Bottlenecked by the time-consuming task of duct-tying dogs and waiting several weeks to extract insulin, Banting hit upon the idea of extracting insulin from the fetal calf pancreas, which had not yet developed digestive glands. By December, they had also succeeded in extracting insulin from the adult cow pancreas. Macleod discontinued all other research in his laboratory to concentrate on the purification of insulin. He invited biochemist James Collip to help with this task, and the team felt ready for a clinical test within a month.
Extraction and purification
Insulin
Insulin | Extraction and purification
Yes. Yes Yes Yes.
the primary high energy molecule in human metabolism is
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Adenosine triphosphate (ATP)
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what is catabolism?
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It is the set of metabolic processes that breaks down large molecules.
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which hormones are related to it?
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Cortisol, glucagon, adrenaline, cytokines, orexin and melatonin.
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what is the emotional response due to the third one?
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Fear.
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how does the first hormone related to metabolism of glucose?
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Cortisol stimulates the synthesis of 'new' glucose from non-carbohydrate sources; this is known as gluconeogenesis..
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is process resisted by insulin?
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Yes.
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when was this substance which resists the process discovered?
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1921.
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can it be extracted?
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Progress accelerated through December 1921 as it was clarified that pancreatic extracts could be used without removing the external (digestive) secretion. As the group prepared for clinical trials, biochemist James B. Collip joined the team at Banting's request to help purify the extract for human injection. On January 23, 1922, Leonard Thompson was successfully treated with Collip's extract at Toronto General Hospital. Six more patients were treated by February 1922 and quickly experienced an improved standard of life. Other notable early recipients of insulin included Elizabeth Hughes, Constance Collier, James D. Havens, and Ted Ryder. In April 1922, the Toronto group jointly authored a paper summarizing all work thus far, and formally proposed to name the extract "insulin".
History of diabetes | Insulin
Bottlenecked by the time-consuming task of duct-tying dogs and waiting several weeks to extract insulin, Banting hit upon the idea of extracting insulin from the fetal calf pancreas, which had not yet developed digestive glands. By December, they had also succeeded in extracting insulin from the adult cow pancreas. Macleod discontinued all other research in his laboratory to concentrate on the purification of insulin. He invited biochemist James Collip to help with this task, and the team felt ready for a clinical test within a month.
Insulin | Extraction and purification
In October 1920, Canadian Frederick Banting concluded that the digestive secretions that Minkowski had originally studied were breaking down the islet secretion, thereby making it impossible to extract successfully. A surgeon by training, Banting knew that blockages of the pancreatic duct would lead most of the pancreas to atrophy, while leaving the islets of Langerhans intact. He reasoned that a relatively pure extract could be made from the islets once most of the rest of the pancreas was gone. He jotted a note to himself: "Ligate pancreatic ducts of the dog. Keep dogs alive till acini degenerate leaving islets. Try to isolate internal secretion of these and relieve glycosuria." In the spring of 1921, Banting traveled to Toronto to explain his idea to J.J.R.
Insulin | Extraction and purification
On 30 July 1921, Banting and Best successfully isolated an extract ("isleton") from the islets of a duct-tied dog and injected it into a diabetic dog, finding that the extract reduced its blood sugar by 40% in 1 hour. Banting and Best presented their results to Macleod on his return to Toronto in the fall of 1921, but Macleod pointed out flaws with the experimental design, and suggested the experiments be repeated with more dogs and better equipment. He moved Banting and Best into a better laboratory and began paying Banting a salary from his research grants. Several weeks later, the second round of experiments was also a success, and Macleod helped publish their results privately in Toronto that November.
Insulin | Extraction and purification
Once the trypsin-secreting cells had died, insulin could be extracted from the islets of Langerhans. Banting discussed this approach with J. J. R. Macleod, Professor of Physiology at the University of Toronto. Macleod provided experimental facilities and the assistance of one of his students, Charles Best. Banting and Best, with the assistance of biochemist James Collip, began the production of insulin by this means. As the experiments proceeded, the required quantities could no longer be obtained by performing surgery on living dogs. In November 1921, Banting hit upon the idea of obtaining insulin from the fetal pancreas. He removed the pancreases from fetal calves at a William Davies slaughterhouse and found the extracts to be just as potent as those extracted from the dog pancreases.
Frederick Banting | Isolation of insulin
The letter emphasized that:The patent would not be used for any other purpose than to prevent the taking out of a patent by other persons. When the details of the method of preparation are published anyone would be free to prepare the extract, but no one could secure a profitable monopoly.The assignment to the University of Toronto Board of Governors was completed on 15 January 1923, for the token payment of $1.00. The arrangement was congratulated in "The World's Work" in 1923 as "a step forward in medical ethics". It has also received much media attention in the 2010s regarding the issue of healthcare and drug affordability.
Insulin | Patent
Between 1911 and 1912, E.L. Scott at the University of Chicago tried aqueous pancreatic extracts and noted "a slight diminution of glycosuria", but was unable to convince his director of his work's value; it was shut down. Israel Kleiner demonstrated similar effects at Rockefeller University in 1915, but World War I interrupted his work and he did not return to it.
Insulin | Discovery
Macleod, Professor of Physiology at the University of Toronto. Macleod was initially skeptical, since Banting had no background in research and was not familiar with the latest literature, but he agreed to provide lab space for Banting to test out his ideas. Macleod also arranged for two undergraduates to be Banting's lab assistants that summer, but Banting required only one lab assistant. Charles Best and Clark Noble flipped a coin; Best won the coin toss and took the first shift. This proved unfortunate for Noble, as Banting kept Best for the entire summer and eventually shared half his Nobel Prize money and credit for the discovery with Best.
Insulin | Extraction and purification
Attempts to extract insulin from ground-up pancreas cells were unsuccessful, likely because of the destruction of the insulin by the proteolysis enzyme of the pancreas. The challenge was to find a way to extract insulin from the pancreas prior to its destruction. Moses Barron published an article in 1920 which described experimental closure of the pancreatic duct by ligature; this further influenced Banting's thinking. The procedure caused deterioration of the cells of the pancreas that secrete trypsin which breaks down insulin, but it left the islets of Langerhans intact. Banting realized that this procedure would destroy the trypsin-secreting cells but not the insulin.
