Erythrokinetics in the Megaloblastic Anemia of Tropical Sprue

Blood production and destruction were measured in 10 patients with themegaloblastic anemia of tropical sprue. Methods employed included the determination of the erythroid/myeloid ratio of the marrow, plasma iron turnover, red cell utilization of Fe⁵⁹ and Cr⁵¹ red blood cell survival. Rates ofproduction and destruction were compared to normal.

Patients with the megaloblastic anemia of sprue were usually not irondeficient. Total bone marrow erythroid activity did not approach the maximal response seen in other hemolytic anemias, and there was a marked decreasein the delivery of erythrocytes to the peripheral blood. The rate of red bloodcell destruction was increased, but as the red cell volume decreased, the totalmass of erythrocytes destroyed per day varied from less than normal totwice normal. Bilirubinemia was not marked, because the amount of hemoglobin destroyed daily was usually not excessive and excretory function wasnot impaired. The severity of the anemia was largely related to the erythrocyte production defect.

     

Erythrokinetic Studies in Severe Bone Marrow Failure of Diverse Etiology

1. Erythropoiesis was studied in seven patients with refractory anemia.

2. In all seven patients the total red cell volume was low and the plasmavolume elevated.

3. The serum iron was elevated and iron binding protein saturated in sixof seven patients.

4. The T for clearance plasma Fe⁵⁹ was decreased in one, normal in two,and increased in four patients.

5. The red cell iron turnover was decreased in six of seven patients.

6. Radioiron accumulated in liver and spleen.

7. Red cell life span was difficult to measure but probably shortened.

8. Androgen therapy in two cases was ineffective, and was associated witha slight effect in one patient.

Submitted on April 18, 1961 Accepted on September 14, 1961     

The mechanism of the anemia associated with Hodgkin''s disease

1. The survival of autotransfused Cr⁵¹-tagged erythrocytes was shortenedin a group of five patients with the anemia of Hodgkin’s disease, but since thenormal marrow is believed to be capable of compensating for destruction ratesof six to eight times greater than normal, it is felt that the increased rate ofhemolysis was not sufficient to account for the development of anemia in anyof these patients.

2. There was evidence of an increased rate of erythropoiesis in all of thepatients with Hodgkin’s disease. This was manifested by normoblastic hyperplasia of the marrow, a moderate reticulocytosis and increased plasma ironclearance rates. The shortened red cell survival times were associated withthe increased plasma iron clearance rates. However, in view of the fact thatall of the patients were anemic, it is evident that these patients were incapableof increasing the rate of red cell production sufficiently to compensate for theincreased rate of destruction.

3. Intravenously injected Fe⁵⁹ citrate was incorporated more rapidly intothe circulating red cell mass in the patients with Hodgkin’s disease than inthe normal subjects.

4. The tissue iron stores of the liver and spleen were greatly increased,but no iron was demonstrated in the marrow of a group of seven patientswith Hodgkin’s disease.

5. The plasma iron was low and the UIBC normal in a group of 12 patientswith active Hodgkin’s disease.

6. It is suggested that one of the factors which limits the ability of themarrow to produce hemoglobin in the anemia of Hodgkin’s disease may be arelative hypoferremia caused by a defect in the mobilization of iron from tissuestores.

Submitted on April 17, 1958 Accepted on February 2, 1959     

Excess Hemolysis in Subjects with Severe Iron Deficiency Anemia Associated and Nonassociated with Hookworm Infection

An excess hemolysis was found in subjects with iron deficiency anemia associated with hookworm infection. Red cell survival, measured with Cr⁵¹ andDFP³² in the subjects before deworming, showed a marked disproportionbetween the decrease of the survival and the amount of daily intestinal bloodloss in most cases. Excess of hemolysis was still present after more than 90 percent of the parasites were removed. Red cell survival became normal aftercorrection of anemia through iron treatment. Excess of hemolysis was alsopresent in noninfected subjects with iron deficiency anemia due to othercauses.

