The Discovery of Insulin

by Michael Bliss

  It was apparently the discussion of this experiment among Banting, Best, Macleod, and Collip which brought the liver/glycogen issue to a head. The discussion was along the following lines: An important process in carbohydrate metabolism is the conversion of glucose into glycogen in the liver. In the diabetic condition very little of this conversion takes place, so the glycogen content of the liver is low. Banting had wondered from time to time whether the pancreatic extract would enable the diabetic dog’s liver to start storing glucose. This would be an important demonstration of its potency as an anti-diabetic agent.

  Why wasn’t the extract effective on the anesthetized dog? It was known that volatile anesthetics inhibited the glycogen-forming action of the liver in normal animals. Perhaps the extract did not work on the anesthetized diabetic dog because its liver was similarly inhibited by the anesthetic. If that were so, the reasoning went (apparently during discussions the four were having over lunch daily), it might be that the key to the extract’s overall effect was in the liver. Whether or not that was true, it was certainly time to turn to the liver and find out what the extract did to it.

  Collip undertook to measure the extract’s effect on the glycogen-forming function of the liver, as well as to make observations on the extract’s effect on the amount of ketone bodies excreted in diabetes. He first confirmed, or thought he did, that the extract had no effect on ether-anesthetized normal dogs as well as ether-anesthetized diabetic ones, reinforcing the hypothesis that the liver was critical to its action.45 On Tuesday, December 13, Banting and Best depancreatized a large Airedale in the surgical operating room and Collip took it to his lab in the pathology building to await the development of diabetes.46 Meanwhile he continued working on his rabbits and tinkering with batches of extract.

  About this time the team had agreed that another highly desirable experiment to test the potency of the extract would be to measure its effect on the respiratory quotient of diabetic subjects. This measurement of the ratio of carbon dioxide breathed out to oxygen absorbed was thought to be a reliable guide to whether or not carbohydrates were being burned in the body. It involved complex apparatus and gas analysis, however, and it was agreed to delay this work until January when another researcher would be available to work on the problem with Best.47

  XI

  While Collip was starting work in his lab, Banting and Best spent the week of December 12–16 working on their newly discovered extract of whole pancreas. That first injection of whole dog pancreas had worked on December 11. On the 12th an alcoholic extract of whole pancreas seemed to work when administered through a stomach tube (apparently causing a blood sugar reduction from .42 to .28 in four hours). Whole cow pancreas injected intravenously also seemed potent. On December 13 and 14 Banting and Best administered extracts of liver, spleen, thyroid, and thymus, all made the same way as pancreatic extracts, to the test dog. None of them was effective. To try to make their pancreatic extract purer, they experimented with dialysis (the use of a semi-permeable membrane to filter out small molecules from colloids in a solution) and also found they could wash the residue with toluene after alcoholic evaporation to rid it of more of the fat-like lipid impurities. On the evening of December 15 they gave their second talk on pancreatic diabetes, addressing a group at Toronto General Hospital.48 On either that day or the next49, dog 35 was given by injection a piece of dried extract the size of a match which had been washed twice in toluene and redissolved in ten cc. of saline. In four hours its blood sugar went from .37 to .06, a spectacular result.50

  Then things went downhill. At week’s end Banting and Best made up the largest batch of extract yet, some seven hundred cc. of tissue and pancreatic juice with five hundred cc. of acid alcohol. They tested it on December 18 and found it had no potency whatever. They concluded that they had probably used too much acid. On the 19th or 20th another batch was made up, using just alcohol, no acid (the notebooks tend to be vague on the quantities used in these mixtures). A subcutaneous injection from this new batch had no effect. A little bit of acid was added to the final saline solution of extract and an intravenous injection was tried. The only effect was to cause the dog to vomit.

  They still had some extract on hand that they knew to be potent. Perhaps in reaction to the week’s disappointments, they decided, apparently without telling anyone, to try it on a human diabetic. One of Banting’s classmates, Joe Gilchrist, had become diabetic in early 1917 a few months after their graduation. He took the Allen treatment and was able to carry on with gradual downhill “progress” until October 1921 when a bout of influenza shattered his carbohydrate tolerance. All the symptoms returned and he began to deteriorate rapidly.51

  An index card in Banting’s papers is the only record of Banting and Best’s first “clinical” test of their extract.

  Clinical Use

  Dec. 20. Phoned Joe Gilchrist -

  gave him extract that we knew to

  be potent. – by mouth – empty stomach

  Dec. 21 – no beneficial result.52

  It was not yet known that no pancreatic extracts ever work by oral administration (they are, in fact, “digested” by the proteolytic digestive enzymes). Only a few days earlier, on December 12, Banting and Best had had that apparent success giving extract via a stomach tube to one of their dogs. It might work on Gilchrist, they must have thought. But it was still too risky to inject the extract into the blood stream or under the skin of a human.

