George Whipple (1954) - The Dynamic Equilibrium of Body Proteins Hemoglobin, Plasma Proteins, Organ and Tissue Proteins

Distinguished guests, ladies and gentlemen. I cannot tell you how much I regret my inability to present this communication in your own German, but I am unable to do that, so you must bear with me. These remarks and experiments were done by animals, they relate to animals. But they also relate to the human diseases of anaemia. The problems of transfusions and parenteral nutrition. So they're not purely abstract in the sense that they relate to physiology alone. The dynamic equilibrium of body proteins covers this area and includes the important proteins, haemoglobin, plasma proteins, tissue proteins and reserves. The main theme of this paper then relate to all these various body proteins. And experiments show that plasma protein can contribute to the store of these other proteins when need arises. That is, plasma protein can meet all the needs of the body for the proteins, whether haemoglobin, reserve proteins, plasma proteins or others. Haemoglobin, likewise, can contribute effectively to the protein pool of the body. Not only when it is released from destroyed or obsolescent red cells. Reserve stores of protein are found in many organs and tissues, for example the liver, where it is most labile and large concentrate in striated muscle, for example, where the total mass is considerable. This reserve can be used for maintenance of these tissues if emergencies arise, for the construction of new red cells and plasma proteins. Body protein reserves can be depleted by demands for new haemoglobin and plasma protein. We have spoken of this reaction as the reading of body protein. That means, by continued anaemia and hypoproteinemia, we see large production of new haemoglobin and plasma proteins with rapid loss of weight, and this may go on to a fatal termination. That is, tissues are so robbed of their protein that they cease to function. During all these exchanges of proteins within the body, there is no evidence for protein cleavage to the amino acid level and subsequent reconstruction. Rather the evidence that these proteins are modified within the cells to meet the needs of the moment. That proteins can pass in and out of cells without breakdown must be accepted. And much evidence is at hand to support this statement. Convincing evidence is found in experiments using radiocarbon 14 to label the amino acid glycine. The glycine is fair and labels the plasma proteins of the amino, which can then be removed from the donor dog and given as plasma intravenously to the test dog. The pathways of the plasma protein can be studied in detail, as they relate to other body proteins under various experimental conditions. Experiments from this laboratory indicate that the normal dog, given a protein free diet, practically protein free, of fat, carbohydrates, minerals and vitamins can be maintained in nitrogen and even weight equilibrium in perfect health by means of adequate plasma, given intravenously or intraperitoneally over periods of one to three months. The dog receives no nitrogen, or practically none, except plasma proteins, which obviously supply the body's requirements for proteins over this period. No surplus of any one of plasma proteins appears at any time during these long experiments, indicating that all types of proteins in the plasma, under these conditions, can be used by the body for its maintenance and the requirements that maintenance demands. It is accepted that most of the plasma proteins, that is albumin, fibrinogen, prothrombin and other globulins derived from the liver. The liver, therefore, is the master organ in plasma protein production and metabolism. The technical details are given in the listed publications and are important. Some workers, who have repeated certain experiments of ours, have had difficulty, because they used oxalate to prevent coagulation of the donor blood. Heparin must be used or some other harmless product, the use of oxalate intoxicates the test dog and terminates the experiment. After these protein stores have been depleted by these large doses of plasma, given intravenously, about 200 or 300 cc of plasma intravenously each day during the observation. This means that hyperproteinemia develops and may elevate or come up to a level of nine or ten grams, almost double normal grams percent. This will be associated with some proteinuria. There is a threshold for the normal dog between nine and ten percent hyperproteinemia. Then the plasma escapes through the kidney, that does no harm, except for the loss of that amount of plasma protein. When the plasma injections cease, the proteinuria clears rapidly and there is no abnormality of the kidney resulting. First slide, please. This slide shows experiments, the longest, 92 days, first one, the shortest, 15 days. The nitrogen in the plasma given by vein, you see, is considerable. The nitrogen in the diet, the next column you see, is just trifling, except in the third experiment. Nitrogen in the urine shows a positive balance. You can see there's a very slight amount of weight loss, probably fat. These experiments are uniform, they're of sufficient duration to indicate that no hypothetical reserve is adequate to explain the situation, as was claimed by some when the experiments lasted but two weeks. This table, that you have just seen, summarises the results in seven long experiments, in which plasma injections continued over 15 to 92 days. There is very little protein in the basal diet, most of that being in the accessories, including the vitamins. And whether it's in the form of protein, although some of it is, but it's very small in amount. The dog received parenterally two to four grams of nitrogen daily as plasma protein, that is 200 to 300 cc. In whole normal dog plasma it may be given intraperitoneally or intravenously, usually intravenously. There is a positive nitrogen balance, weight loss is minimal. The length of experiments, the clinical condition, in spite of all this, is excellent, they are normal dogs, in action, in appearance and in every respect. It came as a surprise to us, when we tested globin and haemoglobin, to see what effect it would have on this picture. Instead of using plasma protein we used globin or haemoglobin obtained from late red cell of dog, given parenterally, intravenously or intraperitoneally. And we're able to show that these dogs can take this material and, as you would expect, can make new haemoglobin. They can also