The reduction in the survival of the erythrocytes from infected subjectstransfused into normal recipients shows that the hemolytic process is due toan intrinsic defect of the red cells. The low values of hemoglobinemia andthe presence of haptoglobins in the plasma indicate that hemoglobin has notbeen liberated in excess intravascularly. Finally, the fact that the red cellsfrom an infected patient taken after deworming survived normally in splenectomized recipients indicates that the spleen is probably the principal siteof the red cell destruction. The clinical and autopsy findings suggest thatsplenic function is not pathologically increased, but rather that this organ isacting physiologically at a more rapid rate, "culling" the abnormal circulatingred cells and thus leading to a decrease in red cell survival.

The studies presented here also indicate that the hookworm infection perse does not induce hemolysis.

Submitted on January 13, 1964 Accepted on April 24, 1964     

Radioactive Iron Studies in Patients with Iron Deficiency Anemia with Concurrent Abnormal Hemolysis

A report is presented on two women suffering from severe iron deficiencyanemia, concomitant with abnormal hemolysis. The life span of the patients’red cells was shortened; transfused normal red cells were more rapidlybroken down. Extracorpuscular factors seem to be responsible for the pathologic hemolysis. There was no pathologic sequestration of red cells labeledwith Cr⁵¹ in the spleen.

After incorporation of radioactive iron into the red cells, the utilizationpercentage of Fe⁵⁹ under iron therapy fell to about 15 per cent within a fewweeks. This, too, indicates that the pathologic hemolysis was to be ascribedto extracorpuscular factors. The Fe⁵⁹ was apparently not sufficiently reutilized.The constantly decreased serum iron concentration might also indicate adisturbance in the reutilization of iron liberated during red cell breakdown.

Six other patients with less severe iron deficiency anemia and an insufficientresponse to iron therapy were examined in addition. In 5 of these patients,the life span of red cells labeled with radioactive chromium was found shorterthan normal. An insufficient response to iron therapy in patients with chroniciron deficiency anemia may be ascribable, in some instances, to concomitantpathologic hemolysis.

     

Studies of the rate of production and life span of erythrocytes in acute leukemia

The rate of red cell formation and the red cell life span were determinedin six adult patients with acute myelogenous leukemia by the use of Fe⁵⁹,Cr⁵¹ and glycine-2-C¹⁴. One patient with acute monocytic leukemia wasstudied with Fe⁵⁹ alone.

Erythropoiesis was normal or increased in two patients, deficient in fourpatients and absent in one. The data indicate that in the cases of deficienterythropoiesis a decreased number of red cells were produced in the marrow, but that they were released at a rapid rate.

The red cell life span appeared to be normal in one patient, short but finitein four patients, and short owing to random destruction in one patient. Onestudy did not include the measurement of red cell life span.

The liver removed an abnormally large proportion of the plasma iron whenmarrow erythropoiesis was deficient.

The bone marrow examination may be an unreliable indicator of the rateof erythropoiesis.

Submitted on August 7, 1958 Accepted on February 21, 1959     

Anemia in Experimental Protein Deficiency in the Rhesus Monkey with Special Reference to Iron Metabolism

1. This investigation deals with a study of the anemia of protein deficiencyin Rhesus monkeys.

2. Protein deficiency was induced in 17 rhesus monkeys. Seven animals,given a protein-rich diet, served as controls. The diets of both the groupswere identical in all respects, except protein. All animals were tube-fed toensure adequate caloric intake.

Hematocrit, hemoglobin, erythroctye count, serum iron, serum iron binding capacity, plasma iron tolerance curves, and iron absorption using theFe⁵⁹ fecal recovery method were studied before and at intervals of theexperiment in both deficient and control groups.

Protein-deficient monkeys consistently developed normocytic normochromicanemia of moderate severity. A striking fall in serum iron binding capacity,total proteins and albumin with a rise in gamma globulin was observed inall deficient animals. A significant and comparable fall in serum iron wasalso observed. The Fe⁵⁹ absorption was depressed and there was flatteningof plasma iron tolerance curves. Two deficient animals, refed a high proteindiet, showed reversal of all these changes. The control animals did not showany of these changes.

