PROTEOSE INTOXICATIONS AND INJURY OF BODY PROTEIN : I. THE METABOLISM OF FASTING DOGS FOLLOWING PROTEOSE INJECTIONS.

Proteose injections in dogs cause vomiting, diarrhea, temperature reactions, low blood pressure, prostration, and, after large doses, an excess of antithrombin with incoagulable blood. A single proteose injection, for example one-half a lethal dose, causes abrupt clinical reactions in a normal dog with apparent complete recovery within 24 to 48 hours. The nitrogen elimination curve in a fasting dog under such conditions shows a great rise in total urinary nitrogen. The apex of the curve usually falls during the second 24 hour period following the injection. This rise may be over 100 per cent increase above the mean base-line nitrogen level. It does not fall promptly to normal but declines slowly in 3 to 5 days or more toward the original base-line (Text-fig. 1). This speaks for a definite cell injury with destruction of considerable protein substance due to a single proteose injection. The disturbance of cell equilibrium is not rapidly or promptly restored to normal. A dog which has received previous proteose injections is partially immune or tolerant to subsequent injections of proteose. These dogs, as a rule, show less intense clinical reactions and less rise in the curve of nitrogen elimination following a unit dose of standard proteose as compared with normal or non-immune controls. The proteose used in these experiments was prepared as described from material obtained in cases of intestinal obstruction or of closed intestinal loops. These experiments explain the sharp rise in blood non-protein nitrogen which follows within a few hours the injection of a toxic proteose. They also point to the correct explanation of the high non-protein nitrogen of the blood found in intestinal obstruction or with closed intestinal loops.     

PROTEOSE INTOXICATIONS AND INJURY OF BODY PROTEIN : II. THE METABOLISM OF DOGS WITH DUODENAL OBSTRUCTION AND ISOLATED LOOPS OF INTESTINE.

Dogs with isolated loops of small intestine show many evidences of intoxication. A study of the total nitrogen elimination shows a great rise above the normal base-line minimum of the fasting period (Table II). This means that the intoxication is associated with a great destruction of body protein, and explains the high non-protein nitrogen of the blood which was observed and reported previously (2). Injection of a proteose obtained from a closed intestinal loop will cause a similar rise in the nitrogen elimination curve. This furnishes more evidence that the intoxication observed in association with a closed intestinal loop is in reality a proteose intoxication. Dogs injected with sublethal doses of proteose will show a definite tolerance to subsequent injection, and will show much less acute intoxication after the isolation of a closed intestinal loop (Table 1). These immune or tolerant dogs show a much less pronounced rise in the nitrogen elimination curve during proteose intoxication of any type. This indicates that the tolerance or immunity to proteose gives more protection for the body proteins against the injury which these toxic proteoses inflict upon the body cells. Complete duodenal obstruction combined with a gastrojejunostomy gives a chronic type of intestinal obstruction associated with little vomiting, which is peculiarly suited to metabolism study (Table IV). Such duodenal obstructions show a definite and sustained rise in the curve of nitrogen elimination above the normal base-line level. These dogs, too, are tolerant to injections of standard toxic proteoses. Control ether anesthesia experiments show little if any rise in the curve of nitrogen elimination (Table VI). Control laparotomy experiments show a definite rise in the curve of nitrogen elimination, but a rise which is small compared with the rise noted in the intoxication of duodenal obstruction or of isolated intestinal loops. It is probable that the tissue injury and disintegration associated with the wound reaction are responsible for the general reaction. We may assume that protein split products from the wound area are absorbed and are responsible for the general reaction observed. We propose to assume that the intoxications here studied are associated with a definite proteose intoxication, which is capable of initiating and continuing a profound injury of tissue protein. One index of this protein injury is the great and sustained rise in the curve of total nitrogen elimination.     

PROTEOSE INTOXICATIONS AND INJURY OF BODY PROTEIN : III. TOXIC PROTEIN CATABOLISM AND ITS INFLUENCE UPON THE NON-PROTEIN NITROGEN PARTITION OF THE BLOOD.