make large amounts of plasma protein. In fact, in some experiments there was a positive nitrogen balance. It's not as easy to demonstrate as with plasma protein, which is so uniform. I suppose there's a little toxicity at times in this globin material. The globin is not used as completely as is plasma protein, but it is used to form new plasma protein and may even maintain the dog in nitrogen balance, as I see. It may be mentioned that a normal ten kilo dog has a mass of haemoglobin of about 180 grams and, given the life cycle of the red cell at 120 days, the dog therefore uses up about a gram and a half a day, and the material is used in the ways of the body. The iron, of course, as is well known, is conserved as gold in some communities. The pigment radical is discarded. It's down to the body to find and to make that pigment easier than it can reclaim it. The tabulated experiment, experiments I cannot show you at this time, but they're required in the literature. But the experiment I think is adequate to show that dog haemoglobin, given parenterally, will be conserved and included in the body protein pool. The utilisation of globin and plasma protein parenterally are compared in the same dogs, and in those experiments it is obvious that the plasma is used a little more completely. But that the globin is used adequately to supply protein needs and the production of new vital protein is very obvious. The next slide shows some of the facts concerning the haemoglobin and the plasma protein in circulation and in body stores. To determine the amount in body stores, they must be exhausted by continuing bleeding or plasmapheresis. At any rate, the circulating mass of haemoglobin in this ten kilo dog, with a volume of approximately 900 cc, would be 180 grams, the plasma protein 30. I'd emphasise that these dogs that we use are in perfect health, their haemoglobin level is somewhat above that of the normal animal, as they are found. The maximal regeneration for haemoglobin is 50 to 70 grams a week for a dog of this sort. And approximately the same amount for plasma protein. In fact, that may be pushed up somewhat in plasma protein. The reserve stores, of course, vary tremendously on various factors, but run from 50 to 200 for haemoglobin and 30 to 100 or more for plasma protein. I would emphasise, as I've stated, that these dogs are in perfect condition. All the dogs that are used in these experiments, they're born and raised in the kennels under our observation. They're accustomed, trained in laboratory procedures. The maximal regenerating capacity is high, but varies somewhat with individual dogs, as you would expect. Just as their muscular capacity and activity varies. Their reserve stores also vary with the individual dog, with its previous dietary and exercise habits. These reserve stores obviously contribute to the production of new haemoglobins and plasma protein when a standard dog is put on a low protein diet and bled regularly. The food protein is made each week and the removed protein also. Haemoglobin and plasma protein is recorded. This is a sample experiment. This continues, you see, for nine weeks, during this continuous bleeding you'll notice that there's a loss from 23.8 kilograms to 19.5. The protein intake, you see, is very small indeed, the total for all this nine weeks being 133 grams. The protein output, due to the bleeding, for haemoglobin, as you see amounts to 335 grams during that time we experienced. The level falls from 14 to ten, sometimes the losses would be lower than that, but in this experiment to that level. Plasma protein levels fell somewhat, but not to an extreme level. The output is given as a total of 95 grams for the nine weeks. The intake, you see, very small, the urinary nitrogen figures are given. Now, with an intake of protein of 133 and a production of 440, that protein must come from somewhere, or the same amount of food obviously. The reserve stores are drawn on. And those reserve stores, presumably in an experiment like this, are reduced to zero. And the function of the animal and its organs is reduced and impaired. The animal may come to a fatal termination by this exhaustion of its proteins. Produced within the body to form new haemoglobin and new plasma protein, and that was removed. So this animal, to repeat, received 135 grams of diet protein and produced new haemoglobin, 335 grams, plus new plasma protein, 95 grams. Obviously, this surplus came from the reserve proteins of the body, but it appeared and was removed as haemoglobin and plasma protein. Now that the important, one of the important things, there was no excess protein breakdown obvious within the body. The nitrogen elimination dropped each week, until it was way below what we would call a normal fasting elimination. If the body protein was broken down into amino acids and reassembled, almost certainly there would be some excess, which does not appear. This indicates the conservation of this important material, and there's a definite decrease in urinary nitrogen. Presumably, blood proteins are formed in the normal way, in the normal organs, and there must be some modification, presumably of the reserve protein in whatever form it is stored, in reserve with production of the specific plasma and haemoglobin proteins. Without any extensive cleavage, the amino acid level and subsequent reconstruction. In this emergence, the demand for new blood proteins is predominant. Three times as much haemoglobin is produced in this experiment compared with plasma proteins. Further information about the dynamic equilibrium of body proteins is obtained by experiments using radiocarbon labelled lysine, the plasma label given to this test dog. Donor dogs are fed lysine, radioactive, which labels the plasma protein of that dog, and then it is removed by bleeding and given to these test dogs. Standardised animals in perfect health. This labelled plasma protein disappears in the circulating plasma rather slowly, about one third in six to seven days, and appears in the body organs and tissues in corresponding amounts during this time. There is very little loss of C14 as carbon dioxide, that is C14O2 in the respired air, and less in the urine. The turnover of globulin is more rapid than for the albumin, that is an interesting fact which applies to many questions. Now, in this slide, the height of the common indicates the concentration of the labelled material in the proteins of the liver, adrenal, gastrointestinal tract and other organs, as you have seen, per gram weight