in a normal dog became lowered by the injections de Paris, acknowledged that Paulescu's results, published in JJR Macleod, 1926 1921, were practically identical to those of the first publica- tion, eight months later, by the Canadian researchers Banting Wilfried Louis Batten Trotter (1872–1939), a promi- and Best [66]. nent British surgeon and a relevant authority in the field Casimir Funk, in addition to his extraordinary work in of biosociology, spoke in the Hunterian Oratio Lecture the field of vitamins, carried out extensive chemical studies (1932) some heartfelt words about Paulescu's contribution on cancer and diabetes. In 1924, he wrote the following: to the discovery of the antidiabetic hormone [26]: “In 1920 and 1921, Dr. Paulescu of Romania decisively 13 Acta Diabetologica the Communist Party in 1947, the figure of Paulescu sank Paulescu’s research was the culmination of years of experimental work of precursors, colleagues and himself. This great advance, into oblivion. Paulescu, a radical antisemite, Orthodox perhaps equivalent in some respect to the discovery of the thera- Christian and member of the Romanian right, was consid- peutic virtue of penicillin, remains unknowledged ered an enemy of the regime. His traces were erased from W.L. Trotter, Huntarian Oratio Lecture, 1932 the history of Romanian science [26]. Paul Georg Trendelelenburg (1884–1931), Professor of Pharmacology at the University of Berlin, with research The discovery of insulin activity focused on endocrine pharmacology, praised Paules- cu's scientific work in 1934 in the following terms: "Shortly The first pancreatic extract (Banting and Best) before the discovery of insulin, Paulescu achieved complete success with pancreatic extracts that reduced blood sugar On February 5, 1922, Frederick G. Banting and Charles H. levels in pancreatectomized dogs within one hour of paren- Best published in the Journal of Laboratory and Clinical teral administration” [69]. Medicine their first manuscript of the Diabetes Project, Uni- versity of Toronto, describing the research activities per- Paulescu died on July 7, 1931 formed by them from June to November 10, 1921 (Fig. 8) [70]. Nicolae C. Paulescu died on July 7, 1931 from kidney com- Banting and Best worked with the hypothesis that after a plications related to a bladder carcinoma with a long and few weeks of having the pancreatic ducts tied off, the pan- painful clinical course. He is buried in the family vault, creas would atrophy but will contain the islands of Langerhans Bucharest National Cemetery [26]. intact. Dogs were initially depancreatized by the method of With the tragic ups and downs of the Second World War, Hédon (1909), pancreatic ducts were ligated under general the political problems in Romania and the rise to power of anesthesia. A 10-week interval was considered necessary for Fig. 8  Left Photographs of the University of Toronto School of Medi- The first article of Frederick Banting and Charles Best (February, cine building and Laboratory of Physiology, University of Toronto; 1922) reported results of at least 75 doses of pancreatic extracts from summer of 1921. Archives of the University of Toronto. https://​ “degenerated pancreas” [70] nobel​prize.​orf/​prizes/​medic​ine/​1923/​banti​ng/​photo-​galle​ry/. Right 13 Acta Diabetologica complete degeneration of the acinous tissue. The removed Organization of diabetes treatment pancreas was sliced into a chilled mortar containing Ringer’s in the Department of Medicine at Toronto General solution. The mortar was placed in a freezing mixture and Hospital, University of Toronto the contents partially frozen. The half-frozen gland was then completed macerated. The solution was filtered through paper Duncan Archibald Graham (1882–1974) was appointed in and the filtrate, having been raised to body temperature, was 1919 as the first director of the John Craig Eaton Chair of ready for intravenous injection. Blood glucose estimations Clinical Medicine at the University of Toronto and Director were made by the Myers-Bailey modification of the Lewis- of the Department of Medicine at Toronto General Hospi- Benedict method [71]. tal (TGH), positions he held until 1947. Graham hired Dr. The article reported results after the administration of at Walter Ruggles Campbell (1891–1981) full-time, whom he least 75 doses of PE from “degenerated pancreas”. The main assigned in collaboration with Dr. Andrew Almon Fletcher graphic of the manuscript showed that the administration of (1889–1964) to the Regular Admission Hospitalization the “degenerated pancreatic extract”, developed by the two Ward for patients with diabetes. These two expert clinics authors, only achieved a partial decrease of the levels of blood initially treated diabetes with a variant of the Allen's starva- and urine glucose in just a fraction of the investigated pancrea- tion diet [76]. tectomized dogs. The authors felt that these results justified to Banting could not hide the anxiety that caused him the state that this extract contained internal pancreatic secretion. delay in starting clinical experiences with the PE. Prof. The experiments revealed that the central idea of Banting, Duncan A. Graham didn’t authorize Banting to have direct inspired to him by an article of M. Barron, “the ligature of the contact with inpatients, considering that a surgeon, not on pancreatic ducts”, was not needed [72] (in fact, in experiments clinical practice, was not qualified for human experimenta- performed later they obtained better results using extracts from tion. Nevertheless, the mediation by J.J.R. Macleod allowed fresh adult and fetal pancreas). Barron’s hypothesis ignored for the administration of the first dose of PE prepared by that Heidenhain