  XII

  While Banting and Best were throwing out batches of impotent extract and seeing their potent extract have no potency on a human diabetic, Collip’s experiment was proceeding smoothly. He was making his own extract, using a technique similar to Banting and Best’s but with various improvements. Instead of evaporating the alcohol in a warm air current, he used a laboratory vacuum still. He did not evaporate all the liquid in the pancreas/alcohol solution, as Banting and Best did, but instead reduced it to about one-fifth of the original volume, giving him a suspension of fine particles in a clear straw-coloured liquid. This was filtered, leaving a liquid filtrate and a residue of solid particles.53 On December 20 Collip was ready to administer pancreatic extract to his diabetic Airedale. First he injected fifteen cc. of the liquid filtrate he had prepared. The Airedale’s blood sugar dropped from .309 to .217 in two and three-quarter hours. Then Collip injected a solution made with the solid particles left after that last filtration. It proved more potent, ten cc. dropping the blood sugar to .085 in 65 minutes and .051 in a further two hours and ten minutes.

  The lesson Collip learned in terms of extract preparation from testing both filtrate and residue-we will see its importance later – was only a bonus in terms of the main aim of the experiment, which was to test the extract’s effect on glycogen formation and ketonuria. As the Airedale became diabetic, Collip had been carefully measuring the amount of ketone bodies in its urine. On the 21st, after the injections of extract, the dog’s urine became completely ketone (and sugar) free. In their notebooks for August 5 and 7, Banting and Best had jotted down two casual, perhaps retrospective observations about their extract causing “acetone bodies” to disappear; they did not mention the subject in their first paper. Collip’s experiment was the first measured demonstration in Toronto that the extract could abolish ketosis.54

  There was much more to be demonstrated in the experiment. Through the 21st and into the 22nd Collip continued to give periodic injections of extract, while allowing the dog to consume glucose and milk freely. He was hoping that the extract would enable the diabetic dog’s liver to start making glycogen again from the carbohydrates it was consuming.

  Banting and Best spent the 21st and part of the 22nd experimenting on normal animals for the first time. On the 21st their extract had no effect on a normal dog. On the morning of Thursday the 22nd another batch had no effect. They tried a normal rabbit. No effect. On that discouraging note, seven failures in a row that week, they quit for the day and for the Christmas holidays. Their notebooks end. Th
e one experiment they kept going over Christmas was the longevity test on dog 33.

  Collip stayed in his lab that afternoon to complete the glycogen experiment. At 6 p.m. he chloroformed the Airedale, cut out its liver, and measured it for glycogen. An untreated diabetic dog’s liver would not have very much, no more than about 1.5 per cent at the very most. This dog’s liver was full of glycogen, so full it could hardly be measured. The liver was an incredible 25.6 per cent glycogen, Collip recorded.55 This was a result beyond anyone’s expectation – a crystal-clear demonstration that the extract enabled a diabetic animal’s liver to form glycogen. “There was thus afforded definite proof,” Collip wrote later, “of the restoration…of a function which was definitely known to be lacking in the diabetic state.”56

  Collip did not see Banting and Best until after Christmas when they all travelled by train to New Haven together for the meeting of the American Physiological Society. He had good news to tell them about the glycogen experiment. Great news, the most solid evidence yet that the group was on the right track, heading towards triumphant success. The “mysterious something,” as Collip described the active principle of their extracts a few days later, worked against diabetes.57

  Banting and Best must have been pleased by Collip’s news. But they must also have been a bit chagrined that it was Collip who had achieved so much with their extract just when their own attempts to make it work at all had resulted in a week of total failure.

  CHAPTER FIVE

  Triumph

  Most of the important people in North American diabetes research came to the Friday afternoon, December 30, session at the American Physiological Society conference at Yale University in New Haven. The program announced a paper by J.J.R. Macleod, F.G. Banting (by invitation), and C.H. Best (by invitation) on “The Beneficial Influences of Certain Pancreatic Extracts on Pancreatic Diabetes.” Among those present were Allen, Joslin, Kleiner, Scott, Carlson, and the Eli Lilly research director, George H.A. Clowes. It had been arranged that Macleod would chair the meeting and Banting would give the paper.

  Everyone who ever described that session (there are no formal records of it, only a half-page abstract of the paper, very similar to Banting and Best’s first long article, published a few months later in the A.P.S. Proceedings)1 remembered that Banting was nervous and spoke haltingly. The best account is Banting’s own (1940): “When I was called upon to present our work I became almost paralyzed. I could not remember nor could I think. I had never spoken to an audience of this kind before – I was overawed. I did not present it well.”2

  I

  The audience was an experienced, tough, and critical group of experts. There would have been many searching questions even if Banting’s presentation had been good. As it was, after Banting spoke, Allen, Kleiner, Carlson, and others, all raised points about the work. As Macleod recalled the meeting nine months later, “it was evident that he [Banting] had not succeeded in convincing all of his audience that the results obtained proved the presence of an internal secretion of the pancreas – the primary object of the work – any more definitely than had those of previous investigators.”3

