Refractory sideropenic anemia in childhood due to an error of iron metabolism

Two cases of essential hypochronic anemia in childhood are reported. Oneof the cases which developed a mild Plummer-Vinson syndrome was completelyrefractory to peroral as well as intravenous iron therapy. In both cases the ironmetabolism has been studied by means of radioiron.

The following characteristics were found: The serum iron level was extremelylow but the iron binding capacity was normal. The absorption of iron from thealimentary tract was defective and intravenously injected radioiron was utilizedfor hemoglobin synthesis at a slow rate. There was a rapid plasma iron turnover.

The hypothesis is offered that the cause of the anemia resided in an abnormalityin the regulation of iron metabolism.

Submitted on March 9, 1955 Accepted on August 2, 1955     

Studies in iron transportation and metabolism; absorption of radioactive iron in patients with fever and with anemias of varied etiology

1. The isotope technic for studying iron absorption has been extended to measurethe unabsorbed isotope in feces as well as the amount synthesized into hemoglobin.The recovery of radioiron from feces was shown to be accurate within 10 per cent.

2. There was suggestive evidence to indicate that, with the 1 mg. per kilogramdose employed, normal subjects may sometimes absorb more iron than is convertedwithin a two week period into hemoglobin.

3. Patients with fever, untreated pernicious anemia, and refractory anemia wereshown to absorb more iron than they use for hemoglobin.

4. Patients with hemolytic anemia may absorb more iron than can be recoveredin the peripheral blood at any one time because isotopic hemoglobin is removedfrom the circulation at a rapid rate.

5. Except in afebrile patients with hypochromic anemia, acceptance of the percent of a given dose of radioiron which appears in circulating hemoglobin as ameasure of iron absorption must be made with caution.

6. The theory that mucosal cells accept iron for absorption or block its assimilation provides the best known explanation for iron absorption; but patients withadequate iron stores may assimilate considerable quantities of the metal and theblock must be regarded as relative.

     

Coexistent chronic lymphatic leukemia and polycythemia vera; morphologic and clinical studies with particular reference to unusual iron metabolism

1. A case of coexistenst chronic lymphatic leukemia and polycythemia verais described.

2. The source of the erythrocytosis was not apparent. Erythroid hypoplasiawas seen in the bone marrow, and there was no apparent extramedullary hematopoiesis in specimens of liver or spleen.

3. Plasma iron contenst was inordinately low, and this was confirmed by acorrespondingly high plasma iron-binding capacity. No stainable iron could bedemonstrated in the tissues examined.

4. The curve for plasma iron clearance was best satisfied by two componentsof different negative slopes. The more rapid had a half-time of 0.2 hours andprobably represented removal of iron for erythropoiesis. The slower had a half-time of 10.2 and probably represented extrahematopoietic iron utilization.

5. Erythrocyte radioiron utilization was greater than normal and approximated 100 per cent within nine days. This could have been a manifestation ofdepleted iron stores, accelerated erythropoiesis, or both.

Submitted on February 25, 1953 Accepted on May 28, 1953     

Hookworm Anemia: Iron Metabolism and Erythrokinetics

Iron metabolism, balance of red cell production and destruction and ironabsorption from hemoglobin were determined in 11 patients with heavy hookworm infection and severe anemia.

The plasma iron, total iron binding capacity, bone marrow hemosiderinand plasma Fe⁵⁹ clearance are in agreement with the idea that the anemia associated with hookworm infection is of the iron deficiency type.

The rate of red cell production measured by the E/M ratio, absolute reticulocyte count and plasma iron turnover showed an increase to about twicenormal, while the rate of destruction estimated by the T erythrocytesurvival showed a destruction about 5 times normal. This unbalance betweenproduction and destruction could explain the severity of the anemia.

The increase of fecal urobilinogen output to twice normal was interpretedas due to the metabolism of the hemoglobin lost into the intestine rather than toan increase of hemolysis.