The acute intoxication following an injection of a toxic proteose is usually associated with a large increase (40 per cent or more) in the non-protein nitrogen of the blood. This increase is found chiefly in the blood urea nitrogen, but the amino and peptide nitrogens also may show small increases. The changes observed in the blood non-protein nitrogen are identical with those which follow the feeding of large amounts of meat (8). These facts indicate that the proteose intoxication causes an abnormally rapid autodigestion of tissue proteins, but that the nitrogenous end-products are, in chief part at least, the same that result from normal catabolism of food proteins. There is no evidence that the autolytic products play any part in causing the intoxication. The possibility of such a part and a resultant vicious circle is not excluded, but from the available facts the autolysis appears more as a result rather than cause of the intoxication. It appears possible that in disease or intoxication tissue catabolism may be enormously accelerated and yet yield the end-products of normal protein metabolism.     

PROTEOSE INTOXICATIONS AND INJURY OF BODY PROTEIN : V. THE INCREASE IN NON-PROTEIN NITROGEN OF THE BLOOD IN ACUTE INFLAMMATORY PROCESSES AND ACUTE INTOXICATIONS.

Sterile abscess formation in the dog is accompanied by a large increase in output of urinary nitrogen and also by a small but definite increase in the blood non-protein nitrogen. All this nitrogenous material of course is derived from body protein injury and autolysis. Septic inflammation in the dog (pleurisy, pneumonia, peritonitis, etc.) likewise shows a distinct rise in the blood non-protein nitrogen. This rise is not often so great as that frequently observed in the intoxication of intestinal obstruction. Many acute infections in man (septicemia, peritonitis, pneumonia, etc.) show a definite rise in the non-protein nitrogen and urea nitrogen of the blood; some cases show a very great rise above normal (over 100 mg. of non-protein nitrogen per 100 cc. of blood). There may be no anatomical change in the kidney beyond the familiar picture of cloudy swelling. This does not exclude the possibility of some transient functional derangement of the kidney epithelium. Certain obscure intoxications in man may show a considerable rise in the non-protein nitrogen of the blood, indicating a large amount of protein disintegration. These findings must be taken into account in any clinical analysis and interpretation of high non-protein nitrogen of the blood in pathological conditions.     

HEMOGLOBIN AND PLASMA PROTEIN : THEIR RELATION TO INTERNAL BODY PROTEIN METABOLISM

Hemoglobin (presumably its essential protein globin), given intraperitoneally to a protein-fasting dog, will be used effectively to supply the protein requirements of the body. Nitrogen balance may thus be maintained for 20 days under favorable conditions. New hemoglobin and plasma protein will be formed related to hemoglobin injections in depleted dogs where there is urgent need for these proteins (anemia and hypoproteinemia). Obviously this calls for supplementary amino acids which in globin are low and we assume these amino acids must be contributed from body protein stores. Plasma proteins (in plasma) tested in the same manner are completely utilized with no loss of nitrogen, positive nitrogen balance, weight balance, and no change in the albumin-globulin ratios. Hemoglobin (globin) is less effectively utilized as compared with plasma protein given parenterally and there is some increase in urinary nitrogen above control periods. The albumin-globulin ratio may be somewhat modified by hemoglobin injections intraperitoneally. Hemoglobin (globin) digests contribute effectively to body maintenance of nitrogen equilibrium. These digests are about as effective as whole hemoglobin in maintaining nitrogen balance but cause a rise in undetermined nitrogen not seen when hemoglobin alone is given intraperitoneally. Pigment radicles derived from hemoglobin given intraperitoneally are thrown away and appear as surplus bile pigment even when there is urgent need for all available nitrogenous material—given protein fasting, anemia, and hypoproteinemia in a bile fistula dog. The body evidently prefers to make rather than conserve the pyrrol aggregate (pigment radicle). We assume that the injected hemoglobin (globin) or hemoglobin digests contribute to the body protein pool and from this pool various proteins emerge to supply protein requirements of tissue or organ cells or to produce new hemoglobin or plasma protein if needed. We have no explanation as to what determines the pattern of this protein flow but new hemoglobin is very high on the priority list.     

