Insulin

INSULIN (from Latin insula-island), is a product of internal secretion of the pancreas. It received its name from the islets of Langerhans, which are considered the place of its formation. First isolated in 1922 by Banting, Best, and Collip. The presence in the pancreas of a substance whose absence or deficiency causes diabetes became undeniable after experiments by Mering and Minkowski (1889), which established that removal of the gland in animals causes diabetes. Numerous attempts to obtain an anti-diabetic substance from the pancreas remained fruitless for a long time. Banting and Best succeeded in solving this problem by selecting experimental conditions under which the gland hormone was not subjected to the destructive action of trypsin, and most importantly—thanks to the fortunate choice of testing method. It was proven that the pancreatic hormone lowers the blood sugar level; the discovery of microanalytical methods for determining sugar in blood played a decisive role in enabling the determination of the sugar-lowering ability of preparations and thus solving the question of the correctness of the extraction conditions selection. The starting material in the first experiments of Banting and Best was a) glands of dogs with atrophy of the part of the organ producing trypsin caused by ligation of the excretory duct, and b) glands of underdeveloped fetuses of cattle at the stage of development when the gland already functions as an internal secretion organ but does not yet produce trypsin. Subsequently, numerous methods for obtaining I. from normal glands of adult animals were developed. Properties of I. Chemically, I., like the overwhelming majority of hormones, has been very little studied. For a time, after Abel obtained crystalline I. with empirical formula C₂₅H₃₈O₇N₅S+3H₂O, it seemed that the problem was close to resolution. Subsequently, however, Dingemanse and Laqueur managed to obtain a preparation three times more active in amorphous form. From this it follows that Abel's crystals do not represent chemically pure I.—Thus, the properties of pure I. cannot yet be studied. Preparations of I. purified to a greater or lesser degree are characterized by certain common features. The nitrogen content is 14%, i.e., close to its content in proteins; upon hydrolysis of I. by acids, amino acids are obtained; I. contains easily cleavable sulfur, is not capable of dialysis. There is an assumption that insulin is a high-molecular albumose. Insulin is easily adsorbed by animal charcoal, kaolin and other adsorbents, easily dissolves in water, acids, alkalis and weak alcohol; insoluble in strong alcohol and fat solvents: ether, acetone, etc. The action of ultraviolet rays, oxidizing agents and reducing agents inactivates insulin. Whether all the described reactions are specific to the I. molecule or they are partially explained by the presence of protein substances in the preparations with which I. is in close connection has not yet been finally clarified.—An extremely important factor for the production and clinical application of I. is its easy destructibility by proteolytic enzymes, of which trypsin acts especially destructively. This property long served as an obstacle to the isolation of the hormone, and to this day it is impossible to apply per os conventional preparations. Destructibility by proteolytic enzymes either indicates the protein nature of I. or points to the easy decomposition of it in the free state as soon as it is freed from its connection with protein. I. has the ability to form water-insoluble compounds with acids (picric, oxalic, trichloroacetic) and to be precipitated from aqueous solution by pyridine and salts [NaCl, (NH₄)₂SO₄]: in high concentration. I. can be isolated from aqueous solutions by precipitating proteins at the iso-electric point (see) at pH approx. 