The estimation of fecal blood loss in the patients whose red cells weretagged with Cr⁵¹ and Fe⁵⁹, showed that the radioactivity counted with Fe⁵⁹was only about 63 per cent of the radioactivity counted with Cr⁵¹. This difference was interpreted as due to iron absorption from the hemoglobin lostinto the intestine.

The mean daily fecal excretion of iron reaches 4.7 mg. Since the ironmetabolism in these patients is in equilibrium, we have concluded that theiron loss is replaced by the iron from food; this is in addition to the 3 mg.hemoglobin iron which is reabsorbed from the blood lost into the gut.

Submitted on January 9, 1961 Accepted on April 2, 1961     

Erythrokinetics: quantitative measurements of red cell production and destruction in normal subjects and patients with anemia

Red cell turnover of 19 normal subjects and 25 anemic patients was measuredwith the following technique: erythroid-myeloid ratio of the marrow, reticulocytecounts, plasma iron turnover, red cell utilization of radioiron, and urobilinogendeterminations. Measurements of blood production and destruction were so expressed as to allow comparison between normal and anemic individuals of different size and different red cell mass. The usefulness and disadvantages of eachprocedure in the study of anemia are discussed.

From studies of various types of anemia, it has become apparent that erythropoiesis must be defined in terms of total quantity of red cells produced and interms of the portion of red cells produced in the marrow which are delivered tothe circulating blood (effective versus ineffective erythropoiesis). A quantitativedefect alone exists when a normal ratio is maintained between effective andtotal erythropoiesis. Here, there are changes of similar magnitude of all erythrokinetic indices, although reticulocyte and urobilinogen values are occasionallydisproportionately high. The normal marrow appears to be able to increase itseffective red cell production to three times normal in acute anemia and six timesnormal in chronic anemia. In many disease states this maximal quantitativeresponse is impaired.

Dyspoiesis of the marrow is characterized by a dissociation of erythrokineticindices. Values which reflect total erythropoiesis (i.e., plasma iron turnover,fecal urobilinogen and erythroid-myeloid ratio of the marrow) are considerablygreater than the reticulocyte level and red cell utilization of radioiron whichrepresent effective erythropoiesis. Such defects may result in the pattern of ahemolytic process or aregenerative anemia, depending on their severity.

Submitted on October 26, 1955 Accepted on December 7, 1955     

The use of reticulocytes with high non-haem iron pool for studies of regulation of haem synthesis

Summary One-hour incubation of reticulocytes with 10^?2m isonicotinic acid hydrazide (INH) and transferrin-bound ⁵⁹Fe changes the normal distribution of radioiron inside the cell. About 10% of ⁵⁹Fe is found in haem and 90% is present in the non-haem iron pool. The accumulated non-haem radioiron may be utilized for haem synthesis. This is demonstrated by the reincubation of washed reticulocytes with a high non-haem radioiron pool induced by INH under optimal conditions. The incorporation of radioiron from intracellular non-haem pool into haem is used as a method for the estimation of the rate of haem synthesis in the presence of various inhibitors. INH reduces haem synthesis from non-haem iron to a greater extent than that from transferrin iron. On the other hand, haemin, which inhibits the incorporation of ⁵⁹Fe from transferrin into haem, does not significantly decrease the utilization of intracellular non-haem iron for haem synthesis. These results are considered as further evidence for the inhibitory effect of haem on the membrane transport of iron. Cells with an artificially increased non-haem iron pool incorporate more [2-¹⁴C]glycine into haem than normal reticulocytes. These results are in accordance with the possibility that the supply of iron to the critical sites of haem synthesis may be a limiting factor controlling the rate of haem synthesis.     

