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
Pancreas. adult cow pancreas. From the islets From the fetal calf pancreas, which had not yet developed digestive glands.
the primary high energy molecule in human metabolism is
user
Adenosine triphosphate (ATP)
assistant
what is catabolism?
user
It is the set of metabolic processes that breaks down large molecules.
assistant
which hormones are related to it?
user
Cortisol, glucagon, adrenaline, cytokines, orexin and melatonin.
assistant
what is the emotional response due to the third one?
user
Fear.
assistant
how does the first hormone related to metabolism of glucose?
user
Cortisol stimulates the synthesis of 'new' glucose from non-carbohydrate sources; this is known as gluconeogenesis..
assistant
is process resisted by insulin?
user
Yes.
assistant
when was this substance which resists the process discovered?
user
1921.
assistant
can it be extracted?
user
Yes.
assistant
from where?
user
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
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
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
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
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
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
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 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
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
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
This yeast then makes the insulin, which is harvested from the bioreactor.
Insulin aspart | Introduction
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
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
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
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
The solution is purified as described above.
Alkaloid | Extraction
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
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
Insulin originates from beta cells located in the islets of Langerhans. Since each islet contains up to 2000 beta cells and there are one million islets in the pancreas it is apparent that pulsatile secretion requires sophisticated synchronization both within and among the islets of Langerhans.
Insulin oscillation | Introduction
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
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
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
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
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
The pump can be removed before a preplanned activity (anything from football practice to water skiing to dancing), and the pump user continues with the reduced basal insulin coming from injected Lantus or Levemir. After the activity is complete, the pump goes back on.
Untethered regimen | Sports and exercise
These are separated and the rest of the blood can be retransfused.
Erythrocytapheresis | Overview
Once an insulin molecule has docked onto the receptor and effected its action, it may be released back into the extracellular environment, or it may be degraded by the cell. The two primary sites for insulin clearance are the liver and the kidney. The liver clears most insulin during first-pass transit, whereas the kidney clears most of the insulin in systemic circulation. Degradation normally involves endocytosis of the insulin-receptor complex, followed by the action of insulin-degrading enzyme. An insulin molecule produced endogenously by the beta cells is estimated to be degraded within about one hour after its initial release into circulation (insulin half-life ~ 4–6 minutes).
Insulin | Degradation
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 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
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
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
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
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
Insulin may have originated more than a billion years ago. The molecular origins of insulin go at least as far back as the simplest unicellular eukaryotes. Apart from animals, insulin-like proteins are also known to exist in the Fungi and Protista kingdoms. Insulin is produced by beta cells of the pancreatic islets in most vertebrates and by the Brockmann body in some teleost fish. Cone snails "Conus geographus" and "Conus tulipa", venomous sea snails that hunt small fish, use modified forms of insulin in their venom cocktails. The insulin toxin, closer in structure to fishes' than to snails' native insulin, slows down the prey fishes by lowering their blood glucose levels.
Insulin | Evolution and species distribution
It is an insulin analogue in which a fatty acid (myristic acid) is bound to the lysine amino acid at position B29. It is quickly absorbed after which it binds to albumin in the blood through its fatty acid at position B29. It then slowly dissociates from this complex.
Insulin detemir | Chemistry
Once the MCS is cut, the gene for human insulin can be added making the plasmid genetically modified. After that, the genetically modified plasmid is put into the bacterial host and allowed to divide. To make the large supply that is demanded, the host cells are put into a large fermentation tank that is an optimal environment for the host. The process is finished by filtering out the insulin from the host. Purification can then take place so the insulin can be packaged and distributed to individuals with diabetes.
Multiple cloning site | Example
Prior to the introduction of biosynthetic human insulin, insulin derived from sharks was widely used in Japan. Insulin from some species of fish may be also effective in humans. Non-human insulins have caused allergic reactions in some patients related to the extent of purification, formation of non-neutralising antibodies is rarely observed with recombinant human insulin (insulin human rDNA) but allergy may occur in some patients. This may be enhanced by the preservatives used in insulin preparations, or occur as a reaction to the preservative. Biosynthetic insulin (insulin human rDNA) has largely replaced animal insulin.
Insulin analog | Animal 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
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
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
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
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
There are phylogenetic examples for such monomeric insulins in animals.
