Irradiated Preparations

IRRADIATED PREPARATIONS, such preparations in which ergosterin (or products containing it) is activated by the action of ultraviolet rays and converted into the antirachitic factor (vitamin D). These include primarily preparations of irradiated ergosterin (see below). In addition, there are preparations of irradiated yeast, irradiated milk, and some other products subjected to the action of ultraviolet rays. A characteristic feature of irradiated preparations is that the ergosterin contained in them, which before irradiation is physiologically inactive by itself and does not possess antirachitic action, under the influence of irradiation by ultraviolet rays (under certain conditions - see below) is activated, acquires extremely strong antirachitic power - it is converted into the antirachitic "vitamin D". Therefore, these preparations are also called "D-vitamin preparations". This group of preparations should be distinguished from those natural products or preparations which, for example, cod liver oil, already possess antirachitic properties by themselves and contain natural, active (so to speak, in ready form) vitamin D; these preparations not only do not need irradiation, but under the action of ultraviolet rays they can even

114 more or less completely destroyed, to lose their active properties (since ultraviolet rays generally decrease or destroy the activity of enzymes _T hormones, etc.; they also destroy the already formed vitamin D, if irradiation is not timely interrupted). There are also preparations of a mixed type: vitamin D, prepared by irradiating ergosterol_u is added, for example, to cod liver oil (containing natural vitamin A and some, variable amounts of vitamin D) or the preparation of irradiated yeast is used, in which in addition to vitamin D obtained by irradiation, there is also vitamin B itself, etc. -The establishment of the fact that under the influence of ultraviolet rays certain substances-ergosterol and products containing it-acquire strong antirachitic action should be considered one of the remarkable discoveries of recent times, extremely important both in practical and theoretical terms; this discovery created a "new era" (in the words of A. Hess) in the doctrine of rickets, in its prevention and therapy, wrote new brilliant chapters in the doctrine of vitamins, forced to change some basic concepts and put forward new ones (e.g. about "hyperavitaminosis"), opened up new possibilities for the so-called "indirect irradiation" (instead of direct, "direct irradiation" of the body, the taking into of substances previously irradiated with ultraviolet rays), created the basis for the successful application of these preparations against such diseases, the development of which depends to some extent on the lack of light, on "light hunger" (Lichtmangelkrank-heit) and many others. History. The discovery that led to the creation of irradiated preparations was the result of 2 series of research and brilliant discoveries in two seemingly completely different scientific fields: in the study of the action of rays, especially ultraviolet ones, and in the study of vitamins and avitaminoses, in particular-successes in the study of experimental rickets. In 1919, on the basis of careful roentgenological and other studies, Huldschinsky showed that by irradiation with ultraviolet rays it is possible to cure children of rickets. The fact itself probably would not have led to further important discoveries if at the same time fundamentally important successes had not been achieved in obtaining and studying experimental rickets. Mellanby and his collaborators (1919) managed to show that the essentially important factor in the occurrence of experimental rickets is the lack in food of a fat-soluble vitamin (vitamin A, as Mellanby mistakenly assumed), and it was possible to obtain typical rickets in puppies. Soon Sherman, Pappenheimer and McCollum with collaborators developed the methodology and technique, with which it is possible to obtain typical rickets in rats with great regularity. This experimental rickets of rats, similar in general to rickets of children (although differing from the latter in some respects), is especially valuable in that the preventive and therapeutic actions-ultraviolet rays, cod liver oil, irradiated antirachitic preparations, etc., which act on rickets of children, have a similar effect on experimental rickets of rats, and in both cases similar changes in the bones, established roentgenologically or on hist. preparations, the same changes in blood (especially Ca and P) etc. are observed. In the further study of the antirachitic action of cod liver oil by McCollum and his collaborators, it was established that there is a special "antirachitic factor", "vitamin D" (see Vitamins), different from the also fat-soluble vitamin A. To clarify how in rickets, characterized by the absence of vitamin D, irradiation of the body with ultraviolet rays can cause healing, i.e. can replace the introduction of vitamin D with food 11« (since according to the basic principle of the then doctrine of avitaminosis can be eliminated only by introducing the missing vitamin into the body), in a short time a large number of diverse studies were carried out.-In 1924 in America Hess and Weinstock and independently of them in the same year Steenbock and Black showed that some products (different oils, spinach, yeast, etc.), themselves antirachitically inactive, after irradiation with ultraviolet rays acquire significant antirachitic properties. Thus a completely new principle of obtaining antirachitic preparations, differing in powerful action on the organism, was discovered. This principle was then tested in relation to rickets of children: in 1925 Kramer in America, Cowell in England, Gyorgy in Germany showed that milk subjected to irradiation with ultraviolet rays successfully cures rickets of children. As numerous further studies have shown, some natural food products can be activated in the antirachitic direction, others-cannot. Hess and others managed to notice a fundamental difference between substances capable of activation and substances devoid of this ability: activation turned out to be in some way connected with the presence of non-saponifiable fat-like substances-with cholesterol, in general with sterols. The further development of these very difficult and complex questions was carried out simultaneously by several groups of united scientists in America, Germany and England. As a result of this work, as early as 1926, almost simultaneously by several researchers, it was indicated that what is activated by ultraviolet rays