Frederick Banting | Isolation of insulin
The researchers sought assistance from industrial collaborators at Eli Lilly and Co. based on the company's experience with large scale purification of biological materials. Chemist George B. Walden of Eli Lilly and Company found that careful adjustment of the pH of the extract allowed a relatively pure grade of insulin to be produced. Under pressure from Toronto University and a potential patent challenge by academic scientists who had independently developed a similar purification method, an agreement was reached for non-exclusive production of insulin by multiple companies. Prior to the discovery and widespread availability of insulin therapy the life expectancy of diabetics was only a few months.
Pharmaceutical industry | Insulin
A series of experiments performed from the late 1800s to the early 1900s revealed that diabetes is caused by the absence of a substance normally produced by the pancreas. In 1869, Oskar Minkowski and Joseph von Mering found that diabetes could be induced in dogs by surgical removal of the pancreas. In 1921, Canadian professor Frederick Banting and his student Charles Best repeated this study and found that injections of pancreatic extract reversed the symptoms produced by pancreas removal. Soon, the extract was demonstrated to work in people, but development of insulin therapy as a routine medical procedure was delayed by difficulties in producing the material in sufficient quantity and with reproducible purity.
Pharmaceutical industry | Insulin
Animal glands were obtainable as a waste product of the meatpacking industry. Insulin was derived primarily from cows (Eli Lilly and Company) and pigs (Nordisk Insulinlaboratorium). The making of eight ounces of purified insulin could require as much as two tons of pig parts. Insulin from these sources is effective in humans as it is highly similar to human insulin (three amino acid difference in bovine insulin, one amino acid difference in porcine). Initially, lower preparation purity resulted in allergic reactions to the presence of non-insulin substances. Purity has improved steadily since the 1920s ultimately reaching purity of 99% by the mid-1970s thanks to high-pressure liquid chromatography (HPLC) methods.
Insulin (medication) | Principles
The work published by Banting, Best, Collip and Macleod represented the preparation of purified insulin extract suitable for use on human patients. Although Paulescu discovered the principles of the treatment, his saline extract could not be used on humans; he was not mentioned in the 1923 Nobel Prize. Professor Ian Murray was particularly active in working to correct "the historical wrong" against Nicolae Paulescu. Murray was a professor of physiology at the Anderson College of Medicine in Glasgow, Scotland, the head of the department of Metabolic Diseases at a leading Glasgow hospital, vice-president of the British Association of Diabetes, and a founding member of the International Diabetes Federation.
Insulin | Controversy
He concluded that there was an internal secretion of the pancreas, but suggested several reasons why it may never be captured in a pancreatic extract. Between 1910 and 1920, techniques for measuring blood sugar (glucose test) were rapidly improved, allowing experiments to be conducted with greater efficiency and precision. These developments also helped establish the notion that high blood sugar levels (hyperglycemia), rather than glycosuria, was the important condition to be relieved.
History of diabetes | Pancreatic extracts before insulin
Further experiments were successful and the three started to present their work at meetings. Macleod was a far better orator, and Banting came to believe that he wanted to take all the credit. Their discovery was first published in the February 1922 issue of "The Journal of Laboratory and Clinical Medicine". Macleod declined co-authorship because he considered it Banting's and Best's work. Despite their success, there remained the issue of how to get enough pancreas extract to continue the experiments. Together, the three researchers developed alcohol extraction, which proved to be far more efficient than other methods. This convinced Macleod to divert the whole laboratory to insulin research and to bring in the biochemist James Collip to help with purifying the extract.
John Macleod (physiologist) | Frederick Banting and the discovery of insulin
The discovery and purification of insulin for clinical use between 1921–1922 by a group of researchers in Toronto—Frederick Banting, J.J.R. Macleod, Charles Best, and James Collip—paved the way for treatment. The patent for insulin was assigned to the University of Toronto in 1923 for a symbolic dollar to keep treatment accessible. In regard to "diabetes insipidus", treatment became available before the causes of the disease were clarified. The discovery of an antidiuretic substance extracted from the pituitary gland by researchers in Italy (A. Farini and B. Ceccaroni) and Germany (R. Von den Velden) in 1913 paved the way for treatment.
History of diabetes | Introduction
The discovery not only significantly precipitated progress on extracting active fractions but also offered the very first concrete information on the structure of insulin—sulfur is an integral part of insulin molecules. Continuing with his research on extracting insulin, on November 1925, Abel finally was able to witness one of the most beautiful sights of his life, "glistening crystals of insulin forming on the sides of a test tube". While at the California Institute of Technology, Abel completed an article in the proceedings of the National Academy of Sciences entitled "Crystalline Insulin" in 1926. Although Abel's work received great compliments from the media and the science community, some doubted the purity of his crystals as preliminary tests revealed that they were in fact proteins.
John Jacob Abel | Crystallization of insulin
However, the criteria advanced to prioritize the pair's early work alone (before the extract was purified) would itself run into challenges in the 1960s and 1970s as attention was drawn to successes in the same year (Nicolae Paulescu) or earlier (George Ludwig Zuelzer, Israel Kleiner). As tends to be true of any scientific line of inquiry, "the discovery of a preparation of insulin that could be used in treatment" was made possible through the joint effort of team members, and built on the insight of researchers who came before them. In 1954, American doctor Joseph H. Pratt, whose lifelong interest in diabetes and the pancreas went back well before the Toronto discovery, published a "reappraisal" of Macleod and Collip's contributions in refining Banting and Best's flawed experiments and crude extract.
History of diabetes | Nobel Prize Controversy
By December 1921, he had also succeeded in extracting insulin from the adult pancreas. Pork and beef would remain the primary commercial sources of insulin until they were replaced by genetically-engineered bacteria in the late 20th century. In spring of 1922, Banting established a private practice in Toronto and began to treat diabetic patients. His first American patient was Elizabeth Hughes Gossett, daughter of U.S. Secretary of State Charles Evans Hughes.
Frederick Banting | Isolation of insulin
The book "Beckman's Internal Medicine" described the methods that Dr. Frederick Banting and Charles Best first used to extract insulin from the pancreases of dogs, calves, and cows in 1921. A Chinese chemist lent them a small laboratory in the basement of a municipal building, where they attempted to extract insulin from pancreata of water buffaloes. After much work, they finally produced a brown-coloured insulin. The insulin was tested on rabbits starved for twenty-four hours and then divided into two groups. One group was injected with the extracted mix, and the other with Eva's insulin. Without equipment to test the rabbits’ urine or blood the best way Victor could test the potency of the insulin was to see if the rabbits experienced the same hypoglycaemic shock as the other rabbits.