The mechanism of anemia and decreased iron absorption observed in theprotein-deficient animals and the relevance of these findings to those inKwashiorkor are discussed.

Submitted on November 20, 1964 Accepted on January 25, 1965     

Erythrokinetics in Cooley''s anemia

Blood production and destructions have been measured in four patients withCooley’s anemia. Methods employed included the determination of erythroid/myeloid ratio of the marrow, reticulocyte count, plasma iron turnover and redcell utilization, Cr⁵¹ survival and fecal urobilinogen. Rates of production obtainedby these measurements have been compared to normal.

Patients with Cooley’s anemia have been shown to have an increased turnover of hemoglobin constituents comparable to the maximal response seen inother hemolytic anemias. There is, howvever, a marked decrease in maximaldelivery of erythrocytes to the peripheral blood amounting to about 50 per centin the mildly anemic patients and 85 per cent in severely anemic patients. Therate of destruction of circulating erythrocytes was similar in the three patientsstudied. The severity of anemia was therefore largely related to the productiondefect.

It was concluded that the defect in Cooley’s anemia is not in total hemoglobin synthesis, but in the fabrications of circulating erythrocytes, which inturn have the associated manifestations of hypochromia, increased percentageof fetal hemoglobin and shortened survival time.

Submitted on May 23, 1956 Accepted on June 25, 1956     

Kinetics of Intracellular Iron in Rabbit Reticulocytes

The kinetics of the intracellular iron were studied in an invitro systemusing reticulocyte-rich rabbit red cells incubated with Fe⁵⁹-labeled plasma.The incubation was stopped at intervals of a few seconds to 70 minutes,and the cells were fractionated by differential centrifugation and by chemicalmeans. In being selectively taken up by reticulocytes, Fe⁵⁹ is first associatedwith particulate fractions and then gradually released to the soluble cytoplasm, when the iron is incorporated into hemoglobin and into a transient nonhemoglobin protein phase. The entire process from the initial binding ofiron by cell surfaces receptors until the incorporation of iron into hemoglobinrequires only 6 to 8 minutes at 37 C. It is probable that in suitable concentrations, lead allows Fe⁵⁹ to accumulate in stroma but blocks its entry into thenonhemoglobin protein iron phase and into hemoglobin.

Submitted on February 3, 1959 Accepted on April 10, 1959     

Reutilization of Iron in Anemia Complicating Malignant Neoplasms

A study of reutilization of iron by means of Fe⁵⁹-tagged hemoglobin solution given to normal individuals yielded a rate of reutilization of 70 per centor more, evidence perhaps that the active storage pool of iron at a givenmoment is not larger than the plasma iron compartment. Reutilization of ironin patients with anemia complicating malignancy disease has shown a significant decrease which correlates well with the severity of the anemia.

Submitted on November 26, 1962 Accepted on February 10, 1963     

The Use of FE59 and CR51 for Estimating Red Cell Production and Destruction: An Interpretive Review

In surveying some of the limitations of studies with Cr⁵¹ and Fe⁵⁹ my purposehas been not to deny their usefulness but to put them in perspective. Thesetechnics have made possible many extensions of the fundamental understanding of red cell physiology and iron metabolism. They will continue to be valuable experimental tools. It is hoped that if some of the limitations of isotopetechnics are considered, the non-experimental use of these isotopes will beconfined to situations in which otherwise unavailable information of diagnosticor therapeutic importance can be obtained. Unfortunately isotope technicsare used when more conventional means would be adequate or even preferable. An extreme example is the suggestion²⁴ that repeated Fe⁵⁹ turnoverstudies might be used to determine the total dose of parenteral iron (as Imferonor saccharated iron oxide) to be given in iron deficiency anemia, pointing outthat in so doing the possibility of iatrogenic hemochromatosis could be avoided.The usual calculations for determining dose²⁵ however are not only safer butmore accurate.