  Macleod found himself in the unhappy position of seeing a presentation of highly promising research from his own lab, to which he had allowed his name to be attached, fall flat. It was a situation all scholars dread when they are students and fear for when their own students give their first papers. There was only one decent thing Macleod could do in the circumstances. Instead of asking the hapless Banting to respond to the criticisms, which would be like throwing the lamb to the wolves, Macleod came to his defence by joining the discussion himself. He tried to answer the critics, “laying stress,” he wrote, “on the frequency of direct relationship between the injections and the lowering of blood sugar and on the prolongation of life of two treated animals.” Elliott Joslin wrote thirty-five years later about the meeting that “Banting spoke haltingly, Macleod beautifully.”4

  Knowing of the work Allen, Scott, Carlson, and Kleiner had done, it is not difficult to imagine the questions they posed to the Toronto people. The most obvious, dealing with the extract’s “beneficial influences,” would be whether it also had toxic effects. Did it cause fever, for example? A hard question to answer in view of there being no temperature records for Banting and Best’s dogs (except one reading, showing fever). Were there other reactions? Well, the dogs did sometimes react to the extract.5 And if the longevity experiment, not yet complete – for dog 33 had been going just under five weeks – was being discussed, it had to be admitted that the first attempt at longevity, with dog 27, had ended abruptly when the dog had died of severe reaction to the extract.

  And what was the precise condition of these dogs? Best’s one memory of the meeting was of Anton Carlson mentioning that his depancreatized dogs sometimes lived for several weeks. Best interjected the comment that if this was so he had probably not taken out all the pancreas. “Young man, you might be right!” Best remembered Carlson responding.6 It was probably a two-edged comment, implicitly raising the question of the completeness of Banting and Best’s operations. Was it their extract or was it pancreatic remnants that kept their dogs alive? Were routine autopsies adequate to prove total pancreatectomy?7 What about the D:N ratios on the experimental dogs? Another embarrassing question, difficult to answer.

  A host of other questions could have been raised. How soundly based in the literature, for example, was the theory of selective atrophy after ligation, which had been so important at the start of the work? How sure were Banting and Best that the early pancreases they used had actually been fully atrophied? How could a fully atrophied pancreas supply as much extract as the figures indicated? Was it really clear that the proteolytic digestive enzymes were the problem in preparing pancreatic extracts?8 Many researchers, including at least three members of the audience, had made extracts that reduced hyperglycemia and glycosuria. Where exactly had Toronto gone beyond them? And so on. Few of these questions, if they were asked, could be answered satisfactorily by the Toronto team. What they could do, as Macleod did, was to keep drawing attention back to the blood sugars and the dogs’ survival. The extract reduced blood sugar; it apparently had kept two diabetic dogs alive (dog 92 back in August, and dog 33 now in December). Macleod might also have referred to the experiments in progress on the new whole gland extract; of course they had to be repeated, but just before Christmas there had been some exciting findings about ketonuria and glycogen formation. The work was going ahead vigorously on several fronts and there would be further reports in the near future.

  Joslin remembered the overall reaction to the session as being “little praise or congratulation, and a moderate amount of friendly but serious criticism of the work.”9 Judging from surviving correspondence, the experts took a cautious interest in the Toronto work. They might be onto something up there in Canada; we’ll look forward to hearing more. After the meeting E.L. Scott walked back to the hotel with Macleod, discussing his 1911 work. A few weeks later he sent Macleod details of his extraction methods, commenting that his extract would not have been likely to cause such sharp reactions as Macleod had described, but on the other hand it was never put to the “severe trials” they were using in Toronto. Writing Macleod about the same time on another matter, Frederick Allen added a last paragraph about their mutual interests:

  I hope your work with the pancreas extract is progressing satisfactorily. With the beginning of our animal experimentation here, I shall probably go ahead with plans I have had for a long time, in the direction of an extract. The methods in view are totally different from yours. You not only have priority, but, if you have solved the initial difficulties, your method is better than mine could ever be. I merely thought out my method as a means of escaping those difficulties, and it may have some value for other purposes at least, so I shall probably give it a trial. It is high time we had some treatment beyond mere diet, though I recognize the difficulties in the way of a practical application of any extra
ct.10

  There was one exception to the experts’ wariness. When Macleod returned to his New Haven hotel room after the session he got a phone call from George Clowes, the Lilly research man, who said that he thought the evidence was convincing and asked whether Eli Lilly and Company could collaborate with the Torontonians in preparing the extract commercially. (“It is true that Banting presented his material somewhat haltingly and certainly very modestly,” Clowes wrote in 1948. “However, anyone who was at all cognizant with the subject must have realized that a great discovery had been made and that provided the work could be brought to fruition there was every prospect that an important means of treating diabetes would be developed.”) Clowes talked the matter over with Banting and Macleod. He was told by Macleod that the work was not sufficiently advanced for commercial preparation. Clowes’ suggestion would be borne in mind.11

  The person most disappointed with the New Haven session was Fred Banting. However good or bad the reaction to the work had been, it was obvious to him and to everyone else how badly he had failed in presenting it. Instead of his idea and his experiments culminating in a great personal triumph, he had endured an embarrassing, humiliating afternoon. And this after all the frustrations of the week before Christmas, when his and Best’s work had gone so badly and Collip’s so well.