The placenta is permeable to B 12 Co 60 . Together with salts, sugar, amino acids, vitamins, and proteins, the B 12 is contributed to the growing embryos from the mother's body stores. At birth, the placenta contains a liberal amount of B 12 , which the mother regains by ingestion of the placentas. Nursing draws a liberal amount of B 12 Co 60 from the mother's stores and contributes it to the body of the pups where it is absorbed and distributed in the various organs, much as noted when adult dogs are injected with B 12 Co 60 . A redistribution is noted as the months pass; but the high values in the heart, liver, and gastric mucosa persist, and the brain usually shows a slow increase. Enterohepatic circulation of B 12 may involve bile and enteric content plus hepatic, gastric, and pancreatic epithelial secretion. This type of body recycling of radioactive B 12 is discussed but not proven. High values of B 12 in the heart, brain, gastric mucosa, and liver indicate that the vitamin is functionally active, not an inert fraction.

During protein depletion produced by plasmapheresis and a very low protein diet there is a proportionately greater decrease in extravascular, extracellular fluid protein than in plasma protein. A shift in the normal ratio of protein in these 2 compartments, approximately 1 to 1 in the dog, to over 2 to 1 as a result of depletion indicates an important, labile source of reserve protein for the plasma in the interstitial fluids. This reserve source is limited since a maximum drop of 50 per cent in the total exchangeable pool and of 75 per cent in the extravascular, extracellular protein occurred after both shorter and longer periods of depletion. Under the rigid conditions of these experiments additional plasma protein removal was associated with loss of weight despite adequate caloric intake. Investigation of the status of the interstitial fluid proteins in other conditions associated with disturbed protein metabolism seems warranted.

Experiments dealing with the distribution of B 12 Co 60 in the dog indicate that with time (9 months after administration) there is a shift in the distribution of the vitamin as compared to the short term experiments, as well as prolonged retention of the vitamin within various dog tissues. The heart, gastric mucosa, liver, spleen, and brain show high concentrations of the isotope in long term experiments. This distribution, in the heart for example, does not fit with an hypothetical breakdown of B 12 Co 60 complex and storage of a physiologically inactive fraction. Repeated periods of anemia produced by phenylhydrazine make it possible to demonstrate radioactive material in red cell stroma of dogs that have previously received vitamin B 12 Co 60 . This radioactive material must have come from other body stores, such as liver and stomach. The high concentration of B 12 Co 60 in the gastric mucosa suggests a relationship between it and the intrinsic factor as described by Castle.

During active blood regeneration in anemia in dogs an increase occurs in the stroma protein of the red cells. When vitamin B 12 with radioactive cobalt is given at the start of this blood regeneration one finds concentration of labeled B 12 in the stroma protein but not in the hemoglobin. After the acute phase of red cell regeneration is ended the concentration of B 12 in stroma protein falls rapidly to very low levels within 2 weeks. Subsequent episodes of red blood cell regeneration seems not to cause remobilization of radioactive cobalt into red cells from other body stores. It appears that the vitamin B 12 is a factor of importance in the first steps of stroma protein formation in the first few days of the life of the red cell in the dog. This response in dogs and the response in pernicious anemia to vitamin B 12 may have some points in common. Distribution of the B 12 -radioactive cobalt in the organs and tissues at autopsy has been recorded. Some very suggestive localizations were noted and some variation 1 week and 7 weeks after B 12 injections. Radioactive cobalt escapes in the urine during the weeks following B 12 injections.

Normal red blood cells in dogs contain stroma in fairly uniform amounts. This red cell stroma is rich in proteins and lipides. Anemia due to blood loss causes an increase in stroma protein. The highest levels of stroma protein are found in the severe anemias. As the anemia is corrected by red cell regeneration, the stroma protein level falls to normal. Anemia due to blood destruction (phenylhydrazine) presents very high levels of stroma protein —almost double the increase noted in anemia due to blood loss. Hypoproteinemia added to anemia due to blood loss causes no significant change on the stroma protein level. Abscesses due to the subcutaneous injection of turpentine during the anemia cause slight decreases in the stroma protein levels. Chloroform poisoning has no effect on the stroma protein levels. The total lipides of the stroma are rather stable and are little influenced by anemia. In certain experiments with hemolytic anemia and with hypoproteinemia, there is a significant rise in total lipide figures.