Insulin analog | Modifications
Additionally, an insert domain (ID, residues 638-756) resides within FnIII-2, containing the α/β furin cleavage site, from which proteolysis results in both IDα and IDβ domains. Within the β-chain, downstream of the FnIII-3 domain lies a transmembrane helix (TH) and intracellular juxtamembrane (JM) region, just upstream of the intracellular tyrosine kinase (TK) catalytic domain, responsible for subsequent intracellular signaling pathways.
Insulin receptor | Structure
DiabetesJournals.org
Insulin port | References
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
Moreover, both human saliva and the intestinal cell-free extract from mice can cause the complete conversion.
Genistin | Metabolism
Clinically testable results are far from realization at this time. Several other non-transplant methods of automatic insulin delivery are being developed in research labs, but none is close to clinical approval.
Insulin (medication) | Pancreatic transplantation
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
The test cannot be interpreted unless hypoglycaemia (< 2.2 mmol/L (or) < 40 mg/dl) is achieved.
Insulin tolerance test | Interpretation
Unlike some other longer-acting insulins, glargine must not be diluted or mixed with other insulin or solution in the same syringe. However, this restriction has been questioned.
Insulin glargine | Mixing with other insulins
coli". Most therapeutic insulin used today derives from this woman's tumor.
Insulinoma | History
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
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
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
As a result, glucose accumulates in the blood. The human insulin protein is composed of 51 amino acids, and has a molecular mass of 5808 Da. It is a heterodimer of an A-chain and a B-chain, which are linked together by disulfide bonds. Insulin's structure varies slightly between species of animals. Insulin from animal sources differs somewhat in effectiveness (in carbohydrate metabolism effects) from human insulin because of these variations. Porcine insulin is especially close to the human version, and was widely used to treat type 1 diabetics before human insulin could be produced in large quantities by recombinant DNA technologies.
Insulin | Introduction
Firstly, the mRNA is obtained and purified from the rest of the RNAs. Several methods exist for purifying RNA such as trizol extraction and column purification. Column purification is done by using oligomeric dT nucleotide coated resins where only the mRNA having the poly-A tail will bind. The rest of the RNAs are eluted out. The mRNA is eluted by using eluting buffer and some heat to separate the mRNA strands from oligo-dT.
CDNA library | mRNA extraction
Insulin glargine is formulated at an acidic pH 4, where it is completely water-soluble. After subcutaneous injection of the acidic solute (which can cause discomfort and a stinging sensation), when a physiologic pH (approximately 7.4) is achieved the increase in pH causes the insulin to come out of solution resulting in the formation of higher order aggregates of insulin hexamers. The higher order aggregation slows the dissociation of the hexamers into insulin monomers, the functional and physiologically active unit of insulin. This gradual process ensures that small amounts of insulin glargine are released into the body continuously, giving an almost peakless profile.
Insulin glargine | Acceptance and repartition in the body
Biosynthetic human insulin (insulin human rDNA, INN) for clinical use is manufactured by recombinant DNA technology. Biosynthetic human insulin has increased purity when compared with extractive animal insulin, enhanced purity reducing antibody formation. Researchers have succeeded in introducing the gene for human insulin into plants as another method of producing insulin ("biopharming") in safflower. This technique is anticipated to reduce production costs. Several analogs of human insulin are available. These insulin analogs are closely related to the human insulin structure, and were developed for specific aspects of glycemic control in terms of fast action (prandial insulins) and long action (basal insulins).
Insulin | Medical uses
It can be extracted from wheat bran and maize bran using concentrated alkali.
Ferulic acid | Extraction
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
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
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
This may be achieved by delivering insulin rhythmically to the portal vein, by light activated delivery, or by islet cell transplantation to the liver.
Insulin | Oscillations
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
Pure phycocyanin extractions can be isolated from algae. The basic segregation order is as follows. The rupturing of the cell wall, with mechanical forces (freeze thawing) or chemical agents (enzymes). Then, C-PC is isolated with centrifugation and purified with ammonium sulfate precipitation or chromatography -either ion or gel-filtration. After, the sample gets frozen and dried..
Phycocyanin | Biotechnology
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
It is possible for a person to live without a pancreas, provided that the person takes insulin for proper regulation of blood glucose concentration and pancreatic enzyme supplements to aid digestion.