had demonstrated in 1875 that fresh pancreas Charles Best to Leonard Thompson, a 14-years old, charity extracts do not have proteolytic activity due to the existence patient with severe diabetes, first diagnosed in December of a zymogen able to generate an active enzyme only under 1919. On January 11, 1922 the resident physician Ed Jeffrey various circumstances [73]. Bayliss and Starling confirmed followed the order of the Senior Consultor Dr. Walter Camp- Heidenhain’s observation, showing that the proteolytic enzyme bell, to administer 15 mL of the Banting-Best preparation trypsin was present in fresh animal pancreas as an inactive pre- of PE (a turbid, light brown fluid), divided in two 7.5 mL cursor (trypsinogen) [74]. Finally, Banting and Best decided injections, one in each buttock. Leonard had been admitted on August 3, 1921 to replace the laborious Hedón’s procedure at the TGH on December 2, 1921. He was on the standard by one step complete pancreatectomy, discontinuing the liga- 450 kcal diet, which included 50 g of lean meat, vegetables tion of the pancreatic ducts, acidulation of the alcoholic extract and fruit, fat-free broth, clear tea and water, with a total (suggested by Macleod), washing with toluol and use of the carbohydrate provision of approximately 100 g. The patient Berkfeld filter. was emaciated (weight, 29 kg) and had extreme glycosuria, The first article of Banting and Best contained formal severe ketonuria, hypotension, and a diuresis of 4 L in 24 h. errors and meaningful mistakes as disagreements between The response to the administration of the PE consisted of the information supplied and figures or lack of accuracy in light reduction in blood glucose from 440 to 320 mg/dL and comments regarding the achieved results. in absolute glycosuria of 24 h from 92 to 84 g; ketonuria John James Rickard Macleod (1876–1935), director of remained unchanged. A “sterile abscess” (characteristic of the research project and well-known authority in the field protein contaminants of the PE) rapidly developed in one of (Professor of Physiology, director of the Department and the injection areas. The extract failed to relief significantly President of the American Society of Physiology) decided the cardinal symptoms of the boy and its administration was not to sign the manuscript as coauthor. discontinued [77]. The physiologist Ffrangon Roberts, University of Cam- bridge, published in the British Medical Journal a letter to Collip’s extract the editor, before the end of the year 1922, expressing a severe critic of the experiments described by Banting and In December 1921, Macleod brought James Bertrand Collip Best. He qualified them as “badly designed, badly conducted to the team. Collip, a 29-years old biochemical expert, then and badly interpreted” [75]. Assistant Professor at the University of Alberta, was on sab- batical at UT with a Rockefeller Fellowship. Macleod asked him to help in the extract development. From the begin- ning, Collip worked with whole beef pancreas in his own laboratory. 13 Acta Diabetologica On the evening of January 19, 1921, he was able to pre- Best. In parallel to the excellent response in the amount of cipitate the active substance of the PE with ethanol at higher glucose excreted, a substantial reduction of blood glucose concentration than 90%. Using this threshold, he was able to and urinary ketones was also recorded (Fig. 9), as well as remove most protein contaminants, obtaining a PE still with clinical improvement. The successful results achieved with impurities but with a much higher potency than previously the case of Leonard Thompson were reproduced with six tested preparations [78]. more patients during the month of February 1922 and pub- lished in March 1922 [79]. “To a small volume of 95 percent ethyl alcohol, freshly minced Prof. Duncan Graham assigned to Drs. WR Campbell and pancreas was added in equal amount. The mixture was allowed to AA Fletcher the implementation of all features related with stand for a few hours with occasional shaking. It was then strained through cheese-cloth and the liquid portion at once filtered. Some the trials with this new therapy. hours later, the precipitate was caught on a Buchner funnel, dis- solved in distilled water, and then concentrated to the desire degree Announcement of the discovery of insulin on May 3, by use of the vacuum still. It was then passed through a Berkefeld 1922 filter, sterility test was made, and the final product was delivered to the clinic” The precipitation of the active substance by 95% or absolute ethanol Between May 1921 and May 1922, research conducted by rendered the extract non-toxic. Essential points of the Collip’s the team led by J.J.R. Macleod in the Department of Physiol- extract were: minimal expression of protein, salt-free, lipoid free, ogy, University of Toronto, demonstrated the actions of the and free of alcohol-soluble constituents PE on rabbit-induced hyperglycemia by various procedures (piqûre, subcutaneous injection of adrenaline, carbon mon- On January 23, 1922, Leonard Thompson was given the oxide, mechanical asphyxia), on glycogen stores in liver and extract made by Collip. Blood glucose decreased from 520 other tissues, on the excretion of ketone bodies, on the res- to 120 mg/dL (Folin-Wu method), absolute glycosuria in piratory quotient in healthy dogs with experimental diabetes 24 h from 71 to 9 g (Benedict method), and ketonuria (Van investigated in metabolic cages) [80]. Slyke procedure) disappeared. The daily registry of absolute In experimental research, Collip used both normal and urinary glucose excretion during the months of January and hyperglycemic rabbits. He could observe that blood glucose February 1922 of L.T., demonstrated