is not cholesterol itself, but some substance difficult to separate from cholesterol, which constantly accompanies it ("contaminates") in very small quantities. Finally, at the beginning of 1997, as a result of the combined efforts of Hess and Windaus, it was proved that the unknown substance that acquires antirachitic strength under the influence of ultraviolet rays is ergosterol-a long-known lipoid described by Tanret as early as 1889 and 1908. Windaus pointed out that ergosterol is the preliminary substance, the "provitamin", which, being activated under the influence of ultraviolet rays, turns into the "antirachitic factor"-vitamin D. Indeed, by irradiating pure ergosterol with ultraviolet rays, as numerous studies have shown, it is easy to obtain extremely strong antirachitic preparations. Irradiation of ergosterol with ultraviolet rays and products of irradiation. To obtain antirachitic irradiated preparations, a solution of ergosterol is usually irradiated with ultraviolet rays. Ergosterol, for which the formula C₂₇H₄₂O (sterol with 3 double bonds) is currently accepted, is constantly present in small amounts along with cholesterol in almost all plant and animal tissues; it is found in relatively large amounts in ergot and in yeast, from which it is obtained for the manufacture of various irradiated preparations sold under different names. Irradiation of the ergosterol solution is carried out in special apparatuses without access to air (e.g. in the atmosphere of N or CO₂); rays with a wavelength shorter than 280 mμ are often filtered out. Under the action of ultraviolet rays, ergosterin is significantly activated already after a few minutes, then the activity reaches a maximum (under certain conditions, e.g. after 22^1/2 minutes of irradiation) and then gradually decreases again. The process of activating ergosterin with ultraviolet rays is still insufficiently studied.-On the basis of modern data, it should be recognized that a solution of irradiated ergosterin does not represent one chemically defined substance, but a variable mixture, consisting partly of pure ergosterin, not yet transformed under the influence of irradiation, of the already formed vitamin D, of intermediate products, of various products of further destruction of this vitamin (already antirachitically inactive), etc. From such a mixture English researchers (Bourdillon, Webster and others) isolated a very strong antirachitic product, provisionally named "calciferol", which they managed to gradually purify and free from accompanying products inactive with respect to rickets-pyrocalciferol and "sterol X" (identical with the inactive component "vitamin D₁" of Windaus; another component of Windaus-"vitamin D₂"-turned out to be identical with calciferol), and finally to obtain it in pure crystalline form. Almost simultaneously in Germany Linsert in close cooperation with Windaus also isolated from the distillation and pyrocalciferol products of irradiated ergosterin a very strong antirachitic substance, which he called "vitamin D₂" and which proved to be identical with calciferol. Absorption spectra of calciferol, crystalline vitamin D₂ product of distillation and pyrocalciferol

300 _Д5^_{B(ШШ (a1>} as well as the absorption spectra of the crystalline vitamin D₂ obtained by Linsert and Windaus, are identical. The question of the chemical nature of vitamin D is still not finally clarified. According to the most recent data, vitamin D₂ (calciferol) is a sterol with the formula C₂₇H₄₄O, i.e. it differs from ergosterin (C₂₇H₄₂O) by two hydrogen atoms. It is assumed that under the influence of ultraviolet rays the sterin ring of ergosterin opens and the resulting compound is vitamin D. However, this hypothesis is not yet confirmed. The most important property of vitamin D is its ability to prevent and cure rickets. This property is determined by biological tests on rats. For this purpose, young rats are fed a diet that causes rickets, and then the preparation to be tested is added to the food. If the preparation contains vitamin D, rickets does not develop or, if it has already developed, it heals. The amount of vitamin D in a preparation is determined by the amount of the preparation that prevents rickets in 50% of animals (ED₅₀). The international unit of vitamin D is defined as the amount of vitamin D that prevents rickets in 1 g of rat weight per day. 1 international unit corresponds to approximately 0.025 micrograms of crystalline vitamin D₂. In addition to antirachitic action, vitamin D also has a general effect on the body, promoting calcium and phosphorus metabolism. It increases the absorption of calcium and phosphorus from the intestines, promotes their deposition in bones and teeth, and increases the excretion of calcium through the kidneys. Vitamin D is found in many foods, especially in fish liver oils, egg yolks, butter, and some vegetables. However, the amount of vitamin D in these foods is usually small. The richest source of vitamin D is irradiated ergosterin. Irradiated preparations containing vitamin D are used for the prevention and treatment of rickets, as well as for other diseases associated with impaired calcium metabolism. They are administered orally, intramuscularly, or intravenously. The dose depends on the age of the patient, the severity of the disease, and the form of the preparation. For the prevention of rickets in infants, the daily dose is usually 400-800 international units. For the treatment of rickets, the dose is higher and can reach several thousand international units per day. Irradiated preparations are usually well tolerated. However, in some cases, they can cause side effects such as nausea, vomiting, diarrhea, headache, and dizziness. In rare cases, they can lead to the development of hypercalcemia, a condition characterized by an excessive amount of calcium in the blood. Hypercalcemia can cause kidney damage, heart arrhythmias, and other serious complications. Therefore, irradiated preparations should be used only under the supervision of a physician. The discovery of vitamin D and the development of methods for its production have made a great contribution to the fight against rickets. This disease, which was once widespread and often fatal, is now rare in developed countries. However, rickets still remains a serious problem in some developing countries, where children do not receive enough vitamin D in their diet and are not exposed to enough sunlight. Further research on vitamin D is needed to better understand its role in human health and to develop new methods for the prevention and treatment of diseases associated with vitamin D deficiency.