The use of Fe⁵⁹ and Cr⁵¹ entails some risk, the main hazards being hepatitis,with the use of donor plasma or cells, and the possibility of untoward effectsfrom radiation. An estimate of the risk of hepatitis can be gained from itsincidence after transfusion. The radiation hazard is more difficult to assess.Leukemia has occurred after large doses of radiation but the extent of the radiation hazard is unknown from the much smaller doses of radiation employedin the usual isotopes studies. Certainly the risk is not such as to preclude theuse of isotopes to obtain information essential for diagnosis. However, whensuch information can be obtained by other means or when results cannot beadequately interpreted, the use of isotopes in clinical medicine appears unwarranted. In considering the use of isotopes in the doubtful case, the dose ofradiation to be delivered should not be thought of as an isolated event butrather as adding to a total radiation dose, which as shown by the British survey²⁸ may be appreciable.

Submitted on December 6, 1960 Accepted on April 3, 1961     

A Quantitative Method for Measuring the Gastrointestinal Absorption of Iron

A simple test is proposed for evaluating the gastrointestinal absorption of iron.It begins with an oral dose of 50 µg. of stable iron labeled with 1 µc. of Fe⁵⁹,to which about 300 mg. of ascorbic acid are added. Feces are assayed for Fe⁵⁹until any single stool contains less than 1 per cent of the dose. Absorption isdetermined on the assumption that all unrecovered Fe⁵⁹ has been absorbed.The test was designed to minimize handling of stools by assaying wholespecimens, and this process has the added virtue of increasing statistical accuracy of the measurement of radioactivity.

The main advantage of the procedure using a 50 µg. dose is that with it oneobtains a normal value for absorption which is about one-half of the dose.This permits measurement of either increased or decreased iron absorption.

With our standard test method, 19 normal women absorbed an averageof 62 per cent of the dose and 22 normal men absorbed 40 per cent.In addition to this sex difference, not explained by the difference in theirblood hemoglobin levels, there seemed to be a tendency toward reducedabsorption with greater age.

Preliminary results of this test in patients with hypochromic anemia andsteatorrhea are reported.

Increased absorption of iron followed the administration of ascorbic acidwhether the iron was in the ferric or the ferrous form. The concept that ferrousiron is better absorbed by man requires re-evaluation.

Submitted on March 2, 1959 Accepted on July 12, 1959     

The Influence of Amino Acids on Iron Absorption

A series of experiments were devised to determine the effect of variousamino acids upon the absorption of iron from the gastrointestinal tract.This was done by introducing single amino acids and Fe⁵⁹ into isolated loopsof small intestine and measuring the serum for radioactivity and the uptakeof Fe⁵⁹ by the liver. It was found that under the conditions of the experimentsall of the amino acids studied effected an increase in the serum iron and irondeposition in the liver.

Submitted on August 13, 1962 Accepted on December 27, 1962     

The Determination of Iron Absorption and Loss by Whole Body Counting

A technic for the study of radioiron absorption and loss is described employing an NaI (T1) crystal-detector whole body counter and 1-10 µc. Fe⁵⁹in 250 µg. elemental iron. Changes in whole body Fe⁵⁹ activity during thefirst few hours and the next 90-100 days after oral ingestion are describedand their significance discussed. Normal absorption with this technic rangesfrom 5.7-24.7 per cent of the administered tracer. In 14 patients with polycythemia vera, 12 previously phlebotomized and 2 with a recent history ofgastrointestinal hemorrhage, iron deficiency as evidenced by increased ironabsorption (20.6 per cent-96.9 per cent) correlates well with the extent ofpreceding phlebotomy, and relatively well with the plasma iron at the timeof study. Although other parameters reflect iron deficiency, none correlatewell with the absorption of radioiron. Next to increased iron absorption, depletion of iron stores in the marrow seems to be the earliest evidence of irondeficiency.