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     

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     

The polycythemia of high altitudes: iron metabolism and related aspects

Observations on the iron metabolism as related to the influence of a lowoxygen tension at high altitudes, and after the disappearance of this factorupon return to sea level, have been made in human subjects. They consistedmainly of studies of intestinal absorption and turnover rate of iron by meansof the radioactive isotope of this metal (Fe-⁵⁹). Additional observations weremade on blood volume, reticulocytosis, bone marrow cytology, life span ofthe red cells and hemoglobin breakdown pigments. The data obtained seemto justify the following conclusions:

1. There is an increase of intestinal iron absorption during the early periodof exposure to an altitude of 14,900 feet. After 48 hours of exposure, this wasestimated to be about 3 times higher than the absorption observed in subjectsat sea level and in native residents at the above-mentioned altitude.

2. There is an increase of plasma and red cell iron turnover rates after 2hours of arrival to 14,900 feet, indicating that the increase in the productionof red cells, to compensate for hypoxia, is a very early response.

3. The highest increase in plasma and red cell iron turnover rate takes place7 to 14 days after exposure to high altitude begins. After six months of exposure, there is still an elevated iron turnover rate. The native residents ofhigh altitudes (14,900 feet) have a red cell iron turnover rate of approximately30 per cent higher than healthy subjects at sea level.

4. A progressive decrease in the plasma and red cell iron turnover rate isobserved in native residents of high altitudes when brought down to sea level,the maximum of which is reached after two to five weeks, indicating a greatdegree of depression on red cell production. After that, a gradual return tonormal rate is observed in the weeks that follow.

5. The degree of reticulocytosis is in close relationship with changes in thered cell iron turnover rates.

6. Changes in the total blood volume, either during ascent or descent, takeplace only after several weeks. The red cell mass variations which occurduring the early periods of environmental change, are compensated by proportional changes in the plasma volume. The increase or decrease of the totalblood volume after this period is due exclusively to red cell mass modifications.

7. The bone marrow cytologic studies carried out in subjects temporarilyexposed or living permanently at high altitudes show a hyperplastic condition.The reverse, or an inhibition of red cell production, takes place when highaltitude polycythemic subjects are brought down to sea level. This constitutesthe cytologic counter-proof for the iron turnover studies.

8. The life span of the red blood cells, after descent from high altitudes tosea level, falls within normal patterns. However, by the method employed itis not possible to determine if there is an increased destruction of red cellsduring the first week. But if there is a greater destruction, this would be of asmall degree, affecting only the older elements. The increase in the hemoglobin breakdown pigments, which occurs under the influence of environmentalfactors, is also discussed.

9. In native residents of high altitudes the amount of free erythroprotoporphyrins is higher than in residents at sea level. The erythroprotoporphyrins innewcomers to high altitudes rise and reach a peak at the end of the secondmonth, followed by a gradual decline. On the other hand, when high altitudenatives are brought down to sea level, a marked decrease in erythroprotoporphyrins is noted. The rate of decrease is highest within the first months.

Submitted on June 11, 1958 Accepted on September 26, 1958     

OBSERVATIONS ON THE EFFECT OF MASSIVE DOSES OF IRON GIVEN INTRAVENOUSLY TO PATIENTS WITH HYPOCHROMIC ANEMIA

Massive doses of iron (from 0.608 to 1.32 Gm. as colloidal terric hydroxide orcolloidal ferric oxide) were given intravenously in single infusions to 8 differentpatients with hypochromic microcytic anemia. One patient was given a secondinjection after an interval of four months, so that nine administrations were made.The following observations were made:

1. The reticulocyte response was higher in each instance than would be expectedin oral therapy. In 3 additional patients in whom injection had to be discontinuedafter 0.070, 0.180, and 0.123 Gm. of elemental iron had respectively been given, thereticulocyte rises were higher than were the average responses reported by Heath¹⁸after optimal oral therapy. This at least suggests that "optimal" oral therapy doesnot provide a maximal stimulus to outpouring of reticulocytes from the bone marrow. Comparable doses of iron given to 3 control subjects with normal hemoglobinlevels did not cause a reticulocytosis.