calciferol. By another way (containing the intermediate products lumisterin and tachysterin) the antirachitic vitamin D was obtained in crystalline form. Purified crystalline calciferol represents the purest preparation of vitamin D. Pure calciferol is characterized by the following properties according to modern data: it forms colorless needle-shaped crystals with a melting point from 114.5° to 117°. Its optical activity [α]_D²⁵ = +122° and [α]_D²⁰ = +102.5° in alcohol. The absorption spectra of calciferol, vitamin D₁ (one of the crystalline products of distillation) and pyrocalciferol are given in the figure. Elementary microanalysis of calciferol gave the following results (Table 1): Table 1. Products investigated Amount of C Amount of H found calculated for C₂₇H₄₂O found calculated for C₂₄H₄₁O₂N₂ Calciferol Calciferil-dinitrobenzoate 81.5 71.1 81.7 70.8 11.1 7.9 -----11.7 7.1 The molecular weight of calciferol was found to be 571 (calculated 576). Further research indicated the presence of three double bonds in it, which shows that it is an isomer of ergosterol. Calciferol, pyrocalciferol and sterin X give color reactions (Table 2). It is essentially important that 1 mg of pure crystalline calciferol, as shown by biological testing on rats, corresponds in its antirachitic strength to 40,000 international antirachitic units (see below). Thus, of all the products isolated by various authors from irradiated ergosterol, precisely this substance—pure crystalline calciferol or identical with it vitamin D₁—possesses the greatest antirachitic strength. The latter circumstance, in connection with a number of additional studies and corresponding calculations, leads to the conclusion that under the name of calciferol or vitamin D₁, it was possible to obtain the antirachitic factor or vitamin D in a pure (at least practically) crystalline form. The obtaining of the antirachitic vitamin in pure form finally made it possible to resolve the controversial question, very important theoretically and practically, whether the toxicity of preparations of irradiated ergosterol depends on the toxicity inherent in the antirachitic vitamin itself, or on harmful impurities. Determination of the toxicity of pure crystalline calciferol (by the generally accepted method of Möny on mice, see below) showed that the minimum toxic dose for pure calciferol is 0.1 mg. Since 0.1 mg of calciferol is equivalent to approximately 4,000 international antirachitic units, the ratio index of the toxic dose (on mice) to the therapeutic dose (on rats) is approximately 4,000:1. Thus it was found that even the pure preparation of the antirachitic factor possesses a certain toxicity, so that in excessively large doses it can cause toxic phenomena, however the interval between the therapeutic (antirachitic) and toxic dose is very large. Antirachitic properties of irradiated preparations and the "calcification factor". Irradiated preparations—especially preparations of irradiated ergosterol—are rightly considered a valuable specific remedy against rickets (Wieland, Adam and many others), possessing both preventive and therapeutic action. But irradiated ergosterol is not only an antirachitic remedy, but generally a preparation with strong pharmacodynamic action: irradiated ergosterol (vitamin D) for example, similar to the hormone of the parathyroid glands (Collip's hormone), generally has a strong influence on the phosphorus-calcium metabolism of humans and animals, increasing the content of Ca and P in the blood. Therefore, the introduction into the body of significant amounts of irradiated preparations can cause toxic phenomena. Nevertheless, when experimenters (Pfannenstiel in rabbits, Kreitmair and Moll in guinea pigs) and clinicians (Degkwitz, Hess and others) described the appearance of harmful (toxic) phenomena from irradiated preparations, this circumstance caused much concern and numerous debates, and the initial enthusiasm was then replaced by distrust and skepticism (cf. Adam and others). At that time, irradiated preparations were used without biological standardization and in the preparation of preparations gross errors were allowed, as a result of which the preparations used at that time not only varied very strongly in their antirachitic strength but also possessed completely different, often very significant toxicity. Constant and very significant overdosing was allowed. As a result, toxic phenomena were often encountered: in children an increase in Ca in the blood (Hess and others), loss of appetite, decrease in weight, pallor, sometimes vomiting (Gyorgy, Adam and others), as well as irritation of the kidneys (Degkwitz and others) were observed. Recently, the same A. Hess, using larger doses of irradiated ergosterol, no longer observed in children either hypercalcemia or other toxic phenomena. Many experimental studies have been devoted to the study of this toxic effect of irradiated ergosterol on different animals. It has been established that when excessively large doses of irradiated