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Infection and Intoxication: Their Influence Upon Hemoglobin Production in Experimental Anemia
Abstract
Infection in human cases is often believed to be responsible for anemia. It is generally believed that lack of absorption and definite blood destruction are responsible for the anemia. Accelerated metabolism due to thyroid or dinitrophenol does not modify hemoglobin production in these standard anemic dogs. Endometritis lasting over many weeks will profoundly reduce the production of hemoglobin in the standard anemic dog. A sterile abscessalso will diminish the production of new hemoglobin in the anemic dog when liver is being fed but particularly during fasting periods when the usual abundant production of new hemoglobin is reduced to zero. Impaired absorption can be excluded as a factor of any significance in certain experiments given above. Destruction of red cells can likewise be excluded as of any significance in certain experiments given above. These experiments point to a disturbance of internal metabolism related to hemoglobin building in the body as responsible for the inhibition of hemoglobin production under these conditions. We believe this same factor is often of importance in human disease.
... It should be noted that the degree of anemia in case 4, impetigo, is only slight or close to a low normal hemoglobin. It has been shown in standard anemic dogs that an infection will not prevent absorption but will delay the utilization of the hemoglobin building material (13). So it is pos-sible that some iron was absorbed but not utilized in the 7 day period between feeding and sampling for the radio iron. ...
Radio iron is a tool which makes iron absorption studies quite accurate in dogs and reasonably satisfactory in human beings. This method is vastly superior to others previously used. Normal human pregnancy without significant anemia may show active radio iron absorption-16 to 27 per cent of iron intake. The pregnant woman as a rule shows 2 to 10 times the normal absorption of radio iron. Diseased states in which iron stores are known to be very abundant-pernicious anemia, hemochromatosis, familial icterus, and Mediterranean anemia -show very little absorption, probably less than normal. This is in spite of a severe anemia in all conditions except hemochromatosis. Chronic infections in spite of anemia show no utilization of radio iron, whether it may be absorbed or not. Leukemia shows little utilization of radio iron in red cells in spite of absorption (autopsy), probably because of white cells choking the red marrow. Polycythemia shows very low values for iron absorption as do normal persons. Two pregnant women showed only normal iron absorption. We believe that reserve stores of iron in the body, rather than anemia, control iron absorption. This control is exerted upon the gastro-intestinal mucosa which can refuse or accept iron under various conditions.
Examinations of single or multiple blood samples have been made in a variety of diseases of domestic animals.
From the heterogeneous group ofhypoplastic-aplastic anemias, several well-defined clinical and hematological entities and distinctive blood responses have been chosen for review. These are normochromic and normocytic syndromes differentiated from other blood dyscrasias by the absence of active blood formation and their resistance to all forms of antianemia treatment except transfusion. These anemias have been placed intothe three general categories of pure red-cell, hypoplastic, and aplastic anemias, in accordance with specific criteria. In pure red-cell anemia the failureof the bone marrow is confined to erythropoiesis, without simultaneous depression of granulocytes or platelets or their precursors. These patients survive long periods, provided repeated transfusions are administered to maintain normal blood levels. An adequate appraisal of therapy is not yet available because of lack of experience with large numbers of cases. However, spontaneous remissions have been observed, as well as recovery with steroids and following splenectomy. It was postulated that pure red-cell anemia and related hypoplastic anemias represent examples of congenital hematopoietic anomalies comparable to other somatic malformations originating from disturbances in embryonic and fetal life. The syndrome of "nonhemolyticanemia of the newborn" was described to include those infants who show a rapid fall of red cells and hemoglobin within the first days of life, without jaundice and erythroblastemia. The factors of incompatibility of blood groups, hemorrhage, hemolysis, and infection are not operative and the outlook based on a limited experience has been favorable. The syndrome probably represents an exaggeration of the aregenerative phase of erythropoiesis which characterizes the newborn period. The designation as "nonhemolytic anemia of the newborn" emphasizes its differentiation from erythroblastosis and probably coincides with some of the cases of anemia of the newborn reported before the discovery of the Rh factor. The mechanism of acute erythroblastopeniaand the aplastic crisis was reviewed in connection with their occurrence in hemolytic states such as sickle cell and spherocytic anemias. Under these circumstances the initial examination without knowledge of a pre-existing hemolytic anemia suggests a diagnosis of aplastic anemia. The bone marrow soon becomes reactive and the underlying condition is manifest. The temporary failure of erythropoiesisaccounts for the observation of protracted anemia in occasional cases of erythroblastosis. This inability to maintain normal blood levels may depend upon an inactive bone marrow as evidenced by a decreased erythroid cellular content. Two cases of chronic hypoplastic anemia were described which were suspected to be intermediate stages