Eva Saxl | Insulin production
Purified animal-sourced insulin was initially the only type of insulin available for experiments and diabetics. John Jacob Abel was the first to produce the crystallised form in 1926. Evidence of the protein nature was first given by Michael Somogyi, Edward A. Doisy, and Philip A. Shaffer in 1924. It was fully proven when Hans Jensen and Earl A. Evans Jr. isolated the amino acids phenylalanine and proline in 1935. The amino acid structure of insulin was first characterized in 1951 by Frederick Sanger, and the first synthetic insulin was produced simultaneously in the labs of Panayotis Katsoyannis at the University of Pittsburgh and Helmut Zahn at RWTH Aachen University in the mid-1960s.
Insulin | Structural analysis and synthesis
Macleod successfully extracted insulin from the pancreas of dogs, fetal calves, and adult cows. In particular, Banting's experimental work with calf pancreas tissue took place at Connaught's farm site, where calves were involved in smallpox vaccine production. FitzGerald had arranged access to Connaught's modest facilities, along with $5,000 from the Labs' reserves, to expedite the team's work. In the months that followed, the researchers worked to refine the extracts to a degree safe for human injection with the help of biochemist James Collip. Tensions mounted during this time between the four "co-discovers" of insulin, exacerbated greatly by Collip's suggestion that he could return anytime to Alberta with his purification work and patent it.
Connaught Laboratories | Peacemaking
The method used by Paulescu to prepare his pancreatic extract was similar to a procedure described by the American researcher Israel S. Kleiner in an article published in 1919 in the "Journal of Biological Chemistry". Using his procedure, Kleiner had been able to demonstrate significant reductions in the concentration of blood and urinary glucose after intravenous injections of his extract. Paulescu then secured the patent rights for his method of manufacturing "pancreine" on 10 April 1922 (patent no. 6254) from the Romanian Ministry of Industry and Trade.
Nicolae Paulescu | Work on insulin
The Nobel Prize committee in 1923 credited the practical extraction of insulin to a team at the University of Toronto and awarded the Nobel Prize to two men: Frederick Banting and J.J.R. Macleod. They were awarded the Nobel Prize in Physiology or Medicine in 1923 for the discovery of insulin. Banting, incensed that Best was not mentioned, shared his prize with him, and Macleod immediately shared his with James Collip. The patent for insulin was sold to the University of Toronto for one dollar. Two other Nobel Prizes have been awarded for work on insulin. British molecular biologist Frederick Sanger, who determined the primary structure of insulin in 1955, was awarded the 1958 Nobel Prize in Chemistry.
Insulin | Nobel Prizes
James Bertram Collip, (November 20, 1892 – June 19, 1965) was a Canadian biochemist who was part of the Toronto group which isolated insulin. He served as the Chair of the Department of Biochemistry at McGill University from 1928–1941 and Dean of Medicine at the University of Western Ontario from 1947–1961, where he was a charter member of The Kappa Alpha Society.
James Collip | Introduction
Insulin which was extracted from animal sources was used as a medicine as early as 1922. These early insulin preparations required multiple daily injections due to the short duration of action and quick degradation of the insulin protein. For this reason, researchers began studying how to prolong the effects of injected insulin. In 1952, a team at Novo Terapeutisk led by K. Hallas-Møller discovered that crystals of various sizes would form when zinc was added to insulin suspensions. Larger insulin crystals take longer to dissolve into the bloodstream when injected into the body, and as such have a much longer duration of action than amorphous or small insulin crystals.
Ultralente insulin | History
Initially, Macleod and Banting were particularly reluctant to patent their process for insulin on grounds of medical ethics. However, concerns remained that a private third-party would hijack and monopolize the research (as Eli Lilly and Company had hinted), and that safe distribution would be difficult to guarantee without capacity for quality control. To this end, Edward Calvin Kendall gave valuable advice. He had isolated thyroxin at the Mayo Clinic in 1914 and patented the process through an arrangement between himself, the brothers Mayo, and the University of Minnesota, transferring the patent to the public university. On April 12, Banting, Best, Collip, Macleod, and FitzGerald wrote jointly to the president of the University of Toronto to propose a similar arrangement with the aim of assigning a patent to the Board of Governors of the University.
Insulin | Patent
Macleod's presentation at a meeting of the Association of American Physicians in Washington, D.C., on 3 May 1922 received a standing ovation, but Banting and Best refused to participate in protest. At that time, demonstrations of the method's efficiency drew huge public interest, because the effect on patients, especially children, who until then were bound to die, seemed almost miraculous. The pharmaceutical company Eli Lilly & Co. took over mass production, but without an exclusive license, as the patent was transferred to the British Medical Research Council to prevent exploitation.
John Macleod (physiologist) | Frederick Banting and the discovery of insulin
In January 1922, after 14-year-old Leonard Thompson suffered a severe allergic reaction to an injection of insulin, Collip achieved the goal of preparing a pancreatic extract pure enough for him to recover and to use in clinical trials. Successful trials were soon completed and the future of insulin was assured. Banting, Best and Collip subsequently shared the patent for insulin, which they sold to the University of Toronto for one dollar. Regrettably, due to disagreements between Banting and MacLeod, there was ill-will generated within the team. The Nobel Prize for Medicine was awarded to Banting and MacLeod in 1923. Feeling that Best had been overlooked in the award, Banting shared his portion with Best.
James Collip | Success of insulin
Hans Christian Hagedorn (1888–1971) and August Krogh (1874–1949) obtained the rights for insulin from Banting and Best in Toronto, Canada. In 1923 they formed Nordisk Insulin laboratorium, and in 1926 with August Kongsted he obtained a Danish Royal Charter as a non-profit foundation. In 1936, Hagedorn and B. Norman Jensen discovered that the effects of injected insulin could be prolonged by the addition of protamine obtained from the "milt" or semen of river trout. The insulin would be added to the protamine, but the solution would have to be brought to pH 7 for injection. University of Toronto, Canada later licensed protamine zinc insulin (PZI), to several manufacturers.
NPH insulin | History
Therefore, when he tested his pancreatic extract on humans on February 25, 1922, he administered the extract rectally. The patients seemed to show some reduction in glycosuria. This apparent success emboldened him to inject his extract intravenously into a diabetic patient on March 24, after which the patient's blood sugar apparently fell to zero (".000", "véritable AGLYCÉMIE"). A blood sugar level of zero should have placed the patient into a diabetic coma, but he made no mention of this effect in any of his papers. In contrast, the Toronto team had known for several months that dogs could be placed into a diabetic coma by an overdose of insulin, so they prepared orange juice and candy for the clinical trials.