Red cell stroma protein and hemoglobin can be labeled by feeding C 14 lysine during periods of active blood regeneration following anemia. Stroma proteins are produced and a maximum concentration of the C 14 label appears 2 to 3 days earlier than with hemoglobin,—which is to say that stroma building precedes hemoglobin construction. The concentration of isotope in stroma protein may exceed its concentration in hemoglobin during regeneration following anemia due to blood loss. Diets favorable for hemoglobin regeneration may force the hemoglobin isotope concentration above that of the stroma protein. In hemolytic anemias great reserves of red cell building material are stored in the body. These stores may modify the curves of isotope concentration in red cells during the recovery periods. When finally formed, the mature red cells show little or no evidence of participation in general body protein metabolism during their life in the circulation.

Plasma proteins tagged in vivo by feeding D-L -lysine-ϵ-C 14 to donor dogs have been administered to pregnant dogs by both oral and intravenous routes. A relatively small percentage of the C 14 activity originally incorporated in these proteins is found to pass from mother to fetus after intravenous injection. The amount transferred tends to increase with the length of gestation period and total number of fetuses. Plasma protein labeled with I 131 does not cross the placenta in the dog, but does in the rabbit. Evans blue dye does not cross the placenta of the dog. After oral administration of labeled plasma protein or lysine, C 14 is transferred promptly and in considerable quantity to the fetus. Labeled plasma proteins disappear more rapidly from the circulation of pregnant than of normal dogs. This increased metabolic turnover occurs without excretion of any excess waste metabolites. The chorionic epithelium , gram for gram, is probably 2 to 3 times as active as the hepatic epithelium in protein metabolism. These findings indicate an important placental function related to maternal and fetal protein metabolism. While the placenta utilizes maternal plasma proteins and amino acids, in a quantitative sense the latter appear to supply the major nitrogen needs of the growing fetus.

Carbon-14-labeled plasma proteins given by mouth to dogs with sterile abscesses undergo decreased absorption, presumably owing to impaired digestion of protein. The turnover of plasma albumin is greatly accelerated but the globulins, excluding fibrinogen, show little change during the acute stage of the sterile inflammation. Fibrinogen shows very rapid production and utilization during acute inflammation. Large amounts of C 14 are incorporated in fibrinogen within a few hours after ingestion of the labeled material. The labeled fibrinogen largely disappears within 2 to 4 days after its production. The appearance of C 14 in new red cells from labeled protein or amino acid sources is reduced by inflammation—evidence of impaired synthesis. The pus of the sterile abscess contains a good deal of C 14 activity which at times is as much as that found in the liver. Pus cell C 14 activity per milliliter is similar after injection of labeled plasma and ingestion of labeled plasma or lysine. However, the pus cell fraction contains 3 to 4 times more C 14 activity per milliliter than does the supernatant fluid when the isotope is fed. In the supernatant fluid the activity is all within precipitable protein, much of which is probably derived from the blood plasma. In spite of increased loss of C 14 as CO 2 in the expired air and in the pus, there is evidence of conservation of protein-building materials for maintenance of new plasma proteins and tissue proteins in the more active organs ( e.g . liver)—a shift of protein C 14 from the less active tissues (muscle and skin).

The metabolism of homologous plasma proteins, labeled with lysine-ϵ-C 14 , after oral administration to dogs has been investigated. The speed of the various processes involved is indicated by the maximum rate of C 14 O 2 excretion which is attained within 1 to 4 hours, the prompt appearance of protein activity in the plasma and disappearance of non-protein activity from it, both virtually complete in 7 to 10 hours, as well as the rapid incorporation of a large percentage of the fed-C 14 into tissues. There are no essential differences between the behavior of labeled plasma and that of an amino acid digest containing ϵ-C 14 labeled lysine when these two materials are given orally. At the end of 48 hours after labeled plasma feeding, a CO 2 elimination of 16 to 28 per cent of the fed C 14 is noted. In contrast, after 48 hours following labeled plasma by vein, a CO 2 elimination of only 2.5 per cent is recorded—almost a 10 to 1 ratio. We believe this, together with the data concerning plasma and tissue protein activity, represents a significant difference in the metabolic process. The evidence favors a complete breakdown of plasma protein to the amino acid level when given orally but not when given by vein.