of aplastic anemia. In each instance the child was known to be allergic and susceptible to severe upper respiratory infections. The anemias resisted all hematinics except transfusion. Both children eventually recovered probably spontaneously although the effect of tonsillectomy in one and cortisone in the other could not be discounted. True aplastic anemia with an acellular bone marrow and pancytopenia is a rare occurrence in infancy and childhood. While search for a myelotoxic agent is usually unsuccessful, chloramphenicol has occasionally been associated with toxic hematopoietic effects. Since this drug has proved the agent of choice in specific clinical conditions, it is important to control its administration by frequent hematologic studies. Precautionary measures including blood and bone marrow examination have been outlined to detect reversible anemia and granulocytopenia. The possible suppressive effect of long-continued transfusions on erythropoiesis in any type of chronic anemia, nonhemolytic as well as hemolytic, may obscure the capacity for inherent blood formation and interfere with the appraisal of a specific blood picture. The optimum amount of blood to be administered and the interval between transfusions require serious consideration. Of major importance is to determine the actual need for transfusion, not in terms of a fixed hemoglobin value, but in relation to the clinical signs and symptoms manifested by the patient, with promise of their relief by this form of treatment.
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).
A major factor in the anemia of infection, inflammation, and malignancy is a relative failure of the bone marrow to increase erythropoiesis in response to a shortened red cell survival. The possible causes for this diminished marrow response are: (a) a reduced production of erythropoietin, or, (b) impaired bone marrow response to erythropoietin. In this report studies were performed on 6 normals, 13 patients with anemia from infection or inflammation, and 18 patients with anemia caused by advanced malignancy. Serum erythropoietin activity was measured using the posthypoxic, polycythemic mouse assay. Assessment of bone marrow response to erythropoietin was made by measuring (59)Fe-heme synthesis in bone marrow suspensions cultured for 3 days with and without the addition of erythropoietin. The results showed that marrow heme synthesis was increased in erythropoietin-treated cultures as compared with saline control cultures by 66+/-8% (mean +/-SE) in normals, 101+/-10% in patients with infection or inflammation, and 31+/-5% in malignancy. Serum erythropoietin levels were consistently diminished relative to expected levels for the degree of anemia in the infection-inflammatory group, but not in malignancy. In these patients, plasma inhibitors to the biological activity of erythropoietin were not detected in vitro. These studies suggest that another factor to consider in the anemia of malignancy is a decreased bone marrow response to erythropoietin. In the anemia of infection-inflammation, marrow response to erythropoietin is normal, but serum levels of erythropoietin are decreased relative to the degree of anemia.
When blood plasma proteins are depleted by bleeding, with return of washed red cells (plasmapheresis), it is possible to bring dogs to a steady state of low plasma protein in the circulation and a uniform plasma protein production on a basal diet. Such dogs become test subjects by which the effect of various factors on plasma protein regeneration can be measured. Dogs previously the subjects of plasmapheresis, during long rest periods appear to increase their stores of plasma protein building materials and their blood plasma protein concentrations above former normal levels. A sterile abscess (turpentine) induces a marked reduction in plasma protein regeneration in these test dogs consuming an ample basal diet. The sharp reduction during the initial 24 hours may in part reflect an extravasation of plasma protein into the injured tissue but there also appears to develop a true disturbance of the mechanism which produces plasma proteins. Digestive disturbances interfere seriously with plasma protein production. Whereas large quantities of live yeast upset digestion and form no plasma protein, autoclaved yeast is well utilized, having a potency ratio of 4.4. Amino acids have been tested inadequately. A mixture of cystine, glutamic acid, and glycine does seem to have a definite effect upon protein metabolism and plasma protein production. Iron, under the conditions of these experiments, does not influence the output of plasma proteins. Liver extract (parenteral) is also inert. The proteins of red blood cells when added to the diet are poorly utilized for plasma protein formation and show a potency ratio of only 10.1. Kidney protein added to the kidney basal diet shows a potency ratio of about 5 as compared with 4.6 for that basal diet. A digest of beef stomach and rice polishings shows a potency ratio of about 7.9. Dried powdered serum shows a potency ratio of 3.5, which is much less than fresh serum (2.6). Powdered thyroid fed in doses sufficient to accelerate body metabolism shows no distinct effect upon plasma protein production not attributable to the protein in the thyroid powder itself. Long periods (25 to 30 weeks) of plasma depletion and basal diet intake remove much protein from body fluids and tissues. Associated with this protein depletion the dog loses its appetite and may vomit some food. There is loss of hair, a tendency to skin ulceration, and a distinct lowering of resistance to infection. The plasma protein output may fall to fasting levels in spite of food intake sufficient to maintain weight. We believe this condition to be a deficiency state related to severe depletion of the essential protein matrix of the body cells.