of organ. And you see, by this measure, the liver is highest as you would expect. But some of the adrenal, some of the ductus glands, adrenal and thyroids are also high. The gastrointestinal tract in the mid-zone, still lower the pancreas and the salivary glands, and lowest the muscle and skin subcutaneous tissue. But the total amount of radioactive protein in the muscle and the skin is highest, because its max is so much greater. This picture shows the relative concentration of carbon, of C14 within the organs. And, as stated, there it varies, the highest concentration noted in the liver and the adrenal and thyroid, as stated. The total amounts in these various tissues varies from 21 to 28% in the muscles, that is a great mass of tissue, you see, although the concentration per gram is less. And the skin and subcutaneous tissue 12 to 13%, the liver 7 to 8%, and the other organs of course are very much lower, because of their size. When C14 lysine plasma protein is fed instead of given parenterally, the picture is very different indeed from that described in this last experiment. The speed of reaction, when fed lysine is given, is quite rapid. The labelled proteins appear in the plasma within seven or eight hours and in the organs within 48 hours in considerable amounts. Large amounts of C14O2 appear in respired air, as much as 16 to 30% of the fed C14 appears in this way within 48 hours. This is in contrast to 2.5%, that is one tenth as much in 48 hours, in the experiments with parenteral labelled plasma protein, it's a totally different picture. When C14 lysine in an amino acid digest is fed to the dog, the picture is precisely the same as with the labelled plasma protein, when fed. In other words, the plasma protein is broken down and so often handled like amino acid mixtures. These experiments listed above are related to normal dogs. When abnormalities appear, the dogs were eliminated from the series. For example, they might have distemper, parasitic infection, acute or chronic nephritis. But we have studied certain abnormalities, which relate to the organs, particularly abnormalities of the liver. The Eck fistula, the bowel fistula have been studied in considerable detail. It may be stated that the Eck fistula liver, in which the portal blood bypasses the liver, does at times show lower production of plasma proteins in depleted dogs. Ascites may be produced experimentally by partial obstruction of the vena cava above the diagram. Then we see a rapid exchange of acidic proteins with a circulating plasma protein. If labelled plasma protein is introduced into the peritoneal cavity of the acidic dog, the labelled proteins appear promptly in the blood plasma. And conversely, if the labelled plasma is given intravenously to the acidic dog, this labelled protein appears promptly in the acidic fluid. A circulation and interchange of protein between the blood and acidic fluid. Now, inflammation is a very important subject, particularly to clinicians and pathologists, and it has been included in our series of experiments. Chance infections, for example, due to bacteria, to viruses, or a sterile abscess, due to an error like turpentine. And the metritis, for example, is not infrequent in some animals, and it may last in a dog with continuing fever and leukocytosis for many weeks. During such time there is a definite lack of production of new haemoglobin, can be demonstrated with ease. Removal of the infected uterus brings the dog promptly back to a normal state of haemoglobin production. And production of new plasma proteins if they are depleted. It is readily demonstrated that synthesis of new haemoglobin is impaired by the inflammation, we usually think of inflammation as destroying proteins, it does, perhaps more importantly, lessen the synthetic ability of the body to build. Radiocarbon labelled plasma proteins enable us to study this condition in considerable detail, and I'll show you the last slide in a moment. Sterile abscesses are used, since the abscess reaction can be terminated promptly at any time by incision and drainage. The appearance of C14 in the haemoglobin with sterile abscesses shows definite delay and impair the synthesis of haemoglobin. The plasma proteins, the turnover of albumin is greatly accelerated in this type of sterile inflammation and abscess. And these curves in this last slide, the upper one, the first top curve shows two normal controls and that shows what a steady down slant appears in the dogs after the plasma protein is labelled. The next curve in the top slide below the control, that dog had one abscess and, you see it, it is different from the control, and as the number of abscesses increase, the turnover of plasma albumin during the week or ten days, you can see, is much more rapid. There's loss of this protein, and it goes somewhere, some of it to the inflamed area. In contrast, the globulins, in the graph below, the control animals, you see, are mixed up with the upper part of the animals that show the abscesses. In other words, there's only a slight change in the globulin turnover, so far as these experiments show. With more severe type of inflammation, or perhaps different type of organisms, the picture might be different, but this is a sterile abscess, or more, affecting the labelled plasma proteins, albumin and globulin. So we can conclude that the globulins show little change during sterile inflammation, excepting fibrinogen. Everybody knows, I think, that fibrinogen has a good deal to do with areas of inflammation. And the fibrinogen shows a spectacular change when followed in this way. The labelled fibrinogen disappears and is used up almost completely within two days, an extraordinarily speed of reaction. The pus obtained in these abscesses shows a good deal of C14, both in the cell debris, that is the wandering cells fallen off the nucleus, a mono nucleus, and the fluid, presumably some plasma, some cell debris, some cell disintegration. As the inflammation continues, the more active organs, that is the liver, will lose much of its C14 as stored in its cell protein, but the largest stores of protein, the C14 stores in the muscle, will be given up to make up this loss in the liver. In other words, where the emergency is, there goes the protein. That is shown very nicely in some experiments which have been published, but cannot be shown in this lecture. All of this, I think, means a dynamic equilibrium, a shifting about of proteins, however we wish to explain it. I wish to thank you for your attention.