preparations, many times exceeding therapeutic doses, are introduced into the animal body, deposits of lime are observed in the arteries, localized mainly in the media (these lesions differ both from true experimental arteriosclerosis and from ordinary human arteriosclerosis), deposits of lime in the kidneys, loss of weight, cachexia, etc. Strong overdosing in pregnant rats causes miscarriage and the appearance of various dystrophic phenomena in the fetus: hyperossification of cartilage, malformations, etc. On autopsy of children who had died accidentally and who had received large doses of irradiated ergosterin for a long time, similar changes, deposits of lime, etc. were also found in some cases (Wiskott and others); however, the interpretation of such cases requires great caution. Regarding the quantitative aspect of the matter, recent careful and long-term studies by Bills and Wiriek have been published, according to which doses 100 times exceeding the usual preventive dose for rats are harmless to them; from doses 1,000 times exceeding the norm, a harmful effect begins to be noticed, and with an overdose of 4,000 times, a quite definite toxic action is already observed. Regarding the cause of toxic phenomena when using irradiated preparations, until recently there were 2 different opinions: according to Hess's view, the above-mentioned phenomena depend on the overdose of the specific antirachitic factor itself; according to Degkwitz's opinion, the cause of these phenomena is non-specific (side) toxic decomposition products of ergosterol (or solvents). If the latter is correct, then the pure preparation of irradiated ergosterol should be free of toxic action. When vitamin D₁ was obtained in pure form (see above), it became possible to finally resolve this controversial question. It turned out that even pure vitamin D (in the form of pure calciferol) possesses a certain toxicity, but it is small, and the interval between therapeutic doses and toxic ones is very large. From incorrect technique in the preparation of irradiated preparations—from too prolonged irradiation, for example, from the use of rays with especially short wavelength, etc.—preparations can acquire particularly significant toxicity, acquire the property of causing sharp hypercalcemia and deposition of lime in organs with relatively weak antirachitic action. The therapeutic index of a good pure preparation of calciferol (and some others) corresponds to 1:4,000. The preparation "Aktinosan", according to the indication of the firm producing it, seems to be completely free of toxicity, but on the basis of verification studies (Laquer and others), it must be considered generally a weak preparation: it has weak antirachitic strength, and naturally also weak toxicity; generally the strongest preparations, for example the German Vigantol, also showed greater toxicity. For testing and evaluating irradiated preparations, determination of antirachitic strength alone is thus insufficient.

Irradiated preparations of ergosterol (the mechanism of their physiological action is still insufficiently studied) possess the ability to cause at least 2 different basic series of phenomena: first, they act anti-rachitically, and second, they cause a complex of phenomena which are attributed to a toxic factor, the so-called 'calcification factor'. Therefore, according to our modern knowledge, the evaluation of irradiated preparations is based on testing both basic factors. The anti-rachitic properties of the preparation being tested are established on the basis of its ability, under certain conditions, to prevent or cure experimental rickets in rats. The 'calcification factor' is best characterized biologically by the fact that it 1) causes deposits of lime in the body and, with appropriate dosage, produces cachexia with loss of weight, which can end in death, and 2) increases the Ca content in the blood and can delay the attacks of parathyroid tetany (on the determination of toxicity-see below). Preparation and biological standardization of irradiated preparations. The majority of modern irradiated preparations are made from ergosterol, which is most often obtained from yeast (or from ergot). Ergosterol is exposed to ultraviolet rays without access to air (in an atmosphere of nitrogen or CO₂) in an ether (or some other) solution, in a thin layer, usually with the aid of special apparatus with quartz tubes (or plates). The already activated ergosterin is transferred to an oil solution; in this form the activity of ergosterin is preserved for years. It is used either in the form of an oil solution (for oral administration it is prescribed by drops) or in the form of pills. Dry products-dried yeast, dry milk, etc.-are subjected to irradiation in special apparatus where ultraviolet rays act on a very thin layer of powder (also in an atmosphere of nitrogen or CO₂). From the foregoing follows the practical requirement that all concentrated preparations containing irradiated ergosterol must be carefully tested and standardized.