Red cell stroma has not been prepared free of iron and/or hemoglobin. It is not certain whether the iron is a contaminant or an essential constituent of the stroma. Extensive washing does not eliminate iron from the stroma but does cause loss of the stroma constituents, protein and lipid.
Red cell stroma protein is increased in anemia due to blood loss in the dog, on the average in severe anemia, almost twice the figures recorded in the pooled normal samples of red cell stroma.
Lipid fractions under the same conditions show minor fluctuations: approximately 90 per cent of total lipids in the red cells are recovered in the stroma by these methods.
The technic for isolation and fractionation of dog erythrocyte stroma is described. Analyses made from the same blood sample by 3 different workers give comparable results.
Red cell stroma deserves careful study in controlled experimental conditions.
It has been shown that the standard anemic dog can use sheep, goose or dog hemoglobin when given by vein and return quantitatively its equivalent as new dog hemoglobin within the red cells. Globin at times can be used when given by vein with a quantitative return of new hemoglobin in red cells in these same anemic dogs. Again the administration of globin by vein will inhibit the expected hemoglobin formation; and we believe the toxic effect of the globin is responsible. A digest of globin may be used by the anemic dog to form new hemoglobin. Globin from both horse and dog have been tested and seem to react in identical fashion. The globin radicle of hemoglobin appears to be an important limiting factor in abundant hemoglobin building in this type of anemia due to blood loss. Globin fed by mouth is well utilized to form new hemoglobin and we may record a 30 to 40 per cent utilization or a return of 30 to 40 gm. new hemoglobin from the feeding of 100 gm. globin. This is to be compared with the utilization of liver protein-an average return of 13 gm. new hemoglobin for the feeding of 100 gm. liver protein.
A low protein intake will cause limited hemoglobin production in standard anemic dogs. It appears that the dog on a limited protein intake is unable to produce the usual amount of globin and therefore of hemoglobin even in the presence of a large excess of iron. Iron given by mouth or by vein shows the same result-the dog made anemic by blood withdrawal cannot produce the expected new hemoglobin related to the iron intake when the protein intake is held at low levels. These dogs can be kept in perfect health and weight equilibrium during these long periods of limited diet intake and anemia. Under the stress of protein limitation the proteins of salmon muscle, banana and carrot are well utilized and it requires only 7 to 8 gm. of these food proteins to produce 1 gm. of new hemoglobin. These experiments show clearly that the iron content of liver is not wholly responsible for its potency in anemia due to hemorrhage.
Spontaneous glomerulonephritis develops not infrequently (11 per cent incidence) in the anemia colony. The course of the nephritis is insidious and usually extends over several years but ends in uremia, often with terminal bronchopneumonia. Hemoglobin production in these standard anemic dogs is well established as related to various standard food factors. These tests are summarized in the tables above to show the changes that appear year by year in the life of each dog. Nephritis causes little or no change in hemoglobin production in anemic dogs in the early stages of the disease. In the late stages of nephritis there may be no change or moderate changes in hemoglobin production in these anemic dogs. The average is 70 per cent of normal hemoglobin production in advanced nephritis. It seems unlikely that this degree of impairment of hemoglobin production in nephritis would result in spontaneous anemia in the dog.