Plasma containing carbon 14 -labeled albumin and globulin, obtained by feeding ϵ-C 14 D,L .-lysine to a donor dog, has been injected intravenously and intraperitoneally into recipient dogs with experimental ascites. The circulation and interchange of total plasma protein between circulating blood and ascitic fluid have been confirmed and the participation of both albumin and globulin in this interchange has been demonstrated. Labeled albumin tends to reach equilibrium in plasma and ascitic fluid in a shorter period of time (1 to 2 days) than does globulin (2 or more days), after administration of labeled plasma by either route. Evidence is presented that the rate of transfer of albumin across the peritoneal membrane is at least three times faster than that of globulin in terms of weight.

Dogs with sustained anemia plus hypoproteinemia due to bleeding and a continuing low protein or protein-free diet containing abundant iron have been used in the present work to test food proteins and supplements as to their See PDF for Structure capacity to produce new hemoglobin and plasma proteins. The reserve stores of blood protein-producing materials are thus largely depleted in such animals and sustained levels of 6 to 8 gm. per cent hemoglobin and 4 to 5 gm. per cent plasma protein can be maintained for considerable periods of time. The stimulus of double depletion drives the body to use all protein building materials with the utmost conservation. This represents a severe biological test for food and body proteins and its assay value must have significance. Measured by this biological test in these experiments, casein stands well up among the best food proteins. The ratio of plasma protein to hemoglobin is about 40 to 50 per cent, which emphasizes the fact that these dogs produce on most diets about 2 gm. hemoglobin to 1 gm. plasma protein. The reason for this preference for hemoglobin production is obscure. The mass of circulating hemoglobin is greater even in this degree of anemia and the life cycle of hemoglobin is much longer than that of the plasma protein. Egg protein, egg albumin, and lactalbumin all favor the production of more plasma protein and less hemoglobin as compared with casein. The plasma protein to hemoglobin ratio is increased, sometimes above 100 per cent. Supplements to the above proteins of casein digests or several amino acids may return the response toward that which is standard for casein. Histidine as a supplement to egg protein increases the total blood protein output and brings the ratio of plasma protein to hemoglobin toward that of casein. Beef muscle goes to the other extreme and favors new hemoglobin production up to 4 gm. hemoglobin to 1 gm. plasma protein—a ratio of 25 per cent. The total amounts of new blood proteins are high. Lactalbumin as compared with casein shows a lower total blood protein output and a plasma protein to hemoglobin ratio of 70 to 90 per cent. Amino acid supplements are less effective. See PDF for Structure Fibrin is a good food protein in these experiments—much like casein. When fed over these 5 week periods it causes a sustained increase in blood fibrinogen . Folic acid in the doses given has no effect on the expected response to various diets. Peanut flour is a very poor diet for the production of new hemoglobin and plasma proteins. Small supplements of casein and beef show a significant response with improved output of blood proteins. Soy bean flour gives a poor response and wheat gluten a good response with adequate output of blood proteins. Visceral products show some variety. Beef heart is not as effective as beef muscle. Beef spleen, kidney, and pancreas give good responses but not up to casein. Pig stomach, beef brain, and calf thymus are below average. The plasma protein to hemoglobin ratio shows a narrow range (40 to 60 per cent) in experiments with visceral products.

Labeled plasma proteins obtained from donor dogs, previously fed ϵ-C 14 - dl -lysine, have been given intravenously to recipient dogs. The disappearance of labeled globulin from the plasma at a rate considerably faster than albumin has been confirmed. Evidence suggesting that the mass of protein in solution in the extravascular, extracellular fluid is approximately equal to the plasma proteins in circulation has been derived from a study of the dilution of labeled plasma protein by repeated injections of non-labeled plasma protein. In a period of 7 days the transfer of C 14 from plasma to tissue proteins amounted to between 30 and 40 per cent of the activity in the labeled plasma protein injected intravenously. The conversion was accompanied by a very small loss of activity in the urine and expired air and the activity remained in the lysine residue of the liver and probably of other tissues. The data presented favor the view that plasma proteins are utilized in the body economy after partial catabolism within the cell area and provide no evidence of complete breakdown to the amino acid level.