In the initial period and in experiments on animals and in application to humans, irradiated preparations were dosed mostly according to the amount (in milligrams) of ergosterin taken for irradiation. However, if one takes into account that only a part of the ergosterin is activated upon irradiation, and moreover, one that varies greatly depending on the irradiation conditions, as well as many other circumstances, it will become clear how inaccurate such dosing is. By giving a patient the same number of milligrams of irradiated ergosterin in the form of different preparations made under various conditions, one was in fact administering very different amounts of the active principle. For example, although the preparation 'Preformin', as indicated by the firm that manufactures it, contains in 1 cm³ 10 mg of irradiated ergosterin, i.e., the same amount as 'Vigantol' (the former), nevertheless the latter preparation is 25 times stronger than the former. Since, with the current state of knowledge, neither chemical nor physical (spectrophotometric) methods are sufficient for the correct and reliable standardization of such preparations, the preparations must be standardized by biological methods (based on their effect on experimental rickets in rats). Moreover, with proper organization of the work to obtain completely uniform preparations, in addition to a certain methodology for biological testing of preparations on rats, it is also necessary to have a certain standard preparation of irradiated ergosterin with constant properties, with which various preparations could be compared (due to the unequal sensitivity of animals, depending on their different origin, the conditions under which they are kept, etc.). The results of biological testing of the preparation are expressed in certain units (see below). The following methods of biological standardization of irradiated preparations are most developed. In England, for standardization, the method developed by Coward is used: young rats are first kept for 3-4 weeks on a rachitogenic diet, and when rickets develops in them, the test preparation is given to them daily for 10 days; after this period, the rats are killed, and with the knee joint of the hind paws or with the distal end of the ulnar and radial bones, a special 'line test' is performed: the joint is immersed for 1-2 minutes in a 1.5% solution of Argenti nitrici, exposed to light, and the width of the blackened line (black strip), indicating freshly deposited lime, is used to judge the cure. This test, as shown by verification studies by various authors, is not sufficiently accurate—since animals, as was indicated, are not always equally sensitive, the effect of the test preparation is compared with that of the standard preparation of irradiated ergosterin; the antirachitic strength of the test preparation is calculated based on the ratio of its found effective dose to one unit (0.1 γ) of the standard preparation. In Norway (and partly in America), the antirachitic strength of preparations is verified based on comparison with standard cod liver oil. According to the method of Poulsson and Lowenskield, rats are kept on a rachitogenic diet for 25 days, then the degree of rickets is established radiologically: the measure is the width (in millimeters) of the epiphyseal line of the proximal end of the femur. For 6 days, the rats are given the test preparation, and another radiograph is taken. The result of the treatment is compared with the result from the smallest therapeutic dose—2 mg of standard cod liver oil (in which 500 units of vitamin D are considered to be in 1 g). Schultz's method is similar to Poulsson's method: rats kept on a rachitogenic diet for 14 days receive the test preparation for 21 subsequent days. The effect is assessed radiologically based on the change in width (in millimeters) of the epiphyseal gap. One unit ('D.V.E.' = D-Vitamineinheit) is considered to be the smallest amount of substance that brings strong rickets (+ + + ^:) to normal (no rickets). The method of Scheunert and Schieblich, in contrast to the previous therapeutic methods, is based on the preventive principle, determining the amount of the test substance that completely protects rats on a rachitogenic diet from rickets. According to this method, one biological (protective) unit (1 'S.E.D.' = Schutz-Einheit für Vitamin D) is considered to be the smallest dose that, when fed daily for 14 days, completely protects young rats (at least 80% of those in the experiment) on a rachitogenic diet from rickets. The complete absence of rickets in the test rats and its presence in the control rats is established radiologically. It is advisable to set up another such series of rats for control; for comparison, they are given a standard preparation (Kreitmair). By this method, Vigantol, the most widespread preparation, is standardized. This method, according to verification (Holtz, Laquer and others) on 12,000 animals and according to data from the department of experimental medicine of the Kiev radiological institute, is the most reliable, expedient, and practical (standardization requires only 14 days instead of 3 weeks or more by other methods). Irradiated preparations of ergosterin, produced in different countries by various firms, are still standardized differently, and therefore their antirachitic strength is expressed in different units, making them extremely difficult to compare with each other. In the near future, some ordering can be expected, since since June 1931, an international unit for antirachitic preparations has been adopted at a special conference of the hygiene section of the League of Nations. The antirachitic strength of one mg of the international standard solution of irradiated ergosterin has been taken as the international unit. The standard preparation of irradiated ergosterin was prepared from yeast at the National Institute for Medical Research (in London), irradiated under certain conditions with a mercury-quartz lamp; the irradiated product is stored in an oil solution in the aforementioned institute. The unit of vitamin D, determined by antirachitic strength, corresponds to 0.0001 mg or 0.1 γ of this preparation. According to the resolution of the Medical Research Council, all irradiated preparations in England have been compared with this standard for several years. The toxicity of irradiated preparations—the 'calcification factor'—is determined by the now generally accepted method of Moll, usually on mice, since mice are the animals most sensitive in this regard, and testing on them is faster and more reliable, and not just one effect is taken into account, but the general picture of toxic action (loss of weight) and calcium deposition in organs is determined for a whole group of mice. The limit toxic dose is considered to be the smallest daily dose (per mouse) that, in half of all mice taken for the experiment (the group of mice must consist of at least 4 adult mice, each weighing more than 16 g), if given 10 times within 12 days under known conditions, leads either to death or to an average loss of weight of 2.5 g or more, and in most mice that received larger doses, calcium deposition in the kidneys should occur, which is detected upon histological examination of them. Based on the data obtained, the 'therapeutic index' of the given preparation is calculated—the ratio of the antirachitic strength of the preparation, established on rats, to the toxicity found on mice. This number serves as a scale for assessing the toxicity of preparations containing vitamin D (Holtz, Laquer, Kreitmair and Moll). For example, if in testing any antirachitic preparation, the limit dose of its antirachitic action (on rats) is 0.1 γ (or 0.0001 mg), and the smallest toxic dose is 0.3 mg, then the index of the test preparation is 1:3,000. Thus, the interval between antirachitically active doses and toxic ones is very large. The index of ergosterin preparations, irradiated under various conditions, according to Laquer's determination, ranged from 1:1,500 to 1:3,000. The index for crystalline pure antirachitic vitamin, obtained as 'calciferol', was 1:4,000. The quantitative determination of antirachitic properties of irradiated preparations has extremely important, and often decisive, significance for the study of theoretical problems and the solution of a number of practical questions. Various types of irradiated preparations. As soon as in 1924 the possibility of activating the antirachitic principle with the help of ultraviolet rays in the most diverse products was discovered, this principle was used for preparing various antirachitic preparations. Special attention was paid to giving milk—this natural and at a certain period of life the only food of infants and children—antirachitic properties. Fresh milk and dry milk (especially the common milk preserves in America) were exposed to ultraviolet rays. Various firms have put on the market preparations of irradiated dry milk under the names 'Ultractina', 'Ra-vix', etc., irradiated powder from chicken egg yolks ('Pleosoma'), irradiated meat extract ('Carnolactin'), crackers from irradiated products ('Bioxinzwieback'), etc.