Iron absorption and erythrocyte incorporation of radioiron was also studiedin several other hematologic disorders, including four heavily menstruatingwomen, three cases of aplastic anemia, and a small number of other conditions.The findings are described and discussed.

Radioiron loss in three normal patients was 0.110 per cent, 0.110 per cent,and 0.182 per cent daily, and in two patients with aplastic anemia 0.103 percent and 0.173 per cent daily, defining the normal range of tracer loss overdays 20-100. Radioiron loss in the polycythemics ranged from 0-0.044 per centdaily. An unusual case of pyridoxine-responsive anemia with increased absorption of radioiron (69.1 per cent), but no red cell incorporation, lost only0.026 per cent/day. Some problems in the interpretation of such data arediscussed.

The results demonstrate the effectiveness of the technic of whole bodycounting in the study of various aspects of iron metabolism.

Submitted on December 26, 1961 Accepted on July 21, 1962     

Influence of Several Hormones on Erythropoiesis and Oxygen Consumption in the Hypophysectomized Rat

Adrenocorticotrophic hormone (ACTH), thyroid stimulating hormone(TSH), adrenocortical extract (ACE), hydrocortisone (F), corticosterone (B),11-dehydrocorticosterone and 3,5,3'-triiodothyronine (T-3) induced a moderate to marked erythropoietic effect in the hypophysectomized rat as indicatedby an increase in both Fe⁵⁹ incorporation in RBC and total circulating red cellvolume. A corresponding increase in oxygen consumption was also observed.Angiotensin increased red cell volume and radioactive iron incorporation inRBC of hypophysectomized rats and stimulated Fe⁵⁹ incorporation in hypophysectomized-adrenalectomized rats, but did not exert a significant effecton oxygen consumption. Cobalt injections resulted in a significant increase inred cell volume and Fe⁵⁹ incorporation in RBC of hypophysectomized rats,but produced a significant decrease in oxygen consumption. The significanceof these findings is discussed.

Submitted on April 3, 1961 Accepted on February 2, 1962     

The Significance of Iron Turnover in the Control of Iron Absorption

Following acute blood loss, there is a 4- to 5-day lag before any demonstrable change in plasma iron kinetics. Then there is a shortened Fe⁵⁹T and transient fall in the plasma iron. Associated with this is a decreasein the intestinal content of iron and an increased absorption of iron. We suggest the iron content of the epithelial cell is important in the regulation ofits ability to absorb iron. The intestinal iron content is in turn closely controlled by the plasma iron turnover.

Submitted on October 7, 1963 Accepted on December 3, 1963     

Abnormal Human Hemoglobins. I. Biosynthesis in Vitro of Fe59-Labeled Human Hemoglobin A and Hemoglobin S

The synthesis of Fe⁵⁹ labeled-hemoglobin during incubation of bone marrow from normal and sickle cell anemia (crisis and non-crisis) patients hasbeen investigated. The in vitro synthesis of hemoglobin S in the steadystate (noncrisis) sickle cell anemia individual was found to proceed ata much faster rate than the synthesis of hemoglobin A from normal individuals. In the crisis of sickle cell anemia, the uptake of Fe⁵⁹ and synthesis ofhemogobin S was approximately at the same level as synthesis of hemoglobin A from the normal individual. The results of this study indicatethat one of the mechanisms involved in sickle cell anemia crisis is a decrease in synthesis of hemoglobin S as compared to the synthesis of hemoglobin S during the steady state of noncrisis.

Submitted on November 20, 1958 Accepted on March 10, 1959     

The effect of acute inflammation on the utilization and distribution of transferrin-bound and erythrocyte radioiron

1. In rats with acute turpentine-induced inflammation, there was a reducedreutilization of radioiron from transfused senescent erythrocytes but a normalutilization of transferrin-bound Fe⁵⁹ after a 40-hour period. There was a pronounced retention of tracer from the nonviable red cells by the livers andspleens of the inflamed animals.