A dog, doubly depleted of blood cells and plasma proteins, was fed dl -lysine labeled with C 14 in the epsilon carbon position. In the first 8 hours 28 per cent of the administered C 14 was excreted in the urine; in the first 72 hours, 35 per cent. Twenty-four hours after feeding, 4.2 per cent of the fed C 14 was circulating in the plasma, decreasing to 1 per cent at the end of 17 days. The C 14 content of the blood cells increased from 1 per cent at 24 hours to 5.5 per cent in 5 days and 6.8 per cent in 22 days. Evidence based on the rate of decrease of the C 14 content of circulating blood cells is presented indicating an average life of 115 days for the erythrocyte protein as an entity not interchanging with extracellular constituents. This corresponds closely to the life span of the dog erythrocyte, 112 to 133 days according to the best evidence otherwise available and indicates that this experiment has actually measured the life span of the dog erythrocyte. Following breakdown of blood erythrocytes the protein comprising them is not used preferentially for the formation of new erythrocytes.

Racemic lysine labeled with C 14 in the epsilon carbon position was fed to dogs. The distribution of C 14 in blood and tissue fractions is recorded. In normal dogs sacrificed at 24 hours, approximately one-third of the C 14 was found in the urine, one-third in expired air, and one-third in the body, mostly in protein, predomantly as lysine residues. The rate of C 14 excretion as CO 2 , hour by hour, paralleled closely the amount of non-protein C 14 in the blood plasma. The liver, kidney, pancreas, and spleen all have high values for C 14 in 24 hour and 17 day experiments. The gastrointestinal tract is significantly high in the 24 hour experiments. Plasma protein from animals previously fed C 14 containing lysine and thus in turn labeled, was transfused into other dogs and the rate of disappearance of albumin and globulin fractions from the circulation of the recipient dog followed. The results lead to the conclusion that as a whole, plasma proteins are utilized and replaced at a rate of at least 10 per cent per 24 hours. This minimum rate is substantially faster than turnover rates commonly accepted and emphasizes the rôle played by the plasma proteins in the protein economy of the body. The exact rate determination is made uncertain by the lack of knowledge of the magnitude of the amount of protein in solution in extracellular and lymph spaces and its rate of equilibrium with circulating plasma proteins. Evidence from these transfusion studies indicates that plasma globulin is metabolized at a significantly faster rate than plasma albumin. This is confirmed by the observation that following the feeding of labeled lysine to dogs, C 14 is first incorporated in globulin in high concentration but that later it also disappears more rapidly from the globulin fraction. These data suggest that the period of bone marrow maturation of the red cell during which time its related hemoglobin is synthesized does not exceed 3 to 5 days.

Sterile inflammation induced by repeated subcutaneous injections of turpentine in non-anemic, non-iron—deficient dogs, leads to a fall in plasma iron concentration, the development of a moderate anemia, and a marked delay in the uptake by the red blood cells of intravenous radioiron. Similar periods of inflammation in anemic, iron-deficient dogs on a diet low in iron cause no increase in the degree of anemia and no inhibition of red blood cell uptake of intravenous radioiron. Radioiron appears only in traces in abscess exudates. Intravenous iron disappearance curves following a single injection are uninfluenced by sterile inflammation in either anemic or non-anemic dogs. The impairment of hemoglobin synthesis caused by inflammation is at most a relative matter, since the anemia that develops is seldom severe or progressive, and since the inhibition can be overcome if the marrow is sufficiently stimulated by the demands of a severe continuing anemia.

Doubly depleted dogs (anemic and hypoproteinemic) respond favorably to all the diet proteins used in the above experiments. Egg products (whole egg, albumin, or egg yolk) are well utilized by these dogs. Egg proteins favor the production of plasma protein and in some experiments the output of plasma protein is actually more than the output of hemoglobin. In contrast fresh beef muscle favors hemoglobin production—the output being 3 or 4 times that of plasma protein. The processed egg albumin fed in Table 4 was not well utilized and there was weight loss. Beef muscle (fresh or processed) gives a total blood protein output about twice that with egg feeding and there is a striking preponderance of hemoglobin output. Beef heart and salmon muscle show a pattern much like beef muscle. The total blood protein output is below that due to beef muscle.

Casein (purified or commercial) in this type of experiment falls in the top bracket as a protein consistently favorable for maximal new hemoglobin and plasma protein production in doubly depleted dogs (anemic and hypo-proteinemic). Lactalbumin is less favorable for total blood protein production and the ratio of plasma protein to hemoglobin is high—that is lactalbumin favors plasma protein production as compared with casein, or is less favorable for hemoglobin production. Peanut flour (purified or commercial) is less than half as effective as casein in promoting new blood protein production. The ratio of plasma protein to hemoglobin is about the same as casein. Wheat gluten as tested is distasteful to dogs. It is neither very good nor very poor for blood protein production when it is eaten. There is nothing unusual about the response. Weight loss usually confuses the picture. Liver stands as a control base line for the above experiments. Its capacity to further hemoglobin and plasma protein production is well established. The production of hemoglobin was about 3 times that of plasma protein in the experiments.

