October, 1924
I N D U S T R I A L A N D ENGINEERING CHEMISTRY
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The Chemistry and Function of the Pituitary Gland‘ By J. J. Abel JOHNS HOPKINS UNIVERSITY, BALTIMORE, MD.
T
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HE whole subject of The posterior lobe of the pituitary gland, or hgpophysis cerebri. F~~~~~~~~ op THE G~~~~ i n t e r n a l secretion contains only one specific extraordinarily pofcnf principle or hormone, which is endowed with various physiological properties, cardioIt is well known that we go e s b a c k many vascular, plain muscle and melanophore stimulating, diuretichave diseases that are years. We have in our antidiuretic, respiratory, etc., and not three or more separate subnarily associated with an bodies a number of organs stances each of which exhibits one or more of these physiological alteration of function in some of which, such as the actions. This single substance with its manifold physiological propt h e s e glands. We have p a r a t h y r o i d glands, are erfies can be prepared in the form of a tartrate, which, at its best, is giantism, which surely is about the size of a hemp1000 to 1250 times more powerful than histamine, which, untilnow, ConnectedinsomewaY with seed, while others, as the has been the most powerful known stimulant for plain muscle f i s this gland itself O r with the p a n c r e a s , w e i g h three Ounces O r more-all of sue such as the guinea pig’s uterus. immediate neighborhood of the floor of the third venwhich are of vital importricle of the brain. Then tance to the body. If one of these organs is removed or destroyed by disease, life cannot we have the condition known as acromegaly, where the hands go on, or, a t best, the individual is crippled to a varying ex- and feet grow to large size and the jaw and head get larger, and the person thus afflicted has to buy a larger hat and shoes. tent in the exercise of his functions. An important structure of this class is the pituitary gland, Or we may have giantism and acromegaly combined. We lying at the base of the brain, safely housed in a small pocket have, too, a condition, more frequently seen in youth than in of the sphenoid bone. This small pocket is called the Turkish the adult stage, known to physicians as dystrophia adipososaddle. The gland is well protected; it projects downward genitalis, which is not infrequently met with wnd which is from the brain. The name “pituitary” is derived from the characterized by an overgrowth of fat sometimes quite enorLatin word pituita, used by Cicero and other writers of the mous, by underdevelopment of the sexual organs, by nervous classical period, and it means mucus, slime, or phlegm; and mental manifestations, and various other mil-conditions. the older physicians for many centuries, from Galen down to This dystrophy, which varies much in different individuals, is the time of Vesalius or later, thought that this little structure ascribed to an underfunction of the gland. Medical men are a t the base of the brain served to carry off from the ventricles often quite in the dark whether to refer the disease in question of the brain excess of mucus and pass it into the upper pharynx to one of the divisions of the gland itself or to the parts of the from whence it could either be ejected or passed into the stom- brain in close proximity to the gland. ach to be digested. These predecessors of ours also called it I n the lower animals, as in the amphibia, the evidence in the “colatorium” or strainer. The term “sentina,” which was regard to the function of the gland is perfectly clear. We used to designate the bilge water in ships, was also employed can remove the anterior lobe in the tadpole and get a cessain reference to this organ with the belief that its function con- tion of the growth. If we feed this stunted tadpole an exsisted in removing excrementitious matter from the brain. tract of the anterior gland we can start his growth again. The more recent term “hypophysis” seems to describe it This is known as replacement therapy. It is not always an better since it connotes no theory of secretion, as the word easy matter to prove that an organ of external secretion “pituitary” does, but merely refers to the location of the gland really gives up its product, the so-called hormone, to the blood. below the brain. Once in the blood, the hormone can play a role in the body and can influence distant organs in various ways, in accordance STRUCTURE OF THE GLAND with its physiological properties. If, therefore, we can actually find the hormone in the blood, as is possible in the case The organ under consideration has two main parts, the ante- of the suprarenal glands, we have made a great step forward. rior lobe and the posterior lobe. The human gland is a small Sometimes it is impossible to trace it in the blood, for anatomistructure about like the tip of the little finger, and weighs cal reasons or because the quantity circulating in the blood a t only 0.6 gram. The bovine hypophysis weighs about 2.4 any time is too minute for detection by our present methods. grams. The anterior lobe is much the larger division, con- It must remain for the future to elaborate more delicate tests stituting about five-sixths of the weight of the gland. I n the for the detection of hormones than are now known to us. case of cattle the posterior lobe weighs 0.4 gram, while the When we cannot show that a given hormone passes into the anterior lobe weighs about 2 grams. The anterior lobe has circulation, we must prove that the gland in question is an the cellular structure of a glandular organ. The posterior organ of internal secretion by means of the replacement lobe is made up largely of so-called “neuroglia” tissue. The therapy. If we take out this little gland and something anterior lobe is richly supplied with blood vessels, the posterior happens-as, for example, cessation of growth-and if we lobe much less so. There is a pars intermedia, which con- then feed an extract of the gland and growth goes on as well as sists of a very narrow strip of tissue, from which chains of cells before, we have a pretty good proof that the little organ pass into the posterior lobe; and it is thought by some that we took out plays a role in the body. The replacement investigators that these cells account for the physiological therapy demonstrates that the gland somehow gives off to the properties of the posterior lobe. blood a chemical principle which is necessary for the functioning of the animal, It is seen, therefore, that in the case of the 1 Presented under t h e title “The Present State of Our Knowledge in amphibia, we have a very clear proof that the anteriorlobeRegard t o t h e Chemistry and Functions of the Pituitary Gland,” before t h e since it can be taken Out without injuring the brain and Division of Chemistry of Medicinal Products a t the 67th Meetina of the the missing hormone can be replaced by feeding the anterior American Chemical Society, Washington, D. C., April 21 t o 26, 1924.