the superior effective- (BG) fell between approximately 25 and 50% within two ness of Collip's EP compared to the previous of Banting and hours after injection. For purposes of physiological assay, it Fig. 9  Portrait of Leonard Thompson (L.T.). Unknown date and ment of Medicine, Toronto General Hospital. Left Daily chart record author. University of Toronto Archives. Recorded effects of the between January 1 and February 20, 1922, of urinary glucose excre- administration of Collip's pancreatic extract to L.T., the first dia- tion. Right Rapid elimination of ketonuria. Reduction of blood glu- betic patient successfully treated in the Inpatient Ward of the Depart- cose and glycosuria fractionated over 24 h. [79] 13 Acta Diabetologica was considered as most satisfactory to estimate the number 1922 were in critical condition awaiting satisfactory insulin of cubic centimeters which lowered the percentage of BG in treatment. the healthy rabbits to 0.045 in 2–4 h. The administration of An important progress in the purification of insulin had 4 g of dextrose in 20% solution was followed by the recov- as its main protagonists Peter Joseph Moloney and Donald ery of the animals. These experiments were critical for the Munro Findlay who, in order to deal with the decrease in concept of unit of the dose of antidiabetic hormone [81–83]. potency of the extract, used as an alternative the precipita- The researchers of the University of Toronto decided to tion with benzoic acid of the active product of the pancreatic name insulin the hormonal preparation, ignoring that the extract, followed by the treatment of the precipitate with name of insuline had already been proposed by the Belgian ether and water, separating the two layers formed (the ben- Jean De Meyer in 1909 [84], and E.A. Sharpey Schafer had zoic acid was contained in the ether layer and the insulin in proposed insulin in 1916 too [85]. the water layer) [86]. On May 3, 1922, at the annual meeting of the American The aqueous solution was subjected to prolonged heat- Medical Association in Washington DC, Macleod summa- ing in a vacuum chamber for complete removal of the ether. rized the research conducted by the departments of Physiol- The use of the Moloney and Findlay method was of critical ogy and Medicine at the University of Toronto, including importance for the availability of insulin in the most difficult animal experimentations and the clinical results achieved stage of the Connaught and University of Toronto laborato- (Fig. 10). ries. More than 250,000 units of insulin made by this method At the end of the lecture, Macleod declared: were used in the treatment of diabetes patients with satisfac- These observations conclusively demonstrate that the pancreatic tory results [87]. extracts that we have used contain substances that exert great Collip was unable to solve the problem of the loss of potency on the control of carbohydrate and fat metabolism in both potency of the pancreatic extract, associated with the need normal and diabetic animals, as well as in people with diabetes to increase its production on an industrial scale. Macleod mellitus and Sir Robert Falconer, President of UT, agreed to negoti- Given the impact created by this announcement, the date ate with Eli Lilly & Co. and its director of research, George of May 3, 1922 has been considered, with scientific criteria, Clowes, in the face of the urgency of manufacturing insulin the date of the discovery of insulin. in large quantities. To do this, they created the Insulin Committee-University Collaboration between the University of Toronto of Toronto (IC-UT), made up by members of the UT Gov- (UT), Connaught Laboratories and Laboratories Eli erning Board, including its Chairman, Colonel A.E. Goo- Lilly & Co. derham and Sir Robert Falconer, among others, an Advisory Committee that included the director of Connaught Labo- Between March and May 1922, attempts by Connaught ratories, Dr. J.G. Fitzgerald, project investigators and mem- Laboratories led by J.B. Collip to produce insulin on a large bers of the University, Drs. J.J.R. Macleod (Secretary), F.G. scale were unsuccessful. Diabetic patients in the spring of Banting and C.H. Best, and on behalf of Toronto General Fig. 10  Between May 1921 and May 1922, the Diabetes Research mentally induced by various procedures. The photographs of the Project led by Prof. JJR Macleod, Chair of the Department of Physi- main authors of this research (JJR Macleod, FG Banting, CH Best ology at the University of Toronto, demonstrated the metabolic and JB Collip) come from the Archives of the University of Toronto. actions exerted by the pancreatic extract on healthy animals and the Public domain therapeutic effects observed in animals with hyperglycemia experi- 13 Acta Diabetologica Hospital, Prof. D.A. Graham. The Committee had the legal He determined in different times the level of blood glucose advice of Mr. C.H. Riches [88]. in normal rabbits injected with extracts prepared from 1-the The agreement was signed on May 31, 1922. According pancreas of representative Elasmobranchs (Squalus -dog to the agreement, Lilly had "an experimental year" for the fish-, and Raja -skate-); 2-Myoxycephalus -sculpi-, and development of the indicators established by the UT and dur- Lophius piscatorius -angler fish-); c-the zymogenous (aci- ing this period the company would enjoy the exclusive rights nar) pancreatic tissue, as free as possible from islets, in the for the production, use, sale and distribution of the PE in the same and other Teleostei. Macleod obtained, at least, 3–4 USA, Mexico, Cuba, Central America and Latin America. units of insulin from one teleostei fish. Control experiments Lilly would provide UT with 28% of each approved