Later, when in 1927 it was discovered that under the influence of ultraviolet rays ergosterol is particularly strongly activated, various firms in different countries began to produce concentrated, potent preparations of irradiated ergosterol, dissolved as a rule in oil (see below). At present in Western Europe and in America, since there the production and sale of irradiated preparations are almost exclusively in the hands of private firms, mainly patented preparations of irradiated ergosterol of the type Radiostol, Vigantol, etc. are widespread, and only in a few places (for example in Frankfurt-on-Main and other cities) are there apparatuses for irradiating milk. Since irradiated preparations are not only an excellent therapeutic agent but are also quite suitable for mass prophylaxis as well as for wide application in poultry farming and animal husbandry, it is hardly expedient to limit oneself in the USSR to the production and use of only concentrated preparations of irradiated ergosterol. Rather, attention should be directed to the various types of I.P. adapted to special needs, and while producing for some purposes preparations of pure irradiated ergosterol, at the same time to develop special preparations, for example the cheapest and most suitable for distribution in poultry farming and animal husbandry, provided however that these preparations are sufficiently potent, tested, and biologically standardized. For all these reasons in the USSR from 1928 in the department of experimental medicine of the Kiev X-ray institute under the direction of Krontovsky, Bronshteyn, Magat and some other scientific research work on irradiated preparations was begun, and then when the laboratory-biological and clinical testing of the preparations gave good results and when in the Kiev X-ray institute the corresponding apparatuses for irradiation were constructed, the production of certain types of preparations was also begun. Soon some experiments with I.P. were also begun in Kharkov in the Ukrainian institute of endocrinology and organotherapy. The Kiev department of the Ukrainian institute of endocrinology and organotherapy together with the Kiev X-ray institute is now proceeding to the wider production of irradiated preparations. a) Preparations of irradiated ergosterol. I.P. prepared from pure ergosterol and biologically standardized are the most concentrated of modern irradiated preparations. Such irradiated ergosterol (most often in the form of an oil solution, also often in the form of dragees) is dispensed in various countries by different firms under the most diverse names: Radiostol (England), Vigantol, Praformin, Aktinosan and others (Germany), L'ergorone, Sterogyl (France), Ultranol (Denmark), etc.; in America (USA) all preparations of irradiated ergosterol of different firms bear one common name 'viosterol' (Viosterol); if in anti-rachitic power this preparation surpasses standard cod liver oil 100 times, it is called Viosterol 100; 250 times - Viosterol 250 D; the preparation of irradiated ergosterol made in Kiev has been given the name 'radiosterin'. The first preparation of irradiated ergosterol (vitamin D), issued precisely biologically (resp. physiologically) standardized, is the English 'radiostol' (Radiostol). In 1 cm³ of radiostol - 10,000 units; therapeutic dose: 2 cm³ (about 30 drops) daily (three times 10 drops each); prophylactic dose - 1 cm³ (8-15 drops) a day (2-3 drops three times). An example of a good modern German preparation of irradiated ergosterol is 'vigantol' (Vigantol). In 1932 vigantol (Vi-gantol of the firms E. Merck and Bayer-Meister Lucius) began to be prepared from pure crystalline preparation of vitamin D, 1 mg of which contains 50,000 international units (see above). Vigantol is now issued 1) in the form of an oil solution (Vigantol-01), 1 cm³ of which contains 0.3 mg of crystalline vitamin D (dispensed in droppers of 5, 10 and 50 cm³), and 2) in the form of dragees (Vigantol-Dragees): 1 dragee contains 0.06 mg of crystalline vitamin D (boxes of 50 pieces and flasks of 250 pieces). It should be borne in mind that although an international anti-rachitic unit has already been adopted, still different firms continue to standardize irradiated preparations by different methods and to express their strength in various units (see above). b) Irradiated yeast. For mass, wide application as an