Pancreas | Removal
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
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
First, genomic DNA (gDNA) is extracted from cells of interest by proteinase K treatment followed by phenol-chloroform extraction and ethanol precipitation. Additional zymolyase digestion is necessary for yeast cells to remove the cell wall prior to proteinase K treatment. gDNA can also be extracted with a variety of other methods, such as column-based methods.
DRIP-seq | Genomic DNA extraction
Pancreatic extracts in the treatment of diabetes mellitus Diabetes mellitus: its differentiation into insulin-sensitive and insulin-insensitive types.
List of important publications in medicine | <a href="Endocrinology">Endocrinology</a>
Regulatory sequences for the insulin gene are:
Regulatory sequence | Insulin gene
This can be followed by phenol extraction to remove proteins and other molecules. Then the DNA can be subjected to ethanol precipitation to concentrate the sample.
Restriction map | Related methods
Minor allergic reactions still occur occasionally, even to synthetic "human" insulin varieties.
Insulin (medication) | Principles
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
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
In essence, this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several hundred micrograms) of very pure plasmid DNA.
Plasmid | Plasmid DNA extraction
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
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
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
Of the approximately 71-minute estimated lifetime of an insulin molecule, over 60 minutes is spent attached to a liver receptor. In addition, circulating unbound insulin is excreted and reabsorbed by the kidneys and broken down in the lysosomes. The remainder of metabolism of insulin molecules is via intracellular proteolysis via insulysin and related enzymes.
Lente insulin | Pharmacokinetics
The latter may even be incorporated into an artificial pancreas system for automated insulin administration.
Microdialysis | Applications
Many of these approaches employ nanoparticle delivery systems and several are being tested in clinical trials.
Insulin (medication) | By mouth
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
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
Normally, endogenous insulin production is suppressed in the setting of hypoglycemia. A 72-hour fast, usually supervised in a hospital setting, can be done to see if insulin levels fail to suppress, which is a strong indicator of the presence of an insulin-secreting tumor.
Insulinoma | Suppression tests
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
The use of a chromatographic technique for insulin assay may be preferable to immunoassay in some circumstances, to avoid the issue of cross-reactivity affecting the quantitative result and also to assist identifying the specific type of insulin in the specimen.
Insulin (medication) | Detection in biological fluids
It contains a syringe-like reservoir with about three days' insulin supply. This is connected by thin, disposable, plastic tubing to a needle-like cannula inserted into the patient's skin and held in place by an adhesive patch. The infusion tubing and cannula must be removed and replaced every few days.
Intensive insulin therapy | Two common regimens: pens, injection ports, and pumps
In some, insulin injections may be combined with other injection therapy such as GLP-1 agonists. Cleansing of the injection site and injection technique are required to ensure effective insulin therapy.
Insulin (medication) | Subcutaneous
The semantic distinction reflects changing treatment.
Intensive insulin therapy | Introduction
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
RNA polymerase can be isolated in the following ways: And also combinations of the above techniques. (Wayback Machine copy)
RNA polymerase | Purification
Recovery of lithium has been demonstrated in the field; the lithium is separated by simple filtration. The process and environmental costs are primarily those of the already-operating well; net environmental impacts may thus be positive.
Lithium | Extraction
If two bands are very close together, it can be hard to separate them without some contamination. In order to clearly identify the band of interest, further restriction digests may be necessary. Restriction sites unique to unwanted bands of similar size can aid in breaking up these potential contaminants.
Gel extraction | Traditional
Here, it is often the case that the carboxylic acid will form a dimer in the organic layer so the distribution ratio will change as a function of the acid concentration (measured in either phase). For this case, the extraction constant "k" is described by "k" = HA/HA
Liquid–liquid extraction | Extraction without chemical change
The pre-mRNA of this protein is edited at two positions. These editing sites occur within the insulin growth factor domain.
IGFBP7 | Editing sites
An important feature is the presence of zinc atoms (Zn) on the axis of symmetry, which are surrounded by three water molecules and three histamine residues at position B10.
Insulin | Structure
Thujaplicins can be produced in plant cell suspension cultures, or can be extracted from wood using solvents and ultrasonication.
Thujaplicin | Occurrence and isolation