Nicolae Paulescu | Nobel Prize controversy
The hormone glucagon was discovered by C.P. Kimball and John R. Murlin in 1923 as a hyperglycaemic (blood-sugar elevating) substance among the pancreatic extracts. The biological importance of glucagon was not known and the name itself was essentially forgotten. It was a still a mystery at the time de Duve joined Bouckaert at Leuven University to work on insulin. Since 1921, insulin was the first commercial hormonal drug originally produced by the Eli Lilly and Company, but their extraction methods introduced an impurity that caused mild hyperglycaemia, the very opposite of what was expected or desired. In May 1944 de Duve realised that cystallisation could remove the impurity.
Christian de Duve | Rediscovery of glucagon
Insulin was the first peptide hormone discovered. Frederick Banting and Charles Herbert Best, working in the laboratory of J.J.R. Macleod at the University of Toronto, were the first to isolate insulin from dog pancreas in 1921. Frederick Sanger sequenced the amino acid structure in 1951, which made insulin the first protein to be fully sequenced. The crystal structure of insulin in the solid state was determined by Dorothy Hodgkin in 1969. Insulin is also the first protein to be chemically synthesised and produced by DNA recombinant technology. It is on the WHO Model List of Essential Medicines, the most important medications needed in a basic health system.
Insulin | Introduction
In 1919, at the Rockefeller Institute, Kleiner was one of the first to demonstrate the effect of extracts from the pancreas on animals, causing hypoglycemia. These were the early efforts which eventually helped lead to the discovery of insulin.
Israel Kleiner (biochemist) | Insulin
Secretary of State Charles Evans Hughes), and future woodcut artist James D. Havens. In 1923, The Nobel Committee credited the practical extraction of insulin to the Toronto team and awarded the Nobel Prize in Physiology or Medicine to Frederick Banting (who shared his prize with Charles Best) and J.J.R. Macleod (who shared his prize with James Collip). The Insulin Committee of the Board of Governors of the University of Toronto was created in the same year to administer the patents (as previously agreed) and undertake licensing arrangements for the manufacture of insulin worldwide. The original committee consisted of John G.
Connaught Laboratories | Peacemaking
Four desired pieces of information are where the reagents used were obtained from, what level of genetic purity was obtained, what yield was obtained, and an electrophoresis gel image for confirmation.
MIQE | Nucleic acid extraction
Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation. After isolation, the DNA is dissolved in a slightly alkaline buffer, usually in a TE buffer, or in ultra-pure water.
DNA extraction | Basic procedure
After Banting and Macleod were awarded the 1923 Nobel Prize in Physiology or Medicine, Nicolae Paulescu wrote to the Nobel Prize committee claiming that he had discovered insulin first. However, his claims to priority cannot be sustained. Paulescu prepared pancreatic extract in 1916 and tested it in dogs, but Israel Kleiner tested pancreatic extract in dogs in 1915, as did George Ludwig Zuelzer in 1906. Zuelzer also wrote to the Nobel Prize committee asserting priority. All of these earlier attempts had produced pancreatic extracts that caused side effects in dogs or humans. The Toronto team had noticed the same side effects with their earlier extracts, but they continued working on the problem until they had purified insulin.
Nicolae Paulescu | Nobel Prize controversy
Hagedorn and August Krogh (1874–1949) obtained the rights for insulin from Banting and Best in Toronto. In 1923 they formed Nordisk Insulinlaboratorium, and in 1926 with August Krogh he obtained a Danish Royal Charter as a non-profit foundation. In the 1930s he became interested in modifying the absorption rate of insulin. He was aware that contaminating proteins slowed the absorption of insulin into the bloodstream, but these caused irritation and side effects. Thus he searched for a protein that would not cause any irritation. He came upon protamine, a protein isolated from fish sperm. Hagedorn discovered that the addition of protamine to insulin caused the insulin to form microscopic clumps.
Hans Christian Hagedorn | Biography
These clumps took longer to dissolve into the bloodstream. This complex of protamine and insulin is known as NPH (neutral protamine Hagedorn) insulin. It is one of the earliest example of engineering drug delivery.
Hans Christian Hagedorn | Biography
Had Noble won the toss, his career might have taken a different path. MacLeod was overseeing the work of Banting, who had no experience of physiology, and his assistant Best. In December, when Banting and Best were having difficulties in refining the pancreatic extract and monitoring glucose levels, MacLeod assigned the biochemist James Collip to the team. In January 1922, while Collip was working on insulin purification, Best and Banting administered prematurely their pancreatic extracts to 14-year-old Leonard Thompson, who suffered a severe allergic reaction. Eventually, Collip succeeded in preparing insulin in a more pure, usable form. Banting, Best and Collip shared the patent for insulin, which they sold to the University of Toronto for one dollar.
Charles Best (medical scientist) | Co-discovery of insulin
In fairness, he decided to give half of his prize money to Best. Macleod on his part felt the biochemist James Collip, who joined the laboratory team later, deserved to be included in the award and shared his prize money with him. Some maintain that Nicolae Paulescu, a Romanian professor of physiology at the University of Medicine and Pharmacy in Bucharest, was the first to isolate insulin, in 1916, although his pancrein was an impure aqueous extract unfit for human treatment similar to the one used previously by Israel Kleiner. When Banting published the paper that brought him the Nobel, Paulescu already held a patent for his discovery (10 April 1922, patent no.
Nobel Prize in Physiology or Medicine | Controversial inclusions and exclusions
When insulin was originally purified from bovine or porcine pancreata, all the proinsulin was not fully removed. When some people used these insulins, the proinsulin may have caused the body to react with a rash, to resist the insulin, or even to make dents or lumps in the skin at the place where the insulin was injected. This can be described as an iatrogenic injury due to slight differences between the proinsulin of different species. Since the late 1970s, when highly purified porcine insulin was introduced, and the level of insulin purity reached 99%, this ceased to be a significant clinical issue.
Proinsulin | Immunogenicity
An article he read about the pancreas piqued Banting's interest in diabetes. Banting had to give a talk on the pancreas to one of his classes at the University of Western Ontario on November 1, 1920, and he was therefore reading reports that other scientists had written. Research by Naunyn, Minkowski, Opie, Sharpey-Schafer, and others suggested that diabetes resulted from a lack of a protein hormone secreted by the islets of Langerhans in the pancreas. Schafer had named this putative hormone "insulin". The hormone was thought to control the metabolism of sugar; its lack led to an increase of sugar in the blood which was then excreted in urine.