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INDUSTRIAL A N D ENGINEERING CHEMISTRY
lobe of another animal-has to do with the control of growth. I n the amphibia the posterior lobe can also be shown to have a function, because if we take it out the pigmentation of the animal is disturbed. Instead of a frog that responds to light and shade we get a frog that remains pale. If now we feed to such a frog the extract of the posterior lobe of the ox, for example, it again regains control of its color. When it comes to the higher animals, as the dog, the cat, and others, we meet with more difficulty in demonstrating sharply the functions of each division of the pituitary. We meet here with the same difficulties that are encountered by the medical man when he attempts to correlate what he calls the pituitary states with the pathological lesions of the gland. Present opinion inclines to the view that in the higher animals and man we cannot always dissociate sharply the functions of the gland from those of the floor of the third ventricle. Here a t the base of the brain are very important centers that appear to influence fat deposition, carbohydrate metabolism, water metabolism, or, in other words, the passage of water through and away from the body, blood pressure, and regulation of body temperature. But, while difficulties are encountered in the case of the higher animals and man in differentiating the relative roles of the gland and the neighboring regions at the base of the brain, most students of this subject nevertheless incline to the view that the anterior lobe of the gland does send a chemical principle or principles into the blood even in man, which influence the growth of our body structures, while in the case of the posterior lobe, which, as we have seen, plays so definite a role in the lower animals, the evidence in regard to the transmission of a hormone into the blood is less well defined. Nevertheless, evidence is accumulating that the posterior lobe in the higher animals and in man also exerts a function, if only in emergencies, as appears to be the case with the medullary principle of the suprarenal glands. CHEMICAL PRIXCIPLE Now what are the chemical principles that h&e to be considered in the case of the gland under discussion? The hormone of the anterior lobe has not been isolated as a chemical individual. Robertson has given the name “tethelin” to the principle that is supposedly present in the anterior lobe, and he has separated from this lobe a mixture of substances which he believes to contain tethelin. This mixture, it is stated, produces striking effects on the metabolism and growth of experimental animals, but it must be borne in mind that thus far no chemical individual, whether it be given the name of tethelin or any other, has yet been isolated from this lobe. P. E. Smith, of the University of California, finds that Robertson’s alcohol-soluble tethelin does not favor the growth of tadpoles whose anterior lobe has been removed. Smith could only get such tadpoles to grow after feeding them the residue from which the tetheli9 apparently had been removed by Robertson’s methods. We cannot, therefore, make definite or conclusive statements in regard to the chemical principles found in the anterior lobe; in a word, we cannot make any statements that will remain unchallenged in regard to the chemical principles of the anterior lobe. The much smaller posterior lobe has been worked a t very intensively during the past twelve years or more, and we are now getting much definite information in respect to its active principle. Physicians employ a simple extract which is furnished in the form of a sparkling clear solution in small, sealed ampules that contain 0.5 or 1 cc. This simple watery extract, which contains all the chemical substances present in the gland except proteids and fats, is of very great service in medicine, and especially in obstetrics where it rivals and often excels that old-time remedy, ergot. Unfortunately,
Vol. 16, No. 10
this highly active pituitary extract is often wrongfully used and has caused the death of a number of women when used by inexperienced physicians or midwives. There are many other medical and surgical uses of this very potent drug. It has been found in recent years that it is the only known substance that will immediately and quickly control the excessive flow of urine in that distressing condition known as diabetes insipidus, a condition which must be differentiated completely from the disease known as diabetes mellitus. Kow what chemical principles are we dealing with here? Is there more than one hormone present in the posterior lobe? Until very recently the belief that there are three or four, or even more, principles has been firmly held by several chemists. Ten years ago the skilled chemists of a great German firm announced that there are four crystalline principles present. These chemists claimed to have isolated these principles in the form of sulfates. Later, not on the basis of chemical isolation, but on evidence supposedly given by solvents and physiological reactions, Dale and Dudley assumed that there were three characteristic principles, and possibly a fourth. It may be definitely asserted that the four crystalline principles of the German chemists and the three or more separate individuals of Dale and Dudley are nonexistent. With the assistance of Dr. Rouiller, the writer has shown that the active principle of the posterior lobe is of enormous potency, and that any number of crystalline residues may be prepared from the pituitary gland, which contain more or less, and often a great deal, of the active principle adsorbed upon their surfaces or included within them, and hence a certain amount of activity, which ten years ago would have been thought quite remarkable, may be shown by such crystalline residues. These investigators claim that, in place of three specific hormones, the posterior lobe of the