PROTEIN METABOLISM AND PROTEIN RESERVES DURING ACUTE STERILE INFLAMMATION : HIGH PROTEIN INTAKE COMPENSATES FOR INCREASED CATABOLISM

Adult dogs were given a proteinless diet plus casein, 80 calories/kilo, 0.4 gm. nitrogen/kilo/day. Sterile controlled inflammation was produced by subcutaneous injection of turpentine. The reaction is characterized by local swelling, induration, and abscess formation, terminated by rupture or incision after 3 to 5 days and by general reactions of malaise, fever, leucocytosis, and increased urinary nitrogen. For 3 to 6 days after turpentine the nitrogen intake was provided in seven experiments by amino acids given parenterally (a solution of the ten essential amino acids (Rose) plus glycine). A normal dog with a normal protein intake showed a negative nitrogen balance after turpentine—urinary nitrogen doubled even as in inflammation during fasting. A protein-depleted dog (low protein reserves produced by very low protein intake) given a normal protein intake after turpentine maintained nitrogen balance—urinary nitrogen rose only slightly. With a high (doubled) protein intake the depleted dog showed strongly positive balance. Normal dogs with high (doubled) protein intakes react to turpentine with doubled urinary nitrogen outputs on individual days and therefore are maintained in approximate nitrogen balance and weight balance. This end may be achieved equally well or better by oral feeding, when such is possible and absorption unimpaired. The increased nitrogen excretion after injury is again shown directly related to the state of body protein reserves. Increased catabolism not inhibition of anabolism best explains the excess urinary nitrogen. Protection during injury of valuable protein reserves appears possible through an adequate intake of protein nitrogen.     

A STUDY OF ACUTE MERCURIC CHLORIDE INTOXICATIONS IN THE DOG WITH SPECIAL REFERENCE TO THE KIDNEY INJURY

A study of the experiments comprising the first group of animals permits the deduction that these animals succumb to the acute poisoning as a result of the shock which the poison induces through its corrosive action in the stomach and intestine. The animals die before the mercury, acting as such during its elimination by the kidney, can induce an acute nephropathy and before the mercury, by inducing an acid intoxication, can lead to an acute kidney injury. The remaining animals of the series, Groups II, III, and IV, have withstood the corrosive action of the poison. These animals have shown the same type of delayed intoxication from the poison. The intoxication, however, has varied in time of appearance, duration, and severity. The animals classified as Group II have developed during the stage of improvement from the gastroenteritis a rapid and severe type of acid intoxication, have become rapidly anuric, and have died either in a state of air-hunger or in convulsions. The animals of Group III, either during or after their recovery from the gastroenteritis, have developed a mild grade of acid intoxication. During the following days of the experiments the animals succeeded in reestablishing their normal acid-base equilibrium. All the animals of this group recovered. The animals of Group IV have shown a recovery from the mercury enteritis. Following a period during which there was an attempt on the part of the animals to return to normal, as indicated by an increase in the alkali reserve of the blood and by an increased output of phenolsulfonephthalein and urine, the members of the group developed a delayed acid intoxication, and, like the animals of Group II, became anuric.     

BLOOD PLASMA PROTEIN PRODUCTION AND UTILIZATION : THE INFLUENCE OF AMINO ACIDS AND OF STERILE ABSCESSES

When blood plasma proteins are depleted by bleeding with return of the washed red blood cells (plasmapheresis) it is possible to bring dogs to a steady state of hypoproteinemia and a uniform plasma protein production on a basal low protein diet. These dogs are clinically normal. Introduction of variables into their standardized life gives insight into the production of plasma protein. Casein retested as the basal protein in the ration may show high yield of plasma protein, equal to 33 per cent of the protein fed. This equals the potency of liver protein (17 to 33 per cent) and approaches the utilization of plasma protein by mouth (40 per cent). Zein has no effect upon plasma protein regeneration but when it is supplemented with cystine, tryptophane, lysine, and glycine, there is a doubling of the liver basal plasma protein production and a retention of the fed protein nitrogen. Threonine does not modify the above reaction. Liver protein supplemented with cystine, leucine, glutamic acid, and glycine in the basal diet yields double the amount of new formed plasma protein compared with liver alone. This combination is then as potent as plasma protein itself when given by mouth—40 per cent utilization. Tyrosine or lysine, arginine, and isoleucine do not modify the above responses. Methionine is not as effective as cystine in supplementing gelatin and tyrosine to produce plasma protein. Cystine, leucine, and glutamic acid appear to be of primary importance in the building of new plasma protein in these experiments. Plasma protein formation is dependent upon materials coming from the body reserve and from the diet. Given an exhaustion of the reserve store there is very little plasma protein produced during a protein fast (3 to 6 gm. per week). A turpentine abscess does not modify this fasting plasma protein reaction. Homologous plasma given by vein will promptly correct experimental hypoproteinemia due to bleeding. It will maintain nitrogen equilibrium and replenish protein stores. Even during hypoproteinemia plasma protein may promptly pass out of the circulation to supply body needs for protein. Perhaps the most significant concept which derives from all these experiments is the fluidity of the body protein (including plasma protein)—a ready give and take between the protein depots—a "dynamic equilibrium" of body protein.     