inexpensive preparation, convenient in respect of the simplicity and speed of its manufacture and especially suitable for application in animal husbandry, irradiated yeast (A. Hess, Krontovsky, Bronshteyn, Magat and others) can serve above all. In brewer's yeast there is relatively much ergosterol (which in this case is the 'provitamin'): in 1 kg of yeast there is about 0.0018 g of 'provitamin' (according to Schubel); with appropriate technique of irradiation under other optimal conditions (as calculated by Schubel) from yeast one can obtain a preparation in which 1 g will contain about 40,000 biological anti-rachitic units. Although difficulties arise during irradiation (since one has to deal with powder instead of a solution) _? still practically satisfactory results are achieved; for example in the apparatus constructed in the Kiev X-ray institute, carefully powdered yeast, prepared in the form of a very thin even layer (in an atmosphere of CO₂) passes with a certain speed under two 'mountain sun' lamps. The activation with such irradiation is so significant that in 1 g of irradiated powder 500-1,000 biol. units according to Sheyner and Shiblih are obtained. As experimental-biological testing on rats with experimental rickets, as well as the clinical application of such a preparation of irradiated yeast ('radiotorulin'), biologically standardized by the method of Sheyner and Shiblih, on children with rickets in the Kiev institute Okhmatdet and some other institutions (Finkel'shtein, Kutsenok), on adult patients with bone (Pavlov) and skin (Bykhovsky) tbc gave good results, and not once was there observed (with daily dosage in adults of 10,000 biological units) any toxic phenomena, any significant hypercalcemia, etc. Unlike the oil solution, irradiated yeast lose part of their activity after several months. Regarding irradiated yeast it should be noted that their action on the organism is probably determined not only by their content of activated ergosterin (vitamin D) but also by the presence in yeast of a considerable amount of vitamin B and perhaps some other constituents. According to the experiments of Pfannenstiel and Scharlau on tuberculosis infection in rabbits (on a special experimentally induced form similar to human tbc) and on staphylococcal infection of the skin, irradiated ergosterol (vigantol) acted much less well than the simultaneous application of vigantol together with yeast (vitamin B). According to these authors vitamin D (activated ergosterol) and vitamin B contained in yeast form together, owing to a special synergism, a particularly favorably acting combination. Correspondingly, according to the observations of Pavlov and Bykhovsky (in Kiev), in patients with bone and skin tbc irradiated yeast had a particularly good effect, whereas irradiated ergosterin alone, judging from literary data, does not have such a good effect on tbc. Judging from the experiments of Magat (on chickens), irradiated yeast should be given special attention in poultry farming and animal husbandry. The question of 'combined' vitamin preparations and their action is still insufficiently studied. c) Irradiated milk. Numerous investigations by a number of authors have established that milk subjected to the action of ultraviolet rays can be activated to such an extent that it proves suitable for the practical fight against rickets (Adam and others). With the former technique milk after irradiation (as a result of oxidative processes; in the presence of the ozone formed) acquired a specific odor and unpleasant taste reminiscent of cod liver oil (why this process was called 'iokerinization'); at present, when irradiation is carried out in an atmosphere of CO₂ (or nitrogen), no unpleasant by-products are formed. Fresh milk with such irradiation does not change in any way either in respect of odor and taste, and probably also in other respects; with brief irradiation the vitamin composition of milk practically does not suffer (Bamberger, Degkwitz and others). For irradiation of milk several apparatuses have been proposed: Scheidt's, Scholl's, Vita-Ray-Apparat, etc. Apparatuses of the Hanau type are issued by the firm Hanau in several sizes, with a capacity from 15 to 200 liters of milk per hour. The effect of irradiation depends on the quality of the milk, on the functioning of the lamp itself, on the speed of feeding the milk, etc.