lot of administering liver extracts showed no endocrine effects insulin for free distribution, and 12% at cost, and batches of (Fig. 11) [93]. the pancreatic extract would be distributed as “tests” at pro- duction cost or free, to physicians and institutions selected Insulin: Canadian and US patent offices by joint decision, After the conclusion of the experimental phase, Lilly would enjoy the license right on the condition On November 10, 1922, the University of Toronto applied of payment of 5% for the real rights of sales to UT. to the USA office for the first patent for insulin with assign- This document represented the first agreement of coop- ment to Best and Collip as investigators. The request was eration between an academic institution, medical profession- rejected on the grounds that it contravened the protection als and a commercial company with the aim of applying a rights corresponding to the patent for acomatol granted on therapeutic advance of great international relevance. Clowes May 28, 1912 to Georg Zülzer. New applications were pro- appointed George B. Walden as supervisor of insulin pro- cessed showing the benefits of the new PE in comparison to duction, under whose direction Harley W. Rhodehamel and the PE of Zülzer: a—possibility of continuous administra- Jasper P. Scott collaborated on the large-scale experimenta- tion; b—lack of serious adverse effects; c—greater potency tion and processing projects [89]. and purity, and d—evidence of benefits for the treatment of Among the first modifications introduced by Lilly we diabetic patients [94]. must mention the use of frozen pancreas, the addition of Patents #234,336 (Canadian Intellectual Property Office; acid in the extraction process and the isoelectric precipita- inventors: Banting and Best) and #234,337 (Canadian Intel- tion of insulin. In the month of October, Lilly was supply- lectual Property Office: inventors P.J. Moloney and D.M. ing the needs raised by 16 Canadian and North American Findlay) were granted on September 18, 1923, and patent clinical teams. The process of isoelectric precipitation of #1,469,994 (United States Patent Office; inventors: F.G. insulin ended in December 1922. In the winter of 1923, Eli Banting, C.H. Best and J.B. Collip) dated October 9, 1923. Lilly finally obtained a more pure and stable insulin, less All patents were assigned in ownership to the Board of Gov- allergenic, in sufficient quantity, between 80,000 and 90,000 ernors of the University of Toronto (Fig. 12). units per week, to meet world needs [90, 91]. In the months of May–June of 1923, the relations between Eli Lilly and the UT experienced serious conflicts which represented a danger for the continuation of the established JJR Macleod definitely established that islet tissue collaboration. Clowes sent several letters to Macleod asking is the source of insulin to accept Lilly's decision to file a new patent application on its own based on the development of a new extraction pro- There was as yet no absolute proof that the hormone of the cedure. Clowes also told him that the company wanted to pancreas was elaborated by the islet cells. In the summer of extend the experimental period and license the patent exclu- 1922, Macleod conducted experiments comparing the meta- sively, claiming the intention to manufacture insulin at a bolic actions of pancreatic extract from elasmobranch and lower cost, sell it at a lower price and facilitate free access to teleost fishes administered to rabbits. He gave recognition indigent patients. Macleod replied that the UT flatly rejected to the previous observation of Rennie that the islet tissue in the proposal and added that the Insulin Committee-UT (IC- the bony fish (Teleostei) is enclosed into nodules, frequently UT) had authorized the British Medical Research Council encapsulated and separated from the acinar tissue. J. Rennie (MRC) to supervise the production of insulin in England and T. Fraser had failed to demonstrate the presence of the [95]. antidiabetic hormone because they preferentially adminis- Despite all of the above, on September 1923, George tered the islets by mouth [92]. B. Walden worked up an American patent for "a new The comparison made by Macleod between the admin- process for the preparation of pancreatic extract" with- istration of the islets and the pancreatic zymogenous tissue out prior notice to the IC-UT. The procedure showed that (containing no islets) leaved little doubt that insulin was the amount of available precipitated hormone depended present only in the islet tissue and not in the acinar tissue. on the concentration of hydrogen ions in the medium and 13 Acta Diabetologica Fig. 11  In the summer of 1922, JJR Macleod investigated alone at encapsulated in nodules and separated from the acinar tissue of bony the Marine Biological Station in St. Andrew's, New Brunswick, the fishes (teleostei). Macleod also performed control experiments with comparative effect of administration of islet-free acinar pancreas liver extracts [93] from elasmobranch fishes with administration of islets of Langerhans that the best results had been observed with a pH between research team led by Shaffer had independently published 4.5 and 5.5. In addition, it reduced the residual nitrogen in 1923 the development of a new pancreatic extract using a content to 0.1 mg per unit of antidiabetic activity and the process similar to that of Walden [98]. purity of the preparation was 10 to 100-fold higher than Given the pressure exerted by the IC-UT, Lilly finally the best previously used pancreatic extract. The process, decided to assign the intellectual property of the Walden pat- developed by Lilly, made the authorized procedure for ent to the UT and keep the name of