5. Stability with respect to temperature varies depending on the medium: in acidic solutions I. withstands boiling for up to one hour, in alkaline solutions it is much less stable. Besides the pancreas, from which I. is obtained, it is also contained in a number of other organs and tissues of the animal body. Apparently in most cases this I. is of pancreatic origin, since after extirpation of the pancreas it quickly disappears from these organs. Insulin-like substances, called glucokinins, have been found in the plant world—yeast, bean husks, blueberry leaves, etc. On the basis of the fact of insulin-like action of agmatine (guanidinobutylamine), an examination of guanidine derivatives was undertaken, which established that particularly active in this respect are diguanidines and among them decamethylene-diguanidine. The latter, named sintaline, is used in the clinic for the treatment of diabetes. Both sintaline and especially glucokinins cannot completely replace insulin. Obtaining I. All the numerous methods for obtaining I. represent various modifications of basic methods, differently using its above-mentioned properties. The usual starting material is the glands of cattle. Due to the rapid destructibility of the hormone by trypsin, the processing of the material begins as soon as possible after slaughter. The main methods come down to the following. 1. In the first, largest group, the initial extraction of finely ground glands is carried out with acid alcohol. The resulting acid alcoholic extract is evaporated in vacuum, defatted and again dissolved in 80% alcohol, from which in turn it is precipitated by ether, absolute alcohol, etc. The raw I. obtained as a result is dissolved in water and can then be purified by various methods based on: a) precipitation of I. from aqueous solution with salts (while some impurities remain in the solution); b) precipitation of I. with picric acid with the formation of picrate; c) isolation of insulin by precipitating proteins at the iso-electric point at pH approx. 5; d) adsorption of insulin by animal charcoal, kaolin, etc. In various methods, different purification techniques are used in different sequences and combinations, often with the aim of obtaining the preparation most economically and advantageously, regardless of the final purity of the product. 2. The second group consists of those methods where the initial extraction of glands is carried out with acids, both inorganic (sulfuric, hydrochloric) and organic (acetic, formic). 3. The third path consists in treating the ground glands directly with picric acid with the aim of obtaining an insoluble picrate, which is then extracted with 70% acetone; picric acid is taken either in crystalline form or in saturated solution. The purification methods of initial extracts of the 2nd and 3rd groups of methods are also based on the same principles as the methods of the first group. I. can be isolated both in the form of a base and in the form of salts. Of the latter, the most common is hydrochloride. Recently, bile acid compounds of I. have been proposed, the main advantage of which is allegedly their stability with respect to enzymes of the gastrointestinal tract. If this assertion is true, new broad horizons open up before insulin therapy, based on the possibility of applying preparations per os. Physiol. properties and mechanism of action. I., whose role as a pancreatic hormone consists in regulating carbohydrate metabolism by accelerating the burning or conversion into glycogen of excess glucose in the body and other intermediate products of carbohydrate metabolism, causes in normal animals a rapid fall in blood sugar; ₍thus, in rabbits already after 1/2 hour after administration of medium doses—2-3 E.D. (see below) per 2 kg weight—the fall in sugar reaches its maximum. The amount of blood sugar, averaging about 0.1%, falls to 0.05-0.03% and sometimes even lower. Such a fall in blood sugar (hypoglycemia) leads to disturbance of respiration, general restlessness, fall in temperature and is often accompanied by convulsions. The mechanism of toxic phenomena and in particular the immediate cause of convulsions, despite the large number of explanations proposed, cannot be considered fully clarified. The action of I. not only on different animals but also on different individuals of the same species of animal is extremely variable. The susceptibility of rabbits to I. is connected with the previous nutrition, breed and even the color of the animals' coat. In some rabbits convulsions occur already from doses of 1/2-1 E.D. per animal; in some even large doses do not cause convulsions. Hypoglycemic phenomena can be eliminated by the administration of glucose, which in emergency cases is administered subcutaneously or even intravenously. I. upon intravenous administration does not act faster than upon subcutaneous. The administration of insulin causes an immediate decrease in the amount of glucose and other reducing substances not only in the blood but also in the tissues of the body.