2. The average rate of hemoglobin regeneration per 100 cc. of blood per day was0.224 Gm.; the lowest value was 0.16 Gm. and the highest 0.27 Gm. These figureswere calculated for the rise that occurred from the day of iron administration to thetime at which the rate of hemoglobin increase was obviously becoming slower.Since correction was not made for blood loss in 3 of the patients during the periodof regeneration, the figures for the rate of hemoglobin formation are lower thanthey otherwise would have been. Even so they are distinctly greater than thoseusually obtained following oral therapy (table 2), but no greater than is found in anoccasional patient given iron by mouth. The data suggest that the fastest rate ofhemoglobin regeneration that can be stimulated by iron in subjects with hypochromic anemia approximates 0.3 Gm. per 100 cc. per day.

3. Calculations indicated that from 71.8 to 99.7 per cent of the injected iron wasapparently used for the synthesis of hemoglobin. These figures are likewise lowerthan they would have been if several of the patients had not lost blood during therecovery period. The observation of other workers that parenterally administerediron is almost completely retained by the body and converted into hemoglobin wastherefore confirmed.

4. Toxic reactions to the injected iron are described in detail. They were severein all but two instances, and in 3 patients were so alarming that injection of ironhad to be discontinued. There can be no doubt that the reactions to iron parenterally administered in large doses are great enough to contra-indicate use of thismeasure as a therapeutic procedure.

     

Studies of Plasma Fe59 Disappearance--A Manifestation of Ineffective Erythropoiesis and of Hemolysis

1. Studies of the plasma radioiron disappearance data have revealed arelative "elevation" of plasma radioactivity after the second day in normalsubjects, and after the first day in patients with hemolytic anemia, megaloblastic anemia and thalassemia. This elevation in plasma radioactivity in normal subjects may be interpreted as due to the return to plasma of radioironfrom those cells that are "ineffectively" produced and are hemolyzed in themarrow. Other interpretations are also presented.

2. Dyspoieses involving ineffective red cell economy, but not detectably affecting iron economy are contrasted with disorders involving ineffectiveutilization of iron and of pigment alone.

3. After the first day plasma radioiron activity shows a diurnal variationsimilar to the diurnal variation in plasma iron concentration.

Submitted on July 17, 1964 Accepted on September 29, 1964     

Metabolism of Heterogenic Hemoglobins: Fe59 Incorporation in Sickle Cell Trait

The relative rates of incorporation of Fe⁵⁹ into heterogenic hemoglobinswas studied in four patients with sickle cell trait. Three of the patients werefree of superimposed disease, while one had active pulmonary tuberculosis.In all subjects there was a significantly greater incorporation of radioiron, permilligram of hemoglobin, into hemoglobin S than into hemoglobin A.

The data indicate that in sickle cell trait the rates of synthesis of theheterogenic hemoglobins are not proportional to their circulating concentrations. Two interpretations appear possible. Since the size of the intra-marrowpool of hemoglobin S was not known, it is possible that there exists a smallerpreformed pool of the abnormal hemoglobin, with the isotope making itsappearance first in hemoglobin S. However, it is also possible that hemoglobinS is synthesized at a rate which is greater than that reflected by its circulatingconcentration. This implies that the relative concentrations of hemoglobin Sand hemoglobin A vary from erythrocyte to erythrocyte, and that those cellswith the greatest proportion of hemoglobin S are selectively destroyed.

     

Absorption of iron as a problem in human physiology; a critical review

1. Evaluation of methods for determination of absorption or retention

It is felt by the present author, although agreement will not be universal, thatbalance studies constitute the standard by which the results of other methodsshould be judged. There are two ways in which iron balance has been determined.1. The older chemical method which determines the difference between the ironingested and that excreted in the feces. 2. The more recent method using radioactive iron which depends on determining the difference between the radioactivity ingested and that excreted in the feces. The chief drawbacks of the firstmethod are (a) the difficulty and now the expense of carrying it out, and (b) theopportunity for inadvertent errors especially in the determination of intake; itsadvantage lies in the possibility of continued and repeated periods of observation. In spite of some stated opinions, there is no reason to believe that themethods for determining iron usually used are not as accurate as those using theradioactive technique. The radioactive method has the advantage of greater easewith the knowledge that one does not have to consider the possibility of thepresence of traces of unaccounted iron; the chief disadvantage is the necessityto confine the determination to one observation in each subject. As a result therewould be greater scatter and consequent uncertainty.