its Iletin brand (Fig. 13) patents granted to UT obsolete. With the granting of this [99]. patent, Lilly's monopoly for the production and sale of insulin in the countries of the American continent was “While we consider ourselves legally and morally entitled under our foreshadowed [96]. agreement with the committee to take the strongest possible patents on our own discoveries and whilst we are not in the least concerned George Riches, legal representative at the IC-UT, wrote about Shaffer’s claims as our process is superior to and differs a letter to Macleod on April 3, 1923, informing him of his essentially from his and we are satisfied of our priority, neverthe- concern about the risks posed by the granting of the Walden less, we would not consider doing anything that may embarrass patent to the Eli Lilly firm against the interests of the Uni- Toronto University.” versity of Toronto and the validity of the Banting, Best and (Clowes, 1923. Telegram to Professor Macleod, April 8, 1923) Collip insulin patent [97]. The IC-UT addressed the problem through two initiatives. The first was to inform the Council Both parties agreed to extend the Walden patent licenses on Pharmacy and Chemistry of the American Medical Asso- to foreign countries, considering it more effective than the ciation that it did not accept the use of Iletin by the Lilly one initially granted to Banting, Best and Collip [100]. firm. The second was to contact P.A. Shaffer, director of the Biological Chemistry Laboratory at Washington University in St. Louis with the proposal to apply for a new patent. The 13 Acta Diabetologica Fig. 12  Insulin Patents. Patents 234,336 were issued to inventors FG substance and its clinical formulation, the patented product (extract Banting and CH Best and 234,337 to inventors PJ Moloney and D.M. obtainable from the pancreas of mammals or fish) was defined by a Findlay (Canadian Intellectual Property Office) on September 18, sufficient degree of purity that surpassed that of the preceding prepa- 1923. Patent 1,469,994 (United States Patent Office) was granted on rations, as well as its physiological effects and beneficial therapeutics October 9, 1923. All of them were assigned ownership to the Univer- for patients with diabetes [94] sity of Toronto Board of Governors. In the absence of a fully purified Unit of Insulin: standards (1922–1923) researchers proposed considering the unit of insulin as the amount of extract (in cc) capable of causing a 45% The University of Toronto Insulin Committee organized decrease in the normal value of blood glucose in a healthy on November 25, 1922 a round table in the library of the 2 kg rabbit (subcutaneous administration). Faculty of Medicine with the participation of Canadian The IC-UT decided on December 30, 1922 to adopt a and American experts in the treatment of diabetes to criterion for the "clinical unit of insulin" and another for discuss the organization of clinical trials and the insulin the "physiological unit of insulin" [101]. unit standardization plan (Frederick M. Allen, Rockefel- ler Institute of Medical Research, New Jersey; H. Rawle “The unit adopted for insulin shall be approximately one-fifth that of Geyelin, Columbia University Presbyterian Hospital, New the original Toronto unit, which is the amount of insulin required to lower the blood sugar of a 2 kg. rabbit to 0.045% within four hours, York; Elliot P. Joslin, Harvard University, Boston; Russell and caused symptoms M. Wilder, Mayo Clinic, Rochester, Minnesota; JR Wil- The small unit would be more practical in cases were small amounts liams, Rochester, New York; Roland T. Woodyatt, Uni- of insulin would have to be prescribed. By using the original versity of Chicago; William D. Sansum, Potter Metabolic (Toronto) unit, fractions would have to be prescribed, and this Clinic, Santa Barbara, California, among others). would be undesirable.” The discovery by J.B. Collip of insulin-induced hypo- (IC-UT, December 1922) glycemic seizures in the rabbit allowed an initial approach to measuring the dosage of the new hormone. The UT 13 Acta Diabetologica Fig. 13  In the summer of 1923, a conflictive situation arose between the standard product. The pressure exerted by the Insulin Committee Eli Lilly and the University of Toronto due to the intention of G. of the University of Toronto finally decided to assign the intellectual Clowes, Director of Research at Lilly, to apply for a new patent property shared with the University of Toronto: US Patent 1,520,673. based on the development of a new exclusive extraction procedure, Inventor George B. Walden. Owners: The Governors of the Univer- invented by G.B. Walden, showing that isoelectric precipitation mark- sity of Toronto (December 23, 1924). Photographs of G.B. Walden edly reduced the residual nitrogen in the preparation and increased and G. Clowes are from the Lilly Archives [99] the purity of the preparation between 10 and 100 times compared to The Standards Commission, created by the Health of W.R. Campbell about the first fourteen cases of diabetic Organization of the League of Nations met in Edinburgh in coma attended [102, 103]. July 1923. It approved the IC-UT definition of the "physi- Campbell provided an outstanding lesson about the treat- ological unit of insulin", and agreed that the "clinical unit ment of ketoacidosis, which, in many aspects remain valid of insulin" corresponded to one third of the value of the today. He pointed out the extraordinary tolerance to insu- physiological unit of insulin [26]. lin in coma and the key role of water, insulin, glucose and alkali in the treatment. Pros and cons of the administration Insulin treatment in the months which followed its of bicarbonate were discussed [104]. discovery (short report) The third manuscript, signed by A.A. Fletcher and W.R. Campbell described the adrenergic and neurogenic stages of A special issue of the Journal of Metabolic Research, pub- insulin-induced hypoglycemia. They observed severe reac- lished a series of manuscripts covering the main experimen- tions with a blood sugar of 60 mg/dL. A blood sugar value tal and clinical contributions dealing with insulin research of 35 mg/dL was associated to unconsciousness. Different in the months following its discovery. treatments of hypoglycemia were tested: orange juice (when A detailed presentation of nine insulin diabetic subjects the patient could swallow), intravenous glucose (5–20 g), treated at TGH was followed by highly relevant observations epinephrine (1/1000 sol., 1 mL) when the patient was uncon- scious [105]. 