Determining the total amount of sugar that disappeared from the fasting organism under the influence of I. and comparing it with the amount of blood sugar that disappeared shows that the main masses of glucose disappear from the tissues. Thus, the disappearance of sugar from the blood is an indicator of the process taking place throughout the body. When I. acts on a fasting organism, the ratio of the amount of CO₂ excreted to the amount of O₂ consumed hardly changes. The essence of I.'s action on carbohydrate metabolism consists in accelerating the oxidation of glucose and the synthesis of glycogen. These data were obtained from experiments on the administration of glucose to fasting animals, followed by the determination of glycogen, glucose, and intermediate breakdown products with and without the simultaneous action of I. The amount of glucose burned was controlled by determining the gas exchange, which was carried out in parallel with the chemical analysis. In experiments with I., the amount of glucose burned and the newly formed glycogen increases compared to control experiments. Glycogen accumulation occurs mainly in the muscles. Dale (Dale) with collaborators, who studied carbohydrate metabolism and gas exchange in isolated striated muscle in decapitated and eviscerated (i.e., with internal organs removed) cats under the influence of I. and without it, established: a) that basic fact that the main site of I.'s action is the striated muscle, where the amount of glycogen increases; b) that I. increases the rate of oxidative processes compared to normal in the ratio of 122:100; c) that I. increases the amount of sugar disappearing from the blood compared to normal by 5-6 times; d) that of the sugar disappearing from the blood, 24-52% turns into glycogen, whereas without I. this transition does not occur at all. The assumption of the predominant action of I. on the liver is refuted on the basis of both Dale's direct experimental data and because the action of the pancreatic hormone on animals with the liver removed manifests itself even faster than in normal ones. There are no firmly established consistent data on the action of I. on the isolated liver. It is believed that I. causes a delay or even complete cessation of sugar formation from glycogen in the liver, thereby preventing the organism from naturally combating the phenomena of hypoglycemia. Along with this, there apparently goes on the process of enhanced accumulation of glycogen by the liver, which can explain the suppression by insulin of the formation of acetone bodies in the liver (glycogen-fat antagonism of the liver).-The essence of the mechanism of the above processes of increased glucose oxidation and glycogen synthesis remains unexplained to this day. It is assumed that insulin either a) chemically causes corresponding changes in the glucose molecule, or b) enhances the enzymatic process of glucose breakdown and in particular its oxidative phase, as indicated by the experiments of Neuberg and Gottschalk (Neuberg, Gottschalk), who proved an increase in the amount of acetaldehyde during the breakdown of glucose in vitro by ground tissues in the presence of I., or c) in one way or another directly affects the animal cell. Changes in carbohydrate metabolism under the influence of I. are very short-lived and quickly cease if the administration of the drug is not repeated. In this respect, I. behaves like all hormones, whose action on the organism is always symptomatic. According to the assumption of the author of the application of bile acid I. (Stephan), this drug promises to produce a revolution in the treatment of carbohydrate metabolism disorders, because its action will a) manifest extremely slowly and persistently due to gradual entry into the blood through the liver and b) affect not only the direct regulation of carbohydrate metabolism but also the influence on the cells of the islets of Langerhans. Thus, by stimulating the organism to form its own I., there are grounds for the causal treatment of diabetes.-The action of I. on protein and fat metabolism has not been studied. The brilliant therapeutic effect of I. in diabetic coma confirms that the secondary disorders of fat metabolism resulting from liver glycogen depletion can be eliminated with the help of I.-The relationship of I. to other hormones has been insufficiently studied. It is firmly established that adrenaline, which accelerates the conversion of liver glycogen to glucose, eliminates the symptoms of hypoglycemia. It cannot be answered affirmatively whether adrenaline is a direct antagonist of I. in terms of its mechanism of action on the liver, because the action of adrenaline is not delayed by I. The hormones of the posterior pituitary gland apparently act in a manner opposite to I. The opinions of authors on the relationship of I. to other hormones, in particular to the thyroid hormone, are extremely contradictory. Testing of I. The complexity of the action on the organism and the danger of using I. in clinical practice necessitates caution in dosing. The absence of chemical reactions necessitates the use of biological evaluation to determine the activity of preparations. Initially, a simple investigation on a few animals has now developed into a developed biological standardization of I. The activity of I. is expressed in units of action (E.D.). Initially, 1 clinical E.D. was considered 1/3 of the amount of the drug that was capable, in rabbits fasted for 18-24 hours with an average weight of 2 kg, to cause after 4 hours a drop in blood sugar to 0.045% (i.e., to half the normal amount, taken as 0.09%). The instability of such an experiment, depending on the random fluctuations in the reaction of individual animals, and the divergent results obtained with it have led to the proposal of various methods of evaluation and concepts of E.D. At the present moment, the question has been resolved at the international level. The Hygiene Commission of the League of Nations has chosen several methods that give equally good results, taking not the abstract concept of E.D. as a basis, but comparison with a standard preparation of constant activity. The latter is produced in England by the Medical Research Council and is sent in dry form to laboratories that deal with the control of I. in their country. Similar functions in the USSR are performed by the laboratory of the Insulin Committee under the Scientific Council of the People's Commissariat of Health in Moscow. The international standard preparation is the hydrochloride salt of I., contains 8 E.D. in 1 mg, and is easily soluble in water. All commercial preparations must be compared with this standard. 1 E.D. of the preparation is taken as the amount that corresponds in effect to 0.125 mg of the standard. Two methods of evaluating I. are accepted. 1. Evaluation by the sugar-lowering effect, which is given in 2 modifications: a) in the most commonly used, activity is calculated by a ready-made formula, which includes data on the determination of blood sugar in rabbits before the experiment and after 1, 3, and 5 hours after the administration of different doses of I.; b) in the less common modification, activity is calculated by the expressed in % drop in sugar from the same dose for all rabbits. 2. Testing preparations by their convulsive action on mice, which are in an incubator at t^c 30-38°. It has been proven that under such conditions mice are particularly sensitive to I. and that the latter's ability to cause convulsions in such animals runs parallel to its sugar-lowering ability. By comparing the convulsive action of the standard and the tested preparation, the activity of the latter is determined.-Preparations. The usual commercial preparations of I. are weakly acidic aqueous solutions, preserved mostly with phenol or tricresol. The usual form of packaging is ampoules of 5 cm³. The activity of various preparations ranges from 20 to 60 E.D. in 1 cm³. In connection with cases of a decrease in activity of liquid preparations over time, dry preparations for dissolution immediately before use have recently come into use. Tablet preparations for sublingual use have not received wide distribution.