A third method is that of inferring absorption from the amount of iron utilizedfor hemoglobin formation, depending on the assumption that if iron is not utilizedfor hemoglobin formation it will not be absorbed. Since this assumption is trueonly in limiited circumstances, the method based on it has only a limited application. About 15 or more years ago it was used extensively without proper control and as a result considerable error was introduced.

A fourth method, that of determining the curve of serum iron following a testdose given by mouth, does not pretend to determine actual absorption, but ratheris a method for comparison between groups. In the individual it is often used todetermine capacity to absorb. While a high post absorptive curve could onlymean good absorption a less high or flat curve does not necessarily mean a lessgood absorption unless it could be known that there was no diversion to thetissues. From a study of the rate of disappearance of intravenously injected ironit is concluded that differences in rate of diversion into the tissues is a factorthat cannot be disregarded.

2. Factors influencing the absorption of iron.

(a) Local factors. These factors, including such things as the effect of reducingagents, gastrointestinal acidity and motility, presence of phosphates, etc., werenot specifically reviewed, although information pertaining to them will be foundscattered through the review, and in the final section a few specific references torecent work have been included, especially where these might modify previouslyaccepted notions.

(b) General factors. 1. Diet. There is no section devoted particularly to diet,but a discussion of many of the dietary factors, especially those of importance inthe feeding of infants, will be found in section B and C. A fact that has beenknown for the past twenty years is confirmed by recent work, that inorganic ironsupplementation is a more effective way of increasing iron retention than givingiron in the form of vegetables. Supplementation with inorganic iron is often inadvertently accomplished in the commercial preparation of processed foods, sothat these foods may be a factor in effectively increasing the iron intake.

The most potent factor in increasing absorption of iron is iron deficiencyanemia. For a time it was generally believed that storage depletion was responsible for the result; while storage plethora caused reduction in absorption. Morerecently, however, the evidence has favored hemoglobin reduction rather thanstorage depletion as the responsible factor. This has been confirmed by recentwork indicating the importance of anoxia in mobilizing iron from ferritin storage.A third possibility, that of increased hemopoietic activity, has also beensuggested.

Whether or not there is actually such a thing as a "mucosal block" acting asa mechanism for excluding excess iron is not to be decided by any review. Therehave been found so many exceptions to any such mechanism that there hardlyseems any area of activity left except possibly in explaining the unimpeded absorption of iron in the presence of anemia, compared with the slower absorptionin the normal.

3. Pathways of distribution and , final disposition of absorbed iron.

Iron entering the body by absorption takes certain definite pathways whichinitially differ from those taken by injected iron. It enters by way of the mucosalcell in which its combination in ferritin is a form of temporary storage from whichit is passed on to the plasma at a rate that ordinarily does not cause increase inthe level of plasma iron. At such a rate it apparently is taken up almost entirelyby the bone marrow to be used to replace the small quota of iron derived fromdaily breakdown that ordinarily escapes the "hemoglobin cycle." When absorbedin amounts sufficient to raise the plasma iron level appreciably, or when hemopoietic activity is reduced, it is taken up by the liver where it is stored or reroutedto the bone marrow dependinig on the latter's capacity to use it. If hemopoieticactivity is raised, the absorbed iron seems to by-pass the liver completely(physiologically, not anatomically) and to go directly to the marrow, in spite ofa rise in plasma iron level that would ordinarily lead to a considerable depositionin non-hemopoietic tissue.