Frederick Banting | Isolation of insulin
This yeast then makes the insulin, which is harvested from the bioreactor.
Insulin aspart | Introduction
Recently, another approach has been used by a pioneering group of Canadian researchers, using an easily grown safflower plant, for the production of much cheaper insulin.
Insulin | Structural analysis and synthesis
In 2006, two teams attempted to extract DNA from a tooth discovered in 2003, but both teams were unsuccessful. It has been suggested that this happened because the dentine was targeted; new research suggests that the cementum has higher concentrations of DNA. Moreover, the heat generated by the high speed of the drill bit may have denatured the DNA.
Homo floresiensis | DNA extraction attempt
The solution is purified as described above.
Alkaloid | Extraction
In the early 1900s, Georg Ludwig Zuelzer experimented extensively with pancreatic extracts. After initial tests on rabbits, he injected his extracts (which he called "acomatol") on humans to clear but inconsistent success and severe side-effects. He nonetheless took out an American patent on his yet-problematic extracts. Unfortunately, Zuelzer was ultimately unable to purify the extract due to difficulty obtaining pancreases, a lack of funding, and interruption by World War I. Ernest Lyman Scott, studying at the University of Chicago between 1911–12, also obtained some promising results but was discouraged from continuing. In 1913, John James Rickard MacLeod, at the time several years into research in the area of carbohydrate metabolism and blood sugar behaviour, synthesized the state of research in "Diabetes: Its Pathological Physiology".
History of diabetes | Pancreatic extracts before insulin
He controlled it with diet until 1927, when insulin treatment became available.
Edmund Clifton Stoner | Biography
In the winter of 1922–23, his diabetes deteriorated badly after an attack of bronchitis and the end of his life seemed nigh. In early-1922, Banting, Best, Collip and Macleod in Toronto, Canada made the discovery and isolation of insulin. Supplies were initially in short supply and slow to reach the UK, but in May 1923, Harrison cabled Lawrence – "I've got insulin – it works – come back quick". By this point, Lawrence was weak and disabled by peripheral neuritis and with great difficulty; drove across the continent and reached King's College Hospital on 28 May 1923. Following some preliminary tests, he received his first insulin injection on 31 May.
Robert Daniel Lawrence | Diabetic patient
Commercial preparations of lente insulin are standardized to 30% semilente (amorphous precipitates of insulin), and 70% ultralente (crystallized insulin). In early versions, the semilente insulin was extracted from pigs, and the ultralente insulin was extracted from cows.
Lente insulin | Manufacturing
In 1913, researchers in Italy (A. Farini and B. Ceccaroni) and Germany (R. Von den Velden) reported the anti-diuretic effect of the substance extracted from the posterior lobe of the pituitary gland. The hormone responsible for this effect was later isolated and named vasopressin. Even while the pathophysiology of diabetes insipidus was being further clarified, these findings made possible a relatively simple and effective treatment such that physicians could begin to control the disease. Various preparations of the extract were produced and made commercially available by the pharmaceutical industry through the 20th century. In 1928, Oliver Kamm and his colleagues posited two active principles in the pituitary extract: one with antidiuretic and pressor properties (vasopressin), and another with uterotonic properties (oxytocin).
History of diabetes | Diabetes insipidus
Following further concern regarding Eli Lilly's attempts to separately patent parts of the manufacturing process, Connaught's Assistant Director and Head of the Insulin Division Robert Defries established a patent pooling policy which would require producers to freely share any improvements to the manufacturing process without compromising affordability.
Insulin | Patent
Macleod, professor of physiology at the University of Toronto, and asked Macleod if he could use his laboratory to isolate pancreatic extracts from dogs. Macleod was initially sceptical, but eventually agreed before leaving on holiday for the summer. Before leaving for Scotland he supplied Banting with ten dogs for experiment and two medical students, Charles Best and Clark Noble, as lab assistants. Since Banting required only one assistant, Best and Noble flipped a coin to see which would assist Banting first. Best won and took the first shift. Loss of the coin toss proved unfortunate for Noble, given that Banting decided to keep Best for the entire summer and eventually shared half of his Nobel Prize money and a part of the credit for the discovery of insulin.
Charles Best (medical scientist) | Co-discovery of insulin
The researchers were hesitant to work with a commercial drug firm, particularly since they had the Connaught Laboratories' non-commercial facilities at hand. Nonetheless, as limits were reached at the scale to which Connaught could produce insulin, Clowes and Eli Lilly met with the researchers in 1922 to negotiate an agreement with the University of Toronto scientists to mass-produce insulin. The collaboration greatly accelerated the large-scale production of the extract. In 1923, Lilly began selling Iletin (Insulin, Lilly), their tradename for the first commercially available insulin product in the U.S for the treatment of diabetes. Numerous objections were registered by the Insulin Committee of the University of Toronto in regard to Lilly's use of the term "Iletin", although production continued under this name and the objection was later dropped "as a concession".
Eli Lilly and Company | Modernization:1900–1940
He received his Ph.D. in Uppsala 1957 with the thesis "Zone electrophoresis in columns and adsorption chromatography on ionic cellulose derivatives as methods for peptide and protein fractionations: application to the study of posterior pituitary hormones". The separation method for which Porath is most well known is gel filtration, which he developed together with Per Flodin. Flodin worked with dextran research at Pharmacia. In 1957, Porath discovered that columns filled with dextran gel could be used as "molecular sieves" to separate biomolecules by size. After patenting had been initiated, Porath and Flodin published their discoveries in "Nature" on 13 June 1959.
Jerker Porath | Introduction
A lot of people require insulin therapy to manage their blood sugar levels and keep them within a target range. In 1916, Nicolae C. Paulescu (1869-1931) succeeded in developing an aqueous pancreatic extract that normalized a diabetic dog. In 1921, he published 4 papers in the Society of Biology in Paris centering on the successful effects of the pancreatic extract in diabetic dogs. Research on the Role of the Pancreas in Food Assimilation by Paulescu was published in August 1921 in the Archives Internationales de Physiologie, Liège, Belgium. Initially, the only way to obtain insulin for clinical use was to extract it from the pancreas of another creature.
Insulin (medication) | Principles
Synthetic crystalline bovine insulin was achieved by Chinese researchers in 1965. The complete 3-dimensional structure of insulin was determined by X-ray crystallography in Dorothy Hodgkin's laboratory in 1969. The first genetically engineered, synthetic "human" insulin was produced using "E. coli" in 1978 by Arthur Riggs and Keiichi Itakura at the Beckman Research Institute of the City of Hope in collaboration with Herbert Boyer at Genentech. Genentech, founded by Swanson, Boyer and Eli Lilly and Company, went on in 1982 to sell the first commercially available biosynthetic human insulin under the brand name Humulin. The vast majority of insulin used worldwide is biosynthetic recombinant "human" insulin or its analogues.