K. Sargin. Clinical Application of Insulin.

By the time of the discovery of insulin, two aspects of diabetic metabolism could be considered clarified: on the one hand, diabetes was characterized by the excretion of sugar in the urine, and on the other, it had been established that with very rare exceptions, diabetic patients have an elevated glucose content in the blood (for more details, see Diabetes mellitus). Both of these symptoms, as it turned out, are susceptible to the effects of insulin. Since the administration of insulin also satisfied the theoretical concepts of diabetes, it initially seemed that the task of treating diabetes with the new drug had been solved. Subsequent years showed that the task was by no means fully solved, but that insulin is the most important aid in the treatment of diabetes, without at the same time eliminating the need for dietary application. The harmlessness of prolonged insulin administration to the organism can be considered established on the basis of numerous cases of insulin use in the clinic. In addition, Macleod and Hedon showed that even with complete removal of the pancreas, with sufficient administration of insulin, dogs live for a long time without showing pathological symptoms (in Macleod's case - 6 years, Hedon's dog - 57 months). On human material, it has been determined that although insulin is a specific agent for diabetes, different patients still react to insulin injections with a decrease in urinary and blood sugar to varying degrees. There are patients, admittedly very few, in whom it is never possible to achieve the disappearance of sugar from the urine with the help of insulin, and in very rare cases, insulin may not have any effect at all. Insulin has a beneficial effect on the disturbed carbohydrate metabolism itself, as well as on all sorts of consequences of this disorder. One of the most frequent concomitant phenomena in diabetes - ketonuria - yields very well to insulin treatment, and with this means, the prevention and even treatment of coma can be achieved in many cases. It is difficult to say whether habituation to insulin occurs. Often, to destroy glycosuria, it is necessary over time to resort to ever larger and larger amounts of insulin units. Such observations, however, do not indicate a habituation to insulin, but rather the progressive nature of the disease. In other cases, patients before treatment and at the beginning of insulin treatment tolerate fewer carbohydrates than after insulin treatment. In exactly the same way, it is very often possible to later reduce the dose of insulin necessary at first to destroy glycosuria. Apparently, the aglycosuria achieved with insulin acts on the strength and tolerance of patients just as favorably as the aglycosuria achieved with diet. However, in this case as well, the question of the mechanism of increasing tolerance is by no means clear. In any case, the basic rule about the need to avoid general overfeeding of diabetic patients must be observed even during insulin application. Otherwise, under the cover of insulin, one may not notice the harmful effects of excessive nutrition (Joslin) on tolerance and may contribute to the progression of the disease. The indication for insulin therapy and the technique of insulin administration are still far from final development; each case of diabetes primarily requires the prescription of a specific diet. All authors agree that in severe cases, in addition to diet, the use of insulin is absolutely necessary. In exactly the same way, no one disputes that in mild cases, the patient should, if possible, do without insulin. But even in mild cases, the use of insulin may be temporarily useful if additional complicating factors are added to the diabetes (acute infection, operation, complication). In cases of moderate severity, the prescription of insulin, its dosage, and distribution by the hour are indicated differently by various authors, and there is no unity in this regard. Depending on their individual views, some doctors pay more attention to diet in treatment, others prefer larger doses of insulin. The technique of insulin administration usually consists of 1-2 daily injections. The number of units needed in each case is determined by careful trial. The morning dose is usually taken larger than the evening dose, and the injection is not later than 10 a.m. The usual starting dose is 5-10 units twice a day. An excess of injected insulin leads to the so-called hypoglycemic condition, which was also known earlier from experiments with liver extirpation. In animals, in these cases, convulsions