Intravenous iron is in general more rapidly and completely utilized for hemoglobin formation than is iron given by mouth even when this is adequatelyabsorbed. The reason for this may be in differences in the primary distributionof iron depending on the portal of entry. Iron absorbed by the gastroinstestinalroute in amounts larger than can be immediately utilized in hemoglobin formation is deposited predominantly in parenchymal liver cells, whereas intravenouslyinjected iron is deposited principally in reticuloendothelial cells, along with theiron derived from breakdown of hemoglobin. It is this latter iron that is generallyavailable for the day to day synthesis of hemoglobin. It is suggested that theiron deposited in liver parenchyma mainly in the form of ferritin, has a differentfunction, namely that of serving as a reserve, mobilized under conditions of anoxiaand specifically sensitive to reduction in level of hemoglobin.

Submitted on January 29, 1957 Accepted on July 16, 1957     

Ferrtin and ferruginous micelles in normal erythroblasts and hypochromic hypersideremic anemias

1. All normal erythroblasts contain some iron in the ferritin form. It maybe present in either a dispersed state or in compact clusters. When largeenough, these clusters may be observed in the optical microscope: they arethe granular particles of the siderocytes.

2. Iron can be found in the mitochondria. It may exist either in the formof ferritin granules or as ferruginous micelles.

3. In thalassemia, large quantities of iron accumulate in the erythroblastsand are even found in the erythrocytes as ferritin, in cluster formation ordispersed. Occasionally, iron is present in great quantities in the mitochondriaas ferritin or micelles. It seems that the various disorders encountered inthalassemia may thus be ascertained; the disturbance in hemoglobin synthesisresults in the accumulation of the unused iron in the hypochromic erythrocytes.

4. In diseases very similar to thalassemia and in which no fetal hemoglobinis found, i.e., the hypochromic-hypersideremic anemias (sidero-achestic anemia,hypochromic hypersideremic anemia, lead-poisoning ), similar findings areobserved.

5. Normally, it is probable that iron metabolism occurs in the mitochondria.In thalassemia and hypochromic hypersideremic anemias, on the other hand,iron metabolism often appears to be "blocked" in the same areas.

Submitted on June 19, 1958 Accepted on August 10, 1958     

A Direct Method of Determining Red Cell Lifespan Using Radioiron: An Application of the Occupancy Principle

Summary. Existing methods of measuring directly the lifespan of red cells are inconvenient or technically difficult. In the present study, plasma iron clearance and red cell utilization curves were followed after the intravenous injection of radioiron as for routine ferrokinetic measurements; chemical determinations of stable iron in plasma and red cells were also made. The mean red cell lifespan and the flow rate of iron into erythropoiesis were derived from these data by applying the occupancy principle (Orr & Gillespie, 1968).
Using this method, the red cell lifespan in two normal subjects was found to be 104 and 115 days respectively. Five patients with various haematological disorders were also studied; in one of these a normal lifespan of 107 days was obtained, and in four others there were shortened lifespans ranging from 25.8 to 49.6 days. In five of the subjects, the survival of in vitro ⁵¹Cr-labelled red cells was also determined. A good correlation was obtained between the red cell lifespan measured by the radio-iron occupancy method, and estimates of red cell survival using ⁵¹Cr-labelled red cells ( P < 0.05). The plasma iron flow rate derived from radioiron occupancy, and the conventional plasma iron transport rate, were also calculated and these are compared in relation to the clinical data.     

Urinary Iron Excretion and Renal Metabolism of Hemoglobin in Hemolytic Diseases

Quantitative and qualitative studies of urinary iron excretion were performed in 12 patients with hemolytic disease and in one normal subject givenan intravenous infusion of hemoglobin. In 9 patients with significant intravascular hemolysis, increased urinary excretion of nonhemoglobin iron wasobserved with amounts as high as 10.75 mg. pen 24 hours. In 7 of 8 patients inwhom fractions of the urinary iron were studied, the majority of the iron wasin the sediment (hemosiderin). Ferritin was demonstrated in the urine byimmunologic and electrophoretic technics and accounted for a significant percentage of iron excreted. In several patients, day-night variations in hemolysiswere associated with parallel fluctuations in iron excretion.