2. During inflammation, the plasma iron turnover fell by about 50 per cent,while the fraction of plasma iron removed per hour was increased. However,there was no marked change in the relative distribution of transferrin-boundFe⁵⁹ to the liver, spleen and bone marrow (after perfusion). Transferrin-boundFe⁵⁹ initially appeared at an increased rate in the circulating red cell mass.

3. Following administration of ferric ammonium citrate in order to raise theplasma iron level, there was a rise in the plasma iron turnover of the inflamedrats, in contrast to the control animals. Diversion of radioiron to the liver andspleen was not markedly increased under these conditions.

4. It is concluded that the immediate fall in plasma iron turnover andhypoferremia during acute turpentine inflammation results mainly from aninhibition of the release of iron derived from senescent red cells into theplasma. An increased avidity of the liver, and of marrow red cell precursorsand/or reticulocytes for plasma iron may accentuate the fall in plasma ironlevels. There appeared to be no inhibition of the bone marrow capacity to turnover larger amounts of plasma iron during inflammation. These results mayhelp in the interpretation of disturbances of iron metabolism during the acuteinflammatory state.

Submitted on July 9, 1965 Accepted on April 4, 1966     

Studies in Iron Metabolism. IV. Iron Absorption in Experimental Iron Deficiency

1) Whole body counting by means of a large phosphor well scintillationcounter has been used to measure the absorption of Fe⁵⁹-tagged inorganic iron,and shown to compare favourably with other methods.

2) There is a delay in the fecal elimination of the unabsorbed portion ofthe dose of Fe⁵⁹ by iron-deficient rats on iron-deficient diet. The cause of thisdelay is unknown but it may be associated with the marked cecal enlargementwhich exists in these animals.

3) It is confirmed that iron deficiency is associated with striking enhancement of absorption of ferrous and ferric inorganic iron.

4) When a series of doses of ferrous iron of increasing size from 5 to 1,000µg. was given, there was a progressive increase in absorption for each increasein dose in both iron-supplemented and iron-deficient rats. The relationshipbetween amount of iron given and amount absorbed suggests that two processesmay be involved: 1) simple diffusion, and 2) a carrier mechanism.

5) The effect on iron absorption of a sudden change in iron intake hasbeen investigated. Switch from a low to high iron diet reduces absorption,and from a high to a low iron diet increases absorption, too rapidily for hemoglobin level or body iron stores alone to be the most important governingfactors and this finding emphasizes the importance of local changes in theintestine.

Submitted on April 23, 1962 Accepted on June 25, 1962     

Studies on the pathogenesis of splenic anemia

Splenomegaly was produced in rats by repeated intraperitoneal injections ofmethylcellulose. Anemia and marked reticulocytosis resulted. Coombs tests werenegative.

Comparative measurements of red cell life with Cr⁵¹ in normal, methyl-cellulose treated, and splenectomized animals showed a marked decrease of red cellsurvival in the methylcellulose rats. Slight but statistically insignificant increasedsurvival could be demonstrated in the splenectomized animals.

Excessive red cell destruction occurred in the spleen as evidenced by rapidlocalization of Cr⁵¹-tagged cells in this organ. Direct correlation between severityof hemolysis and splenic size could be demonstrated. The hemolytic processdecreased significantly after splenectomy.

Affected spleens showed red cell congestion of the pulp microscopically. Hemochromogen determinations revealed approximately twice the normal amount ofhemoglobin per unit splenic mass.

Quantitative measurements of splenic phagocytosis showed a marked increasesof total phagocytic mass with no significant increase of phagocytes per unit ofspleen mass.

The mechanism of splenic anemia in these animals is thought to be a combination of an enlarged pulp compartment with resultant stasis and cell destruction.

Methylcellulose-induced splenomegaly may be considered as an experimentalmodel for the study of the hyperfunctional spleen in human disease.

Submitted on March 28, 1956 Accepted on June 17, 1956