pituitary gland contains but a single hormone endowed with a number of distinct physiological properties. A chart has been prepared which gives an outline of the chemical methods which with his collaborators, Dr. Rouiller and Dr. Geiling, the writer has been following for several years. They have found, for example, that the extract in the little ampules previously mentioned contains three substances, each of which is characterized by marked physiological properties. Let us call these three substances A , B, and C. B and C are of minor importance, physiologically speaking, although from a scientific point of view considerable importance attaches to them. C is histamine. Now this histamine is a very extraordinary base derived from histidine, one of the useful but quite innocuous amino acids produced in the digestive tract and found in our blood and tissues. Bacteria can change this amino acid, by the simple expedient of removing from it a molecule of carbon dioxide, into this base histamine, the constituent C of pituitary extracts. The action of this histamine, a substance which has played a great role in experimental medicine for many years, is in many ways typical of that seen in anaphylactic shock and in similar states of depression. This histamine, or C constituent, is split off in small amounts by mild treatment with acids from a complex precursor which is present in the posterior lobe tissue. It is present in very small amount in all commercial preparations, and when tested by itself causes a drop in the blood pressure, stimulates the uterus and other structures containing plain muscle, such as the smaller bronchioles in our lungs. Fortunately, it is present in small amounts only in gland extracts, and this holds also for the second constituent, B, which does not pass from a dry carbonate powder into dry chloroform as does histamine. This B substance also stimulates the bronchi to contraction and appears to have the same general physiological properties as C. B and C, therefore, may be set aside a t the present as of no interest to us as they are present in small amount only and since their
I N D U S T R I A L A N D ENGINEERING CHEMISTRY
October, 1924
physiological action is entirely counterbalanced by the more important, more powerful substance, A . This substance A constitutes the truly important principle of the posterior lobe and it will be referred to as the hormone of this lobe. I n using this term we take it for granted that it does pass into the blood, although we cannot a t the moment give a conclusive proof for this statement in the case of the higher animals. SEPAR.4TION O F THE PRINCIPLE
Horn does one separate a chemical principle of this sort from the many other substances contained in an extract of the posterior lobe? At the slaughterhouse the posterior lobe is separated from the anterior lobe of the chilled glands by trained operators. A great many of these small lobes have to be shelled out much as a small acorn might be removed from its woody cup, before enough material to work with is secured. The freed posterior lobe is then finely ground to a very fine paste and each 100 grams of paste is thoroughly mixed with 0.35 per cent hydrochloric acid containing 4 grams of mercuric chloride. There can be no decay or putrefaction as no bacteria or spores can develop in the presence of an excess of corrosivt: sublimate. When the material reaches the laboratory finely powdered corrosive sublimate is added in excess. The flask is then fastened in a shaking machine and the material is kept in agitation for a couple of hours. At the end of this time it is found that the protein material has coagulated and that it quickly settles to the bottom of the flask. We have now at the bottom of the flask what we may call the mercuric chlorideprotein cake with a clear, supernatant fluid. This top fluid contains all of the B and C depressor substances as well as almost all of the soluble crystalloid material contained in the posterior lobe, while the A substance is adsorbed by the surfaces of the mercuric chloride-protein precipitate. At one stroke, 1herefpre, we have almost completely separated from our active A principle, or hormone, the other constituents of the posterior lobe. The next step is to separate the mercuric chloride cake by means of pressure filtration, disintegrate it, and wash with cold, sal urated corrosive sublimate solution. This washing process is repeated and the cake is then disintegrated, almost neutralized to litmus paper with sodium hydroxide, treated with hydrogen sulfide in excess, then with a certain amount of a saturated sodium chloride solution (in order to flocculate the precipitiite of mercuric sulfide), and filtered. The filtrate is aerated to remove the excess of hydrogen sulfide, and is then completdy precipitated with phosphotungstic acid. The phosphotungstate precipitate is then decomposed according to a method previously describedG2 The next step consists in preparing a tannate which, after washing, is converted into a tartrate. The tartrate, after appropriate treatment, is subjected to a purification process by means of picrolonic acid. After this treatment the tartrate is carried through a number of stages of purification by the appropriate and consecutive use of various organic solvents, such as pyridine, absolute ethyl alcohol, n-butyl alcohol, and precipitating agents in which the tartrate is insoluble, such as ether and acetone. By such processes the tartrate is finally obtained in the form of a dry, white precipitate which is extremely soluble in water and also in 93 to 94 per cent alcohol. The final product then is a highly active tartrate. This has not yet been obtained in crystalline form, but it is hoped that this mill soon be accomplished. PHYSIOLOGICAL PROPERTIES S o w what are the physiological properties of this tartrate? In the first place, it is an extraordinarily powerful stimulant 2
J Pharmacol , 22. 290 (1923).