13 Acta Diabetologica A recent publication associates clinical reports, incidents The animosity and confrontation of Banting and his and biographical anecdotes related to the introduction of admirers against Macleod persisted for a long time. Finally, insulin therapy in North America and the European conti- Macleod accepted the nomination of Regius Chair of Physi- nent [106]. ology at the University of Aberdeen, his alma mater, and Walter R. Campbell and Andrew A. Fletcher of the TGH left Toronto in 1928. One friend reproduced the last words Department of Medicine (directed by Duncan A. Graham) of Macleod before departure: “I am shuffling my feet on the successfully treated more than fifty patients with insulin floor to wipe away the dirt of this city”. At the University of before the end of 1922 at the Diabetes Ward of TGH [107]. Aberdeen, Macleod carried out a notable teaching activity By early 1923, more than 1000 diabetic patients were and resumed his research on the influence of the nervous treated with insulin or iletin in 60 clinics of United States system on hepatic glycogenolysis. In 1935 he updated the and Canada [26]. edition of his book Physiology and Biochemistry in Modern Medicine. Progressively debilitated by rheumatoid arthritis, The disclosing of documents explaining the true he died in 1935 at the age of 59 years with symptoms of story of the discovery of insulin cardiorespiratory failure [109, p. 234]. His passing tempo- rarily coincided with the sudden death of another professor A group of friends and admirers of Banting decided, from at the university. A joint funeral was held in King's College the first months of 1923, to orchestrate a campaign aimed Chapel. Macleod's remains rest with those of his wife, Mary at providing maximum credit for the discovery of insulin Watson, in Aberdeen's Allenvale Cemetery. Macleod was to F.G. Banting, Canadian hero of the First World War, relegated to oblivion, as was his leadership in the discovery wounded at Cambrai, relegating the Scottish emigrant J.J.R. of insulin. Macleod, shy and aloof physiologist. Banting's friend and colleague, Dr, George W. "Billy" Banting, responsible for and organiser Ross, son of Sir William Ross, who had been Premier of of the Anglo‑Canadian germ warfare programme Ontario, with strong political connections, led the initia- (1938–1941) tive. Ross was aided by Velyien Henderson, Professor of Pharmacology, UT, Sir William Mulock, Vice-Chairman of After the discovery of insulin, Banting supervised research the UT, and Charles Evans Hughes, previous Republican on silicosis developed by scientists of the Banting Research candidate for the USA presidency against Woodrow Wilson, Department but never had real success in his own research Secretary of State in the administration of USA President initiatives. Warren Harding (1921–1925), and later appointed president On 11 September 11, 1937, Banting warned General to the Supreme Court between 1930 and 1941. At all times, Andrew McNaughton, Chairman of the National Research Fred Banting had the support and favors of William Lyon Council, that Germany was prepared to initiate biological Mackenzie King, the Prime Minister of Canada. warfare (which never could be demonstrated). In 1938 he With Banting's consent, Ross wrote to all known Cana- was elected the first Chairman of the Associate Commit- dian expert diabetologists asking for their support to Bant- tee on Medical Research of the National Research Council ing and the miracle of the discovery of insulin. As a conse- and rejoined the Royal Canadian Army Medical Corps with quence of this campaign, the Canadian Medical Association the rank of major. With McNaughton’s encouragement, he passed a resolution on March 23, 1923 (also approved by the prepared a detailed memorandum of biological warfare, Toronto Academy of Medicine) dictating that the internal describing the various air-borne, water-borne and insect- secretion of the pancreas, named insulin, was discovered borne agents which could be used for military purposes. His by Banting and Best in the summer of the year 1921 [108]. confidential program (paid with 1.3 million CAD) to elimi- The UT decided to create the Banting and Best Chair of nate the German enemy consisted of creating epidemics, Medical Research, a special professorship to Banting with famine and economic misery through the attack on livestock, an annual grant of 10,000 CAD to pay for his salary and sterilization of crops, food poisoning and contamination of research support, plus an additional amount of CAD 10,000 water and fields. as reimbursement for the discovery period. Also, on June 27, In September, 1939, Banting entered the Canadian Army 1923 the Canadian House of Commons accepted a resolu- with the rank of major. In November, he updated the germ tion to grant Banting with a lifetime annuity of CAD 7,500 warfare program memorandum and handed it over to Lord