and hypoglycemic coma may occur. Upon closer examination, one cannot speak of coma, but only restlessness, weakness, trembling, and a feeling of hunger are observed. Since cases (Maddock) of prolonged lowering of blood sugar below 0.05 percent without any symptoms have been described, it has been suggested that the matter is less about the low concentration of sugar in the blood than about the rapid drop of the curve from high to low figures. It is possible that the toxic substance in hypoglycemic coma is the intermediate products in the decomposition of glucose (methylglyoxal - Fischler). To eliminate the symptoms of hypoglycemia, it is sufficient to give the patient a small amount of carbohydrates (preferably glucose, fructose, or orange juice). The control of blood sugar after insulin injection, initially recommended, is often practically unfeasible and usually unnecessary. It is much more important not to inject excessively large doses of insulin at once. On the other hand, one should not suddenly deprive the patient of a large number of insulin units. If a patient receiving insulin changes the diet and switches from a carbohydrate-rich diet to a carbohydrate-free diet or even to fasting days, it is not recommended to discontinue insulin at the same time, otherwise one can cause pre-comatotic conditions or malaise in the patient. It should also be mentioned that under the influence of insulin, widespread edema easily occurs, so the increase in weight during insulin application should be taken into account cautiously. In addition to the use of insulin as a specific anti-diabetic agent, it has been recommended for use in some other diseases. Richter observed that diabetic patients gain weight from insulin administration and that a decrease in blood sugar concentration is accompanied by a feeling of hunger. Falta proposed using insulin for more successful fattening. To prevent hypoglycemia after insulin, the patient is immediately given a little food or a drink sweetened with glucose or cane sugar. The insulin dose is 5-30 units twice a day. Along with this, fattening with fats and carbohydrates is carried out. It is not easy to form a correct idea of the significance of insulin in these cases, since the therapy is combined (insulin-fattening), and on the other hand, insulin increases the amount of water in the body and the total weight of the patient. The use of insulin and fattening in tuberculosis has not been very successful. Insulin has not yet become widespread for the treatment of Basedow's disease, although it often gives good results in this disease and is rationally justified by the fact that thyroxine is in many respects an antagonist of insulin (effect on protein, fat, water and salt metabolism). It has been noticed that sugar, as well as glycogen fixed in the liver cell, help maintain the integrity of the liver cell. In jaundice and especially in cases of acute yellow atrophy, glycogen disappears from the liver. A liver poor in glycogen is more susceptible to the toxic effects of various poisons. On the other hand, the liver of diabetics who received insulin is much richer in glycogen compared to those cases where insulin was not used. From this arose the idea of using insulin therapy in connection with the administration of large amounts of glucose in the most diverse cases of liver diseases, in order to increase the glycogen content in the liver cell and thereby increase its resistance. The impression from such treatment (Richter, Eppinger) in acute hepatitis and even in acute yellow atrophy of the liver in its initial stages is undoubtedly favorable. Also other toxicemic conditions have been treated with insulin, primarily vomiting of pregnancy, and later vomiting as a symptom of other ailments. Many authors report successful observations. Finally, in cases of non-diabetic ketonuria, insulin treatment has also been tried (ketonuria after anesthesia and surgical operations). Apparently, non-diabetic ketonuria yields less well to insulin treatment than diabetic ketonuria. The area of surgical ketonuria and its significance for the clinic are far from sufficiently studied, and therefore the results of insulin treatment are extremely difficult and premature to discuss. There are also observations that the healing of wounds sometimes occurs particularly well with insulin injections, even if there is no ketonuria. In all cases of insulin use not for diabetes, it is recommended to simultaneously give carbohydrates or glucose orally or better - intravenously (4-5%). Then one can safely administer 10-20 units of insulin.