Insulin | Structural analysis and synthesis
Even if the results were negative, it was the kind of experiment that ought to have been tried long ago, if only for completeness's sake. Banting, Macleod, and student assistant Charles Best began the first experiment on May 17, 1921. On June 14, Macleod left for Scotland and advised remotely through the summer, returning on September 21. During this time, Banting and Best obtained mixed but encouraging results. Since they began with the hypothesis (months later falsified through their own work) that it was necessary to avoid the external secretion in order to obtain the internal secretion, they first used degenerated pancreas, then used foetal pancreas obtained from slaughterhouses.
History of diabetes | Insulin
Insulin is produced in the pancreas and the Brockmann body (in some fish), and released when any of several stimuli are detected. These stimuli include the rise in plasma concentrations of amino acids and glucose resulting from the digestion of food. Carbohydrates can be polymers of simple sugars or the simple sugars themselves. If the carbohydrates include glucose, then that glucose will be absorbed into the bloodstream and blood glucose level will begin to rise. In target cells, insulin initiates a signal transduction, which has the effect of increasing glucose uptake and storage. Finally, insulin is degraded, terminating the response.
Insulin | Synthesis
Heparin is a blood thinner (anticoagulant), originally discovered in 1916 by Jay McLean and William Henry Howell at Johns Hopkins University. However, the crude substance was toxic and could only be extracted in small quantities. As co-discoverer of insulin Charles Best returned from his postgraduate studies in Europe to continue as Connaught's Assistant Director, he began a program in 1928 to purify heparin for clinical use. This direction of development benefitted from Connaught's experience with insulin production and ongoing arrangements with Canadian meat processors to obtain research material. In 1933, Arthur Charles and David Scott published the first papers on increasing the yield of heparin by rotting source tissues.
Connaught Laboratories | Development of heparin: 1928-1930s
In February 1922, doctor Frederick Grant Banting and biochemist John James Rickard Macleod from the University of Toronto, Canada, published their paper on the successful use of a different, alcohol-based pancreatic extract for normalizing blood sugar (glucose) levels (glycemia) in a human patient. An unsuccessful attempt had been made on January 11, 1922, and a successful administration followed on January 25, 1922. The Toronto team felt confident in the purity of their insulin and injected it intravenously into the patient, clearing up his glycosuria and ketonuria and restoring normal blood sugar. Paulescu's 1921 papers mentioned that the extract caused toxic side effects on dogs, which made it unusable in humans ("qui la rendent inapplicable dans la pratique médicale").
Nicolae Paulescu | Nobel Prize controversy
Toward the end of January 1922, tensions mounted between the four "co-discoverers" of insulin and Collip briefly threatened to separately patent his purification process. John G. FitzGerald, director of the non-commercial public health institution Connaught Laboratories, therefore stepped in as peacemaker. The resulting agreement of 25 January 1922 established two key conditions: 1) that the collaborators would sign a contract agreeing not to take out a patent with a commercial pharmaceutical firm during an initial working period with Connaught; and 2) that no changes in research policy would be allowed unless first discussed among FitzGerald and the four collaborators. It helped contain disagreement and tied the research to Connaught's public mandate.
Insulin | Patent
A partial extraction procedure was developed in 1935 which involved reacting the compound with benzoyl chloride to allow it to be separated from the water-soluble components. The compound was first isolated and purified to crystals by Osamu Shimomura. The structure of the compound was confirmed some years later. Feeding experiments suggest that the compound is synthesized in the animal from three amino-acids: tryptophan, isoleucine, and arginine.
Vargulin | History
In October 1923, Banting and Macleod were awarded the Nobel Prize in Physiology based on a nomination by August Krogh for "the discovery of insulin "and" their exploration of its clinical and physiological characteristics". Banting and Macleod publicly shared the prize with Best and Collip, respectively.
History of diabetes | Insulin
He demonstrated that Lilly's insulin process was contaminated, showing that, when injected into rats, the Lilly insulin caused initial hyperglycaemia and the Danish Novo insulin did not. Following his research published in 1947, Lilly upgraded its methods to eliminate the impurity. By then de Duve had joined Carl Cori and Gerty Cori at Washington University in St. Louis, where he worked with a fellow researcher Earl Wilbur Sutherland, Jr., who later won the Nobel Prize in Physiology or Medicine in 1971. Sutherland had been working on the puzzle of the insulin-impurity substance, which he had named hyperglycemic-glycogenolytic (HG) factor. He and de Duve soon discovered that the HG factor was synthesised not only by the pancreas but also by the gastric mucosa and certain other parts of the digestive tract.
Christian de Duve | Rediscovery of glucagon
Protein extraction often involves degradation of the cell wall. It is common for the degraded and fragile cell wall to be accidentally lysed, releasing unwanted DNA and the desired proteins. The resulting DNA-protein extract is highly viscous and difficult to purify, in which case DNase is added. The DNA is hydrolyzed but the proteins are unaffected and the extract can undergo further purification.
Deoxyribonuclease | Uses
Insulin was one of Hodgkin's most extraordinary research projects. It began in 1934 when she was offered a small sample of crystalline insulin by Robert Robinson. The hormone captured her imagination because of the intricate and wide-ranging effect it has in the body. However, at this stage X-ray crystallography had not been developed far enough to cope with the complexity of the insulin molecule. She and others spent many years improving the technique. It took 35 years after taking her first photograph of an insulin crystal for x-ray crystallography and computing techniques to be able to tackle larger and more complex molecules like insulin.
Dorothy Hodgkin | Insulin structure
The test result was contrary to the dominant view at the time on proteins—proteins were considered as unlikely to have such highly specific physiological activity shown by a hormone such as insulin. In 1927, he was published in the journal Science for this work with a paper entitled "Chemistry in Relation to Biology and Medicine with Especial Reference to Insulin and Other Hormones". At the time, Abel's laboratory was no doubt the center of insulin research in the United States. A lot of young scientists came to his lab and worked under Abel to study the newly crystallized hormone. Abel himself gradually withdrew from the actual experimental work on insulin after the first four years, but he continued to guide the scientists in his lab to unravel more and more about the structure of insulin molecule.
John Jacob Abel | Crystallization of insulin
No such substance has been found as yet, and the existence of such a substance remains speculative.