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for all smooth muscle tissues, as is well illustrated by its power to induce the virgin guinea pig’s uterus to contract. Here we must compare its action with that of the well-known amine, &imidoazolylethglamine, more generally known as histamine (the C constituent of pituitary extracts), which up to the present time has been the most powerful oxytocic or uteruscontracting agent known to medical science. The best and final tartrates are found to be from 1000 to 1250 times more powerful in their action than the acid phosphate of histamine. The tartrate has many other physiological properties. It acts upon the entire cardio-vascular apparatus, constricting the arterioles and capillaries and causing a significant and prolonged rise of the blood pressure. It acts in a remarkable way upon the respiration when the dose is more than a mere minimum, causing the periodic respiration known as CheyneStokes resbiration. It acts upon the kidneys in two ways. I n rabbits, for example, which have been fed for a week on cabbages or other succulent vegetables, it causes after each injection of a minute amount only a sharp but temporary flow of urine. Under other conditions it can cause a cessation or diminution of the urinary flow. This latter antidiuretic action is seen at its best in human beings in the disease known as diabetes i n s i p i d u s . Numerous trials in hospitals have shown that when our tartrate is administered subcutaneously to patients suffering from this form of diabetes the relief is secured as promptly as when a well-prepared aqueous extract of the posterior lobe is administered.3 This tartrate exhibits every known specific physiological property of a good extract of the posterior lobe; for example, it even darkens the skin of tadpoles that have been changed into pale tadpoles by removal of the posterior lobe of the hypophysis. There is further proof of a chemical nature that all the physiological properties described belong to one substance only. In other words, the tartrate, while still perhaps contaminated with some physiologically inactive material, consist3, not, of several active substances, but of only one physiologically active substance, or hormone. The chemical properlies of the tartrate cannot be described here in the proper detail with the proper qualifying statements. The method of isolation rests largely on adsorption methods, and it is interesting to observe that the final product can no longer be precipitated by such agents as mercuric chloride, picric acid, and tannic acid (in the presence of sodium chloride), and only imperfectly by phosphotungstic acid, while all these agents precipitate it in a practically quantitative manner from aqueous extracts of the posterior lobe. So, too, the behavior of the isolated substance towards solvents differs greatly from that observed when it isstill contaminated with other constituents of the gland. Naturally there are many other points relating to the physiological action, the clinical uses and chemical properties of this extraordinary substance which space does not permit considering. 3
J Pharmacol., 22, 317 (1923).
B u s t of P a s t e u r Dedicated O n t h e afternoon of Tuesday, September 16, ceremonies were held a t the American Institute 01 Eaking, Chicago, Ill., in connection with t h e dedication of a portrait bust in bronze of Louis Pasteur. H . E. Earnard, director OF t h e institute, made t h e opening address. The presentation 02 behalf of t h e donors of the bust was ma5e by Max Hexius, president of t h e Wahl-Henius Institute, and Raymond K. Stritzinger, president of t h e American Baker’s Association, made the acceptarce speech. S. P. L. Sorensen, director of t h e Carlsberg Laboratories, Copenhagen, Denmark, delivered an address, taking as his subject, “Louis Pasteur.” T h e Danish and French Consuls located in Chicago also spoke and during t h e ceremonies t h e national anthems for Denmark, France, and t h e United States were played. A lunch followed a t which members of the institute and specially invited guests were present.