THE METABOLISM OF AMINO ACIDS AND CASEIN DIGEST IN PHLORHIZINIZED DOGS

When solutions of sugar-forming amino acids are given parenterally or orally to phlorhizinized dogs, extra sugar and nitrogen are excreted in the urine. The route of administration does not influence the way in which the amino acids are metabolized. 89 percent of the expected sugar was recovered when the acids were fed and 81 per cent when the acids were administered parenterally. Casein digests are metabolized like the amino acids with extra sugar and nitrogen excreted in the urine. When casein digest is given orally there is an average conversion into sugar of 53 per cent, as compared with 56 per cent intravenously. Solutions of amino acids or casein digests are readily available sources to the body of nitrogen and carbohydrate.     

CHEMOPATHOLOGICAL STUDIES WITH COMPOUNDS OF ARSENIC : IV. THE CHARACTER AND DISTRIBUTION OF RENAL INJURY PRODUCED BY ARSENICALS AS INDICATED BY THE PROCESSES OF REPAIR.

1. The processes of repair in the kidneys of guinea pigs after sublethal doses of certain arsenical compounds indicate that all arsenicals do not produce a purely vascular type of renal injury. 2. While some arsenicals produce a predominantly vascular injury and others produce a predominantly tubular injury, both these tissue elements are undoubtedly always affected, although in varying proportion. In addition, the interstitial connective tissue is probably always affected. The diffuse proliferation of this tissue may be relatively conspicuous in the processes of repair after arsenicals that cause but slight vascular injury. 3. All red kidneys do not necessarily show identical pictures during the processes of repair; the same is true of pale kidneys. 4. The mode of action of an arsenical compound as a renal toxic agent is bound up with the chemical constitution of the compound.     

CHANGES IN THE PLASMA PROTEIN PATTERN (TISELIUS ELECTROPHORETIC TECHNIC) OF PATIENTS WITH HYPERTENSION AND DOGS WITH EXPERIMENTAL RENAL HYPERTENSION

The plasma protein pattern of patients with uncomplicated essential hypertension showed only slight variations from the normal while that of patients with severely malignant hypertension showed marked shifts. The fibrinogen and β-globulins were usually elevated beyond the normal range and the albumin decreased. In less severely malignant hypertension, the changes were less marked. In dogs with experimental renal hypertension, the γ-globulin level was greatly elevated, and in one animal exhibiting the malignant syndrome β-globulin and fibrinogen were also increased. Elevation of β-globulin seems in some manner associated with the occurrence of severe vascular disease.     

CELLULAR MECHANISMS OF PROTEIN METABOLISM IN THE NEPHRON : IV. THE PARTITION OF SUCCINOXIDASE AND CYTOCHROME OXIDASE ACTIVITIES IN THE CELLS OF THE PROXIMAL CONVOLUTION OF THE RAT AFTER INTRAPERITONEAL INJECTION OF EGG WHITE

1. Shifts of enzymatic activity have been followed during the formation and evolution of the droplets that form in the cells of the proximal convolution of the nephron of the rat after the injection of a 50 per cent solution of egg white in isotonic saline. 2. Twelve hours after injection there is a 35 to 40 per cent decrease in succinoxidase and cytochrome oxidase activities in the fraction containing the larger particles; i.e. mitochondria and droplets in equal concentration. Although after 30 hours the quantitative proportion of droplets and mitochondria is the same as previously, the activities of the fraction have returned to the normal observed originally in the uninjected rat in a corresponding fraction consisting of mitochondria only. 3. The microsome fraction shows an average increase of 35 per cent in oxidative enzyme activities during the early period following injection, and decreases to the original figure in the later period of droplet formation. 4. It is concluded from the shifting pattern of localization of oxidative enzyme activity within the cell particulates that the absorption droplets arise by the incorporation of the mitochondrial elements, which originally contain the highest enzyme activity, with absorbed protein through the intermediate stage of smaller (microsomal) particles.     