Insulin resistance | Hormones
Macleod, a senior professor of physiology at the University of Toronto, to ask if he might mount a research project on the internal secretion of the pancreas. Banting lacked experience in physiological research and had superficial knowledge of the subject. Nonetheless, Macleod took some interest and accepted Banting's request to work in his lab. On account of what may have interested Macleod, Michael Bliss considers the following:Speculation is in order here and is permissible because we have some idea of Macleod's knowledge of the literature. Whether he and Banting were discussing grafting or extracting, what must have appealed to Macleod as "never having been tried before" was the idea of somebody experimenting with degenerated or atrophied pancreas.
History of diabetes | Insulin
Unlike many medicines, insulin cannot be taken orally at the present time. Like nearly all other proteins introduced into the gastrointestinal tract, it is reduced to fragments (single amino acid components), whereupon all activity is lost. There has been some research into ways to protect insulin from the digestive tract, so that it can be administered in a pill. So far this is entirely experimental.
Insulin (medication) | Methods of administration
Grodsky's study of the perfused pancreas in the 1960s, and then of isolated pancreatic islets in the 1970s, resulted in the discovery that insulin is released from the pancreas in two separate phases, one "fast" and one "slow." This biphasic understanding eventually evolved to accommodate a third, even slower phase of insulin release. These discoveries were significant because they implied that it was not possible to replicate the pancreas's function through insulin injections alone, since injections deliver the insulin all at once. The need was thus identified for a method of pumping insulin into the body in stages. The result was the artificial pancreas, to whose development and refinement Grodsky contributed substantially throughout his career.
Gerold Grodsky | Research Work
Ultralente insulin was considered to be a "long-acting" insulin that could be used once per day to provide a basal level of insulin, similar to some protamine-containing preparations. While originally isolated from bovine or porcine sources, the advent of recombinant DNA technology in the 1980s allowed human insulin to be mass produced in yeast or bacteria. By the mid 1990s, ultralente insulin was being prepared from recombinant human insulin, instead of insulin extracted from animals. The advent of insulin analogues and continued use of NPH insulin led to the discontinuation of ultralente insulin products in the mid-2000s, and FDA approval to be marketed in the US was withdrawn by 2011.
Ultralente insulin | History
His research on soybeans led to techniques that allowed insulin to be dried and later processed into an inhalable insulin, named Exubera by Pfizer.
A. Carl Leopold | Career
Extraction in chemistry is a separation process consisting in the separation of a substance from a matrix. Common examples include "liquid-liquid extraction", and "solid phase extraction". The distribution of a solute between two phases is an equilibrium condition described by partition theory. This is based on exactly how the analyte moves from the initial solvent into the extracting solvent. The term "washing" may also be used to refer to an extraction in which impurities are extracted from the solvent containing the desired compound.
Extraction (chemistry) | Introduction
Christian de Duve, the chairman of the Laboratory of Physiological Chemistry at the Catholic University of Louvain in Belgium, had been studying the mechanism of action of a pancreatic hormone insulin in liver cells. By 1949, he and his team had focused on the enzyme called glucose 6-phosphatase, which is the first crucial enzyme in sugar metabolism and the target of insulin. They already suspected that this enzyme played a key role in regulating blood sugar levels. However, even after a series of experiments, they failed to purify and isolate the enzyme from the cellular extracts. Therefore, they tried a more arduous procedure of cell fractionation, by which cellular components are separated based on their sizes using centrifugation.
Lysosome | Discovery
Secoisolariciresinol diglucoside can be isolated from de-fatted (hexane extraction) flaxseed by extraction of the lignan polymer precursor with a water/acetone mixture, followed by acetone removal and alkaline hydrolysis.
Secoisolariciresinol diglucoside | Extraction
Due to its extreme lethality, as well as it being only commercially available at certain times and then at an extremely high cost, Amanita phalloides had to be retrieved from the wild in order to collect the β-amanitin protein. This was first achieved by collecting "Amanita phalloides" fruiting bodies in New Jersey in 1975. These mushrooms were then dried for 24 hours and then ground in a blender with water. The slurry created was homogenized further to break open any intact cells, and after this a brown syrup extract containing the toxins was collected. This extract was then taken through various separation methods to isolate the toxins themselves.
Beta-Amanitin | Collection and purification
Insulin was introduced by Banting and Best from the University of Toronto in 1921 as an injectable agent. German researchers first introduced the idea of inhalable insulin in 1924. Years of failure followed until scientists realized they might be able to use new technologies to turn insulin into a concentrated powder with particles sized for inhalation. In the 1980s Nektar Therapeutics developed technology to make insulin into small particles, technology then licensed to Pfizer. Alkermes developed a delivery device that they licensed to Eli Lilly and Company. Once concrete methods were developed, human tests began in the late 1990s. In January 2006, the U.S.
Inhalable insulin | History
Abel's work on insulin started with an invitation from his old friend Arthur A. Noyes at California Institute of Technology. Noyes had just received a grant from the Carnegie Corporation for research on insulin, and he thought that Abel would be the right person to lead that research. After some preliminary experiments on the subject, Abel decided to take on the research, and replied to his friend, "Will attack insulin. Writing. J. J. Abel." Abel invested the next few years on purifying insulin. While he was trying various means to purify insulin, he had the idea to measure sulfur content of his extracts and found that the higher the sulfur content, the greater the activity.
John Jacob Abel | Crystallization of insulin
Enzyme separation may be accomplished through solid-liquid extraction techniques such as centrifugation or filtration, and the product-containing solution is fed downstream for product separation.
Industrial enzymes | Recovery
A Soxhlet extractor is a piece of laboratory apparatus invented in 1879 by Franz von Soxhlet. It was originally designed for the extraction of a lipid from a solid material. Typically, Soxhlet extraction is used when the desired compound has a "limited" solubility in a solvent, and the impurity is insoluble in that solvent. It allows for unmonitored and unmanaged operation while efficiently recycling a small amount of solvent to dissolve a larger amount of material.
Soxhlet extractor | Introduction
In October 1920, Frederick Banting took interest in carbohydrate metabolism while preparing a talk he was to give his physiology students at Western University in London, Ontario. He encountered an article by Moses Barron which reported an autopsy of a patient whose pancreatic stone had obstructed the main pancreatic duct, but most of the islet cells had survived intact. From this encounter, Banting had the idea to: "Ligate pancreatic ducts of dog. Keep dogs alive till acini degenerate leaving Islets. Try to isolate the internal secretion of these to relieve glycosurea ["sic"]" On November 8, 1920, Banting met with J.J.R.