Proteinuria in normal dogs can be produced at will by parenteral injections of dog plasma. As the plasma injections are continued the plasma protein concentration rises and at some point protein begins to appear in the urine. The level of plasma protein concentration at which proteinuria appears in normal dogs ranges from 9.6 to 10.4 gm. per cent. This may be termed the renal threshold for proteinuria. Repeat experiments in the same dog show threshold levels to be practically identical. An interval of days (4 to 26 days) has been noted between the start of plasma protein injections and the appearance of the proteinuria. Larger doses of plasma shorten this interval and the critical plasma protein level is attained sooner. Considerable amounts of protein may appear in the urine—298 gm. protein during a 52 day period in one instance studied—yet the urine clears in 1 to 4 days after cessation of protein injections. Autopsy shows undamaged kidneys. Maximal levels of plasma protein concentration range from 10.0 to 11.5 gm. per cent. The highest levels are usually associated with maximal output of protein in the urine. It seems clear that plasma proteins readily pass cell barriers (or membranes) within the body, including the endothelium and epithelium of the renal glomerulus.

Proteinuria in normal dogs can be produced at will by parenteral injections of dog plasma. As the plasma injections are continued the plasma protein concentration rises and at some point protein begins to appear in the urine. The level of plasma protein concentration at which proteinuria appears in normal dogs ranges from 9.6 to 10.4 gm. per cent. This may be termed the renal threshold for proteinuria. Repeat experiments in the same dog show threshold levels to be practically identical. An interval of days (4 to 26 days) has been noted between the start of plasma protein injections and the appearance of the proteinuria. Larger doses of plasma shorten this interval and the critical plasma protein level is attained sooner. Considerable amounts of protein may appear in the urine—298 gm. protein during a 52 day period in one instance studied—yet the urine clears in 1 to 4 days after cessation of protein injections. Autopsy shows undamaged kidneys. Maximal levels of plasma protein concentration range from 10.0 to 11.5 gm. per cent. The highest levels are usually associated with maximal output of protein in the urine. It seems clear that plasma proteins readily pass cell barriers (or membranes) within the body, including the endothelium and epithelium of the renal glomerulus.

Given healthy dogs fed abundant iron and protein-free or low protein diets with sustained anemia and hypoproteinemia, we can study the capacity of these animals to produce simultaneously new hemoglobin and plasma protein. Reserve stores of blood protein-building materials are measurably depleted and levels of 6 to 8 gm. per cent for hemoglobin and 4 to 5 gm. per cent for plasma protein can be maintained for weeks or months depending upon the intake of food proteins or amino acid mixtures. These dogs are very susceptible to infection and various poisons. Dogs tire of these diets and loss of appetite terminates many experiments. Under these conditions (double depletion) standard growth mixtures of essential amino acids are tested to show the response in blood protein output and urinary nitrogen balance. As a part of each tabulated experiment one of the essential amino acids is deleted from the complete growth mixture to compare such response with that of the whole mixture. Methionine, threonine, phenylalanine, and tryptophane when singly eliminated from the complete amino acid mixture do effect a sharp rise in urinary nitrogen. This loss of urinary nitrogen is corrected when the individual amino acid is replaced in the mixture. Histidine, lysine, and valine have a moderate influence upon urinary nitrogen balance toward nitrogen conservation. Leucine, isoleucine, and arginine have minimal or no effect upon urinary nitrogen balance when these individual amino acids are deleted from the complete growth mixture of amino acids during 3 to 4 week periods. Tryptophane and to a less extent phenylalanine and threonine when returned to the amino acid mixture are associated with a conspicuous preponderance of plasma protein output over the hemoglobin output (Table 4). Arginine, lysine, and histidine when returned to the amino acid mixture are associated with a large preponderance of hemoglobin output. Various amino acid mixtures under these conditions may give a positive urinary nitrogen balance and a liberal output of blood proteins but there is always weight loss, however we may choose to explain this loss. These experiments touch on the complex problems of parenteral nutrition, experimental and clinical.