The activity of irradiated milk is periodically controlled experimentally - by biological testing in the usual manner on rats. Milk irradiated in an apparatus constructed on the principle of Scholl and built in the workshops of the Kyiv Röntgen Institute was also tested experimentally on rats and on rachitic children: both general clinical observations and special radiological examinations and blood analyses for phosphorus and calcium content showed a clear anti-rachitic effect of irradiated milk. As for the strength of such milk, the apparatus built by the Kyiv Institute, as shown by control studies, gives milk good activity: 0.5 cm³ of milk when tested by the method of Sheinert and Schiblih completely protects rats from rachitis [according to data from various authors, the effective rat dose of irradiated milk is 0.3-0.5-1.0 cm³ (cf. Adam), and not 0.03 cm³ of milk, as Scheer erroneously indicated at first (cf. Adam, Gyorgy and others)]. Milk is usually used on the same day; when boiled and pasteurized, irradiated milk does not lose its activity. On the basis of clinical observations and theoretical calculations, Gyorgy considers it necessary to have a daily dose of irradiated milk of 500 cm³ and higher (400-600 according to Adam). Irradiated milk, having certain advantages, does not have the strength of concentrated preparations, and in cases of more severe rachitis and other diseases may prove insufficient. d) Preparations of other types. In view of the fact that the content of anti-rachitic factor in ordinary cod liver oil varies greatly, recently irradiated ergosterin (vitamin D) has been added to cod liver oil in a fixed amount. Such preparations by the firms E. Merck and Bayer-Meister Lucius were released under the name "Vigantol-Lebertran". In America (USA), cod liver oil is common, to which irradiated ergosterin is added in such a way that this cod liver oil preparation exceeds the vitamin D content of ordinary (good) cod liver oil by 10-15 times (accordingly, the cod liver oil preparation is marked "10 D" or "15 D"). In addition to those mentioned, there are many other, very diverse preparations. For example, if during mass prevention of rachitis one wants to give children, following Adam's technique, milk with the addition of a certain amount of irradiated ergosterin (instead of irradiated milk), then for the ergosterin to be evenly distributed in the milk and not separate during standing along with the cream or foam, Deutsche Vitamingesellschaft (Hamburg) has put on sale the emulsion "Vitanemulsion", which mixes well with milk. Various firms also offer many other preparations, such as irradiated flour, irradiated crackers, baked goods, etc. Among the preparations offered, there are also many completely inferior preparations, advertised unscrupulously. In any case, it is necessary to ensure that only irradiated preparations prepared according to the requirements of modern methodology and technique, and whose anti-rachitic strength (as well as other properties) have been biologically tested, are used. Application of irradiated preparations in medicine and in animal husbandry. In medicine, irradiated preparations, especially preparations of irradiated ergosterin, have now gained universal recognition primarily as the best preventive and therapeutic agents against rachitis (Wieland, Gyorgy, Shafer-stein and many others). With the help of irradiated preparations, it is possible to carry out so-called "open", explicit prevention, when irradiated ergosterin is openly prescribed to a child as a medicine, or "silent", hidden prevention (laute oder stumme Prophylaxe in Adam's expression), which works imperceptibly, when the child daily (at home or in a special institution) drinks the usual amount of milk (irradiated or with the addition of irradiated ergosterin to ordinary milk). In the USSR, the mass application of irradiated preparations can follow the path of children's preventive institutions, which can link a more or less complete coverage of the population with an individual assessment of the indications for the use of irradiated preparations. Other possibilities for the application of I.P. are also interesting. As experiments and clinical observations have shown (Mc Collum, Hess, Guggisberger and others), preparations of irradiated ergosterin given to pregnant women can affect the fetus and born children, protecting them from rachitis. Milk of lactating women (and animals) receiving irradiated ergosterin also acquires some anti-rachitic strength. Practically this is of little importance, since irradiation of the milk itself gives a more significant anti-rachitic effect (Gyorgy and others). Some authors prescribe irradiated preparations to pregnant women (and nursing mothers), having in mind the benefit to the mother herself (since in pregnant women the calcium-phosphorus metabolism needs support - some authors even speak of "physiological osteomalacia of pregnancy"), as well as the benefit to the child (greater resistance to rachitis). In any case, if irradiated preparations are prescribed to pregnant women with impaired calcium-phosphorus metabolism, their use should be carried out with appropriate control, very cautiously, since, as experiments on animals show, an overdose can have a harmful effect on offspring. Along with rachitis, irradiated preparations are considered one of the best means for tetany (both for tetany of children associated with rachitis and for some other forms of tetany in adults), for late rachitis and for osteomalacia (Hess, Gyorgy, Adam and many others). In the literature there are also indications of the successful use of irradiated preparations in diseases having the character of osteoporosis, for improving the healing of bones after fractures, in the fight with pseudoarthrosis formations under various conditions, etc. There are data on the beneficial effect of irradiated preparations on dental anomalies, on predisposition to carious processes, etc. (Mellanby, Gyorgy and others). A whole series of theoretical and clinical data indicates that irradiated preparations can usefully be applied in certain cases in certain forms of tuberculosis: if the literature data regarding pulmonary tuberculosis are contradictory, then in relation to bone and skin tuberculosis in most cases a favorable, and in some cases directly striking effect of irradiated preparations was noted (Pavlov, Bykhovsky and others). At present, during the period of enthusiasm for irradiated preparations, they are often prescribed for the most diverse diseases, even in cases where there is no basis for their use. Irradiated preparations, besides medicine, find wide application in agriculture; with the help of irradiated preparations (as is seen for example from the experience of the USA) one can successfully fight rachitis in farm animals-in piglets, chickens, etc.- These same preparations can be successfully used against osteomalacia, osteoporosis ("bone wasting"), which often affects pregnant cows giving milk, the most valuable pedigree pigs, etc. The field of application of irradiated preparations is not limited to the fight against diseases, they can also increase and improve agricultural production in other ways. In particular, judging by experiments conducted on many farms in the USA and recently carried out by Magat in the state farm "Feofania" near Kyiv, irradiated preparations can bring significant benefit to poultry farming not only by fighting rachitis, but also by improving the development of chickens, improving egg production, etc.