History of diabetes | Insulin
Prior to analysis, YTXs must be isolated from the sample medium whether this is the digestive gland of a shellfish, a water sample, or a growth-culture medium. This can be achieved by several methods:
Yessotoxin | Extraction methods
Other notable discoveries since the early development of insulin and metformin include: In 1980, U.S. biotech company Genentech developed biosynthetic human insulin. The insulin was isolated from genetically altered bacteria (the bacteria contain the human gene for synthesizing synthetic human insulin), which produce large quantities of insulin. The purified insulin is distributed to pharmacies for use by diabetes patients. Initially, this development was not regarded by the medical profession as a clinically meaningful development. However, by 1996, the advent of insulin analogues which had vastly improved absorption, distribution, metabolism, and excretion (ADME) characteristics which were clinically meaningful based on this early biotechnology development.
History of diabetes | Further developments
Now there was nothing new in the idea of producing degeneration or atrophy of the acinar tissues by ligating the pancreatic ducts—all sorts of researchers had done this. Their interest, however, had been almost entirely in measuring the relative amounts of degeneration that took place in the various components of the pancreas, particularly the relative changes in the acinar and islet cells... Nobody had either tried to prepare a graft or administer an extract using a fully degenerated pancreas. And yet, theoretically, if there was an internal secretion, and if it did come from the islets of Langerhans, and if it was the acinar cells but not the islets that degenerated after the ducts were ligated, and if two or three other conditions held good, then perhaps some interesting results would follow.
History of diabetes | Insulin
It can be extracted from wheat bran and maize bran using concentrated alkali.
Ferulic acid | Extraction
β-glucan extraction from oat can be difficult due to tendency of depolymerization – which often occurs in high pH. Thus β-glucan extraction is usually performed under a more neutral pH and generally at temperatures of 60-100 degrees Celsius. Usually β-glucan is solubilized in the extraction process with residual starch, which is then removed by hydrolysis with alpha-amylase. The residual solution usually contains coextracts of hemicelluloses and proteins which can then be separated through selective precipitation. Through wet milling, sieving, and solvent-extraction, oat beta-glucans can achieve up to 95% extraction purity.
Oat beta-glucan | Extraction
This study can possibly be the insight for developing a new way to treat type-1 diabetes, which currently can only be treated with injection of insulin.
Cellulin | Research
Another measure of insulin resistance is the modified insulin suppression test developed by Gerald Reaven at Stanford University. The test correlates well with the euglycemic clamp, with less operator-dependent error. This test has been used to advance the large body of research relating to the metabolic syndrome. Patients initially receive 25 μg of octreotide (Sandostatin) in 5 mL of normal saline over 3 to 5 minutes via intravenous infusion (IV) as an initial bolus, and then, are infused continuously with an intravenous infusion of somatostatin (0.27 μg/m/min) to suppress endogenous insulin and glucose secretion. Next, insulin and 20% glucose are infused at rates of 32 and 267 mg/m/min, respectively.
Insulin resistance | Modified insulin suppression test
While most insulin pumps are waterproof (at least when they are new), the high cost of the pump often leads to caution on the part of the user, who often removes the pump when planning to get wet. Additionally most pump manufacturers recommend that the pump be removed even if it is waterproof as waterproofing is only guaranteed ideally and cannot take into account wear and tear. However waterproof pump cases for water sports are available. The pumps can always be disconnected for short periods for bathing with little issue. However, for parties in a hot tub or for a day at the beach, removing the pump and "going untethered" can be a welcome vacation.
Untethered regimen | Water activities
In the late 19th century the extraction of peat slowly continued.
Nij Beets | History
Best moved in 1915 to Toronto, Ontario, where he started studying towards a bachelor of arts degree at University College, University of Toronto. In 1918, he enlisted in the Canadian Army serving with the 2nd Canadian Tank Battalion. After the war, he completed his degree in physiology and biochemistry. As a 22-year-old medical student at the University of Toronto he worked as an assistant to the surgeon Dr. Frederick Banting and contributed to the discovery of the pancreatic hormone insulin, which led to an effective treatment for diabetes. In the spring of 1921, Banting travelled to Toronto to visit J.J.R.
Charles Best (medical scientist) | Co-discovery of insulin
George Walden started working for Eli Lilly in 1917, at which time he was paid $85 each month. George Walden was the head chemist at Eli Lilly in 1922. Previous attempts at purification of insulin attempted to keep the insulin in solution and precipitate out the contaminating proteins. Walden found that if he adjusted the pH to produce the maximum precipitation, the precipitate contained highly purified insulin. His process yielded insulin that was 10 to 100 times more pure than previous batches.
George B. Walden | Introduction
This coagulated sample is then digested using proteolytic enzyme to yield an extract from which pure heparin can be extracted. This mechanism proved more effective than the old method of extracting heparin by applying the digestive enzyme to the sample before separating the proteins, increasing the yield of heparin in each sample.
Edith M. Taylor | Career
Before biosynthetic human recombinant analogues were available, porcine insulin was chemically converted into human insulin. Chemical modifications of the amino acid side chains at the N-terminus and/or the C-terminus were made in order to alter the ADME characteristics of the analogue. Semisynthetic insulins were clinically used for some time based on chemical modification of animal insulins, for example Novo Nordisk enzymatically converted porcine insulin into semisynthetic 'human' insulin by removing the single amino acid that varies from the human variety, and chemically adding the human amino acid. Normal unmodified insulin is soluble at physiological pH. Analogues have been created that have a shifted isoelectric point so that they exist in a solubility equilibrium in which most precipitates out but slowly dissolves in the bloodstream and is eventually excreted by the kidneys.
Insulin analog | Modifications
From fluid tissues such as blood, cells are extracted by bulk methods, usually centrifugation or apheresis. From solid tissues, extraction is more difficult. Usually, the tissue is minced and then digested with the enzymes trypsin or collagenase to remove the extracellular matrix (ECM) that holds the cells. After that, the cells are free floating, and extracted using centrifugation or apheresis. Digestion with trypsin is very dependent on temperature. Higher temperatures digest the matrix faster but create more damage. Collagenase is less temperature dependent, and damages fewer cells, but takes longer and is a more expensive reagent.
Tissue engineering | Extraction
This is advantageous as the cells are still living after the extraction has taken place. Further advances now mean that it is possible to extract the secreting cells using a magnetic-based separation system or using a flow cytometer.
Secretion assay | Introduction