to devote his life to medical research. On the contrary, and Maurice Hankey, the British Minister, coordinator of the particularly in Canada, the role in the discovery of insulin subcommittee on germ warfare. McNaughton informed of the Scottish introverted immigrant, Macleod, physiologist Banting of the final agreement between Great Britain and and not clinician, was forgotten [109, pp. 214–222]. Canada on the joint germ warfare program and the decision 13 Acta Diabetologica to appoint him as delegate of the Canadian government for Waller, Ph.D. in History from University College London all purposes. and Research Fellow at the Wellcome Trust Center for the Banting created a core group of Canadian biological war History of Medicine, in his book entitled Fabulous Science researchers including the bacteriologists P.H. Greey and D. [113, p. 232]. Fraser of UT and the virologist James Craigie of the Con- The Anglo-Canadian military steering committee called a naught Medical Research Laboratories, among others. C.J. meeting in London to decide on joint military actions against Mackenzie provided Banting with CAD 50,000 of seed the enemy. Banting's secret mission was deemed "of high money out of the so-called Santa Claus Fund until British scientific and national importance" by the Canadian Prime support was forthcoming [110, pp. 14–55]. Minister. The British military high command decided to install the At around 8 pm on February 20, 1941, a few minutes bacteriological warfare arsenal in Porton Down (England), after taking off from the Gander military airport, the bomber in accordance with the instructions provided by Banting. had mechanical problems. The first pilot, Joseph C. Mackey, The first proposed offensive action on German territory attempted an emergency landing and crashed in the grove would consist of the aerial drop of sawdust infected with near Musgrave, Newfoundland. Both the radio specialist and lethal infectious agents. The preparatory tests were made at the second pilot died immediately. Banting was mortally Balsam Lake, some 50 miles northeast of Toronto, between wounded and died in the snow the next day. Only Mackey October 9 and 16, 1940 [111, pp. 1–18]. survived (Fig. 14). The body of Banting was flown to After these tests were completed, Banting noted in his Toronto. On March 4, the solemn funeral was held in the UT personal diary: “We are beyond the purely experimental Convocation Hall. The casket was carried in solemn proces- stage (…) for obtaining the means by which we can retaliate sion to Mount Pleasant Cemetery where Banting's remains 100-fold if the Germans used bacterial warfare (…) We have rest alongside those of his second wife Henrietta Ball [112, to kill 3 or 4 million young huns—without mercy—without pp. 298–310]. feeling” (October 14–20, 1940) [112, pp. 283–286]. The “Billy” Ross documents reveal the deteriorating rela- tionship between Banting and Best. Banting disparaged his Charles Best reinvented the history of the discovery former aide on several occasions. Charles Best wanted a of insulin (1941–1978) Research Institute to be established and named after him. Banting immediately set about frustrating this plan. “Best After Banting's death, the UT presidency decided that Best is naïve in his abject selfishness”, he said. In 1940, Best should succeed him as director of the department. Best con- offered himself as the Canadian’s government medical emis- sidered that the occasion had come to spread a new read- sary to a besieged Britain. Banting was fully aware of the ing of the discovery of insulin, attributing to himself the dangers involved in the crossing, he elected to go himself. exclusive role of the preparation and administration of the Shortly before leaving, he was heard to say: “If they ever pancreatic extract to the first patient. give that chair of mine to that son of the bitch, Best, I will roll over in my grave”. This was reported by Australian John Fig. 14  Sir Frederick Banting, victim of plane crush (February 20, 1941). News report on the accident in the Alliston Herald. Public domain 13 Acta Diabetologica Fig. 15  Charles Best backdated the discovery of insulin to the first insulin—insulin100.com -, on April 15–17, 2021, advertised with weeks of the summer of 1921. The Banting and Best Center of the only images of researchers Banting and Best University of Toronto celebrated the centenary of the discovery of In his account he backdated the great discovery to the first in falsification which verges on scientific fraud. In the weeks of the summer of 1921, when Banting and he worked later years of his life Charles Best appears to have had a alone [113, pp. 238–239]. profound hunger for recognition, a serious ego-problem, His maneuver prospered to the point that most organiza- which overwhelmed his good sense” [115]. tions celebrate the anniversary of the discovery of insulin in 1921 and not in 1922. As an example, the University of Best’s death (1978) Toronto celebrated the 100th anniversary of the discovery of insulin (insulin100.com) on April 15–17, 2021, that was On March 31, 1978, Charles Best died after suffering a advertised with images of Banting and Best, excluding stroke. He and his wife, Margaret Mahon are buried in Macleod and Collip (Fig. 15). Pleasant Cemetery, in a grave close to that of Frederick Best knew how to position himself at the forefront of and Henrietta Banting [26]. the most important national and international organiza- tions in diabetology. For this purpose, he had the favor The myth of Banting and Best of academics of maximum influence. Among them, Prof. Henry Dale, Nobel Prize in Medicine in 1936, and Dr. Michael Bliss published in 1988 what he described as Robert Daniel Lawrence, diagnosed with