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.
Copper added to a standard diet often effects a moderate increase in hemoglobin production in anemia due to blood loss. The copper response is quite irregular in contrast to the iron response. In these dogs there is no lack of copper held in reserve stores (liver and spleen) so the reaction is not related to an actual deficiency of the element. An effect upon enzyme complexes related to globin and hemoglobin production is to be considered. Cobalt under similar conditions causes no stimulus to hemoglobin production, rather an inhibitory effect when more than minimal doses are given. The claim that cobalt causes a polycythemia in dogs receives no support from our experiments.
Human liver tissue has been assayed to determine the amount of hemoglobin production factors in normal and abnormal states. Standardized dogs made anemic by blood removal have been used in this biological assay. Normal animal liver as control is rated as 100 per cent. Normal human liver tissue as compared with the normal animal control contains more of these hemoglobin production factors-a biological assay ratio of 120 to 160 per cent. Infections, acute and chronic, do not appear to modify these values, the concentration of hemoglobin-producing factors falling within the normal range. Pernicious anemia and aplastic anemia both show large liver stores of hemoglobin-producing factors-a biological assay ratio of 200 to 240 per cent. Therapy in pernicious anemia reduces these liver stores as new red cells are formed. Secondary anemia presents a low normal or subnormal liver store of hemoglobin-producing factors-an assay of 60 to 130 per cent. Hemochromatosis, erythroblastic anemia, and hemolytic icterus in spite of large iron deposits in the liver usually show a biological assay which is normal or close to normal. Polycythemia shows low reserve stores of hemoglobin-producing factors. Leukemias present a wide range of values discussed above. Hypoproteinemia almost always is associated with low reserve stores of hemoglobin-producing factors in the liver-biological assays of 60 to 80 per cent. Hypoproteinemia means a depletion of body protein reserve stores including the labile protein liver reserves-a strong indication that the prehemoglobin material (or globin) is related to these liver stores. Pregnancy, eclampsia, and lactation all may present subnormal liver stores of hemoglobin-producing factors. Exhaustion of protein stores lowers the barrier to infection and renders the liver very susceptible to many toxic substances. It should not be difficult to correct hypoproteinemia under these conditions and thus relieve the patient of a real hazard.
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.
The reactions to inoculation into the teat canal of approximately 100,000 million living Str. agalactiae contained in a volume of 10 ml. were a sharp systemic reaction that lasted two to three days, followed by streptococcal mastitis that was clinically, biochemically, and bacteriologically closely. similar to bovine streptococcal mastitis.
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.
Sterile abscess, pleuritis, and pancreatitis give a clinical reaction in the experimental animal very like the same acute inflammatory processes due to bacterial activity, provided the bacterial agents are limited to the initial location. The curve of urinary nitrogen excretion in the fasting dog shows the same precipitous and sustained rise in sterile and bacterial inflammatory reactions. This indicates that the same type of protein injury and autolysis in the body is produced by the sterile inflammatory reaction as by the bacterial reaction. It is assumed that the primary effect of the chemical agent or of the bacterial growth in the tissues is local cell injury or necrosis. This injured cell protoplasm undergoes prompt autolysis with escape of toxic protein split products. These toxic protein split products may be, in part at least, of the proteose group and are absorbed into the circulation, producing the familiar general reaction. The injury of body protein is obvious from the great increase in elimination of nitrogen in the urine and appears to be the same in sterile and in bacterial inflammation. The injurious agent in the sterile inflammation must be derived from the host protein, and we may assume with safety that much of the injurious material emanating from a septic inflammation must come from the host protein rather than from the bacteria. Acute sterile pancreatitis is one of the purest examples of an acute non-specific reaction where the intensity of the host's intoxication may reach a maximum in 12 to 24 hours. We believe that fundamentally this reaction is very similar to that observed after the production of a sterile abscess or pleurisy. Non-specific intoxication must account for the sterile reactions described above. Septic inflammations show the same acute reaction and injury of body protein. The deduction is obvious-that a great part, at least, of the reaction in septic inflammation is truly non-specific and results from the primary injury of the host's protein and cell autolysis.
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