The results were analyzed in relation to current concepts of glomerularclearance and renal tubular metabolism of hemoglobin. The significance tobody iron balance of the massive "iron diuresis" occurring in some of thesepatients was discussed.

Submitted on January 21, 1966 Accepted on April 5, 1966     

Iron Metabolism in the Bone Marrow as Seen by Electron Microscopy: A Critical Review

High resolution electron microscopy has made possible the visualization oftransport and storage iron in the form of ferritin, both in dispersed form andin aggregates and in the form of "iron micelles" in mitochondria. Hemosiderinwas found to consist either of pure ferritin in crystalline clusters or, more frequently, of ferritin associated with other substances, including a lipid component in the form of myelinic figures and PAS positive material.

In the following paragraphs we have summarized the new morphologicfindings and what appears to us the most likely interpretation in the light ofknown biochemical and isotopic studies. Alternative interpretations have beendiscussed in the body of the paper.

Electron microscopy has established the erythroblastic island as a morphologic and functional unit of the bone marrow. A central reticular "nurse cell"appears to impart nutrients to surrounding rows of erythroblasts by the processof rhopheocytosis. Transfer of ferritin by this process is probably a passive phenomenon, since the amount transferred parallels the amount of iron present inthe central reticular cell. Ferritin is increased both in the reticular cell and inerythroblasts in hemochromatosis. It is absent in iron deficiency, althoughrhopheocytosis remains prominent. Normally all erythroblasts (proerythroblastsand normoblasts) and reticulocytes contain ferritin. Only the larger aggregatescan be visualized by the Prussian blue reaction in sideroblasts and siderocytes.Ferritin generally disappears when reticulocytes mature, even in hemochromatosis and infections, two conditions in which there is an excess of ferritin inerythroblasts. Interestingly, the increase in infections is entirely in form ofdispersed ferritin and cannot be visualized by the Prussian blue reaction; i.e.,sideroblasts are absent, in contrast to hemochromatosis where they are normal or increased.

It appears most likely that ferritin disappears from normal maturing reticulocytes because it is utilized for hemoglobin formation. It persists in maturered cells in Cooley’s anemia, hypersideremia, hypochromic anemia and leadpoisoning where hemoglobin formation is disturbed.

The origin of the ferritin in the nurse cells and the extent to which ferritinrather than siderophilin contributes to hemoglobin synthesis are unsolvedproblems. Isotopic studies indicate that almost all of the iron used for hemoglobin synthesis is derived from siderophilin and hemoglobin synthesis canproceed without any visible ferritin, as in iron deficiency anemia. These factsmust be reconciled with the electron microscopic observations which suggestthat normally some iron reutilization within the marrow proceeds by way oferythrophagocytosis, fragmentation, intracellular hemolysis of red cells, formation of ferritin and ropheocytosis. Iron derived from erythrophagocytosis elsewhere in the body probably reaches the marrow bound to siderophilin. Suchiron can be incorporated into ferritin of reticular cells as may be seen in hyperferremia and following injection of iron compounds. The process of rhopheocytosis would then lead to utilization of at least part of this ferritin iron forhemoglobin synthesis.

In certain pathologic states, accumulation of ferritin and related visibledispersed or conglomerated iron micelles may point to the sites where hemoglobin synthesis or iron transport is blocked. In Cooley’s anemia and the hypersideremic hypochromic (non-thalassemic) anemias, iron accumulates in themitochondria, which are known to be involved in hemoglobin synthesis. Inlead poisoning, the mitochondria are markedly abnormal, and probably correspond to the areas of punctate basophilia. However, the iron accumulatesin other areas of the cell, suggesting a different type of block.

     

Iron metabolism; the pathophysiology of iron storage

On the basis of experimental and clinical observations and a review of theliterature, a concept of the behavior of storage iron in relation to body iron metabolism has been formulated.

Storage iron is defined as tissue