DM in 1920. "An adventure between archives to discover documents Lawrence and the writer George Wells (also suffering on insulin". He interviewed Richard Landon, director of from diabetes), created the British Diabetic Association the Fisher Rare Book Room at the University of Toronto in 1934. Dale, Lawrence, and Best were co-founders of Library. He accessed confidential documentation that he the American Diabetes Association and the International described as “dynamite material”: Banting's moves to Diabetes Federation. feudalize the discovery, his multiple displays of hostility Best convinced the UT authorities to keep secret for towards Macleod, his excessive drinking, and episodes of 56 years Macleod’s personal account, written in Sep- verbal violence against Macleod and Collip and physical tember 1922, about the events and activities carried out violence against Collip were reported in detail. in the Department of Physiology since May 1922 [114]. Reading the biography of Charles and Margaret Best, He achieved most of his proposed objectives. One of the written by their son Henry, he noted his father's frequent most desired became a reality in 1953: the opening of cyclical episodes of severe depression. In several inter- the UT Best Research Institute. The historian Michael views with Bliss, McGill University psychiatrist Robert Bliss claimed that “Best distortions of the historical H. Cleghorn, well informed about the Toronto team of records seem to amount to a deliberate, unethical exercise 13 Acta Diabetologica researchers, did not hesitate to attribute Charlest Best as a Funding Open Access Funding provided by Universitat Autonoma de megalomaniac [116]. Barcelona. The research activities of this work were supported by the Institute of Biomedical Research, Hospital de Sant Pau-Universidad Bliss and other researchers have dismantled the myth of Autónoma de Barcelona and Fundación DIABEM. Banting and Best and have contributed to the rehabilita- tion of Macleod. Two articles on this subject, published in Declarations 1993 and 2013, deserve special mention [115, 117]. Human and Animal Rights disclosure The animal and human rights consent statements are not required, due to the historiographical char- Concluding remarks acter of this research. (1) The European researchers G. Zülzer (1908) and N. Paulescu Conflict of interest The authors declare that they have no conflict of (1921) meet the requirements of the priority rule in the discovery of interest. the antidiabetic hormone (2) Socioeconomic and political factors related with the First World Open Access This article is licensed under a Creative Commons Attri- War and the inter-war period delayed the process of purification of bution 4.0 International License, which permits use, sharing, adapta- the antidiabetic hormone in Europe tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, (3) The Canadian scientist J. B. Collip, University of Alberta, tempo- provide a link to the Creative Commons licence, and indicate if changes rarily assimilated to the research team of the UT, and the American were made. The images or other third party material in this article are chemist and researcher G. B. Walden, employee of the pharmaceu- included in the article's Creative Commons licence, unless indicated tical company Eli Lilly, were the main authors of the purification otherwise in a credit line to the material. If material is not included in process of the antidiabetic hormone the article's Creative Commons licence and your intended use is not (4) The scientific evidence allows to assert that the basic research permitted by statutory regulation or exceeds the permitted use, you will carried out by the Department of Physiology of UT, directed by J. J. need to obtain permission directly from the copyright holder. To view a R. Macleod, in conjunction with the clinical research undertaken at copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. the Department of Medicine of the UT, directed by D. A. Graham, made it possible in record time the successful treatment of patients suffering diabetes mellitus, that was until then a deadly disease References Acknowledgements We are indebted with a numerous group of col- 1. Zülzer GL (1893) Über alimentäre Glycosurie in Krankheiten laborators. The following list is only a selection. À. Martínez-Vidal and und über puerperale Lactosurie. Dissertation, University of JL Barona (Univ. of Valencia and Institute López Piñero) and C. Pérez- Humboldt, Berlin Aguado (F. DIABEM) provided insightful comments on the manuscript 2. Blum F (1901) Über Nebennierendiabetes. Dtsch Arch Klin draft. C. Labaune (Collège dr France) and S. Vervynck (Univ. of Gand) Med 71:146–147 facilitated documents on the scientific biography of E. Gley, R. Hoff- 3. 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Bliss M (2013) The eclipse and rehabilitation of JJR Macleod, 110. Avery D (2013) Pathogens for war-biological weapons, Cana- Scotland’s insulin laureate. J R Coll Phys Edinb 43:1–7 dian life scientists, and Nort American biodefence. University of Toronto Press, Toronto Publisher's Note Springer Nature remains neutral with regard to 111. Prasard SK (2009) Biological war. Discovery Publishing House jurisdictional claims in published maps and institutional affiliations. Pvt Ltd, New Delhi 112. Bliss M (1992) Banting—A biography. University of Toronto Press, Toronto 113. Waller J (2002) Fabulous Science. In: Fact and Fiction in the history of scientific discovery. Oxford University Press, Oxford Authors and Affiliations Alberto de Leiva‑Hidalgo1,2 · Alejandra de Leiva‑Pérez2 2 * Alberto de Leiva‑Hidalgo Fundación DIABEM., Barcelona, Spain aleiva@fdiabem.org 1 Universidad Autónoma de Barcelona, Barcelona, Spain 13