George Whipple (1954)

The Dynamic Equilibrium of Body Proteins Hemoglobin, Plasma Proteins, Organ and Tissue Proteins

George Whipple (1954)

The Dynamic Equilibrium of Body Proteins Hemoglobin, Plasma Proteins, Organ and Tissue Proteins

Comment

When George Whipple received the Nobel Prize in Physiology or Medicine together with George Minot and William Murphy in 1934, it was the first time that this particular Nobel Prize was shared by three. In this trio of Nobel Laureates, Whipple represented Physiology and the other two Medicine. Whipple had studied how the production of the oxygen carrying molecule haemoglobin depends on the food given to dogs suffering of anaemia. The other two had tried out his findings on hospital patients. They found that they could cure the deadly disease pernicious anaemia by letting their patients eat large amounts of raw liver, thus saving thousands of lives even before 1934. The reason that raw liver is effective has much later been shown to depend on the liver containing vitamin B12. The synthesis of this vitamin can thus be said to have led to a much more agreeable cure for the disease! The medical doctor William Murphy gave a talk on the history of the treatment of pernicious anaemia at the very first Lindau Meeting, which was intended for other medical doctors. When George Whipple was invited in 1954, Count Lennart Bernadotte had begun the transformation of the meetings into meetings for young scientists and students. Whipple writes in his acceptance letter to Lindau: “I understand that this is to be presented to an audience consisting of many lay people so that the language will be directed to lay people rather than to the professional investigator. … I understand that lectures occupy about 3/4 of an hour.” As you can hear from the recorded lecture, Whipple really lives up to his acceptance letter. He speaks slowly and clearly and uses a language that can be readily understood. Even the time span of the lecture, which quite often is a problem even for a Nobel Laureate, gives him no problem, he needs only about 35 minutes. The investigation that he reports is a logical extension of the work performed 30 years earlier, but one important new technique has been added: Radioactive tracers had been introduced and with these one could follow the pathways of different proteins injected into the dogs. For the inventor of the radioactive tracer method, George de Hevesy, who received the 1943 Nobel Prize in Chemistry for his work and who was present at this Lindau meeting, this must have felt quite nice!

Anders Bárány

Cite


Specify width: px

Share

COPYRIGHT

Cite


Specify width: px

Share

COPYRIGHT


Related Content