HEMOGLOBIN REGENERATION IN THE CHRONIC HEMORRHAGIC ANEMIA OF DOGS (WHIPPLE) : I. THE EFFECT OF IRON AND PROTEIN FEEDING

1. A group of dogs on a standard salmon bread diet with a slowly regenerating anemia were studied. The addition of liver to this diet during a 2 week period promoted a definitely greater regeneration of hemoglobin than did the addition of an amount of inorganic iron which was equivalent to that contained in the added liver. The more effective result attained with liver cannot, therefore, be attributed solely to the iron intake. 2. The greater response to liver is not due to its content of amino acids which are present in casein, since a diet containing an exactly similar amount of calories, iron and protein nitrogen, made up of inorganic iron and casein does not cause a greater response than that obtained by the addition of that amount of inorganic iron alone to the standard basal diet. 3. Furthermore, the salmon bread diet does not produce a deficiency of the amino acids represented in casein, since dogs eating the high protein (casein) Cowgill dog ration show the same basal hemoglobin regeneration rate and a similar greater response to liver than to inorganic iron. The Cowgill ration, however, supplies some non-ferrous factor involved in hemoglobin regeneration which is not contained, to as great a degree at least, in the salmon bread. 4. Whipple''s chronic hemorrhagic anemia of dogs serves as an accurate assay method for measuring the hemoglobin producing power of a substance. Quantitatively reproducible responses can be obtained.     

THE INFLUENCE OF DIET UPON THE REGENERATION OF SERUM PROTEIN : II. THE POTENCY RATIOS OF SERUM PROTEIN,LACTALBUMIN AND CASEIN,AND THE EFFECT OF TISSUE PROTEIN CATABOLISM ON THE FORMATION OF SERUM PROTEIN

1. By the technique of quantitative plasmapheresis the effects of single proteins in artificial synthetic diets were studied with respect to their value in promoting the regeneration of serum protein. 2. The ratio of (a) the amount of serum protein per week removed by bleeding above that regenerated by the dog when eating the protein-free diet, to (b) the dietary protein increment (i.e., above that required for nitrogen equilibrium) was termed the potency ratio. The results indicated that serum protein was slightly superior to casein and lactalbumin in promoting the regeneration of serum protein. However, the respective potency ratios, varying from approximately 0.51 to 0.36, were comparable and not widely divergent as those reported by others. It was concluded that, whereas in some individuals dietary proteins may be able to produce a significant increase in the serum protein concentration, the potency ratios are not sufficiently different to warrant the administration of any one protein in preference to another. 3. The inhibitory effect of the basal protein-free diet with respect to serum protein regeneration in the dog was also demonstrated by the inability of the protein concentration to attain the normal level in spite of discontinued plasmapheresis. However, a subsequent fasting period resulted in a progressive rise in the serum protein concentration until the normal value was approximated. These observations are interpreted as indicating that the products of tissue protein catabolism can be utilized in the formation of new serum protein. 4. The experimental production of what seems to be an inhibition of the serum protein regenerating mechanism was described. This observation together with the hypothetical evidence presented by Bloomfield (17) and Weech and his associates (9) suggests that the most profitable line of approach to solution of the problem of hypoproteinemia lies not so much in the evaluation of dietary factors but in finding a way for stimulating internally the serum protein regenerating mechanism, which seems to involve in some manner the capacity of the tissue to furnish protein for the needs of the plasma. 5. A hypothesis explaining the mechanisms responsible for serum protein formation was presented and the experimental support for it discussed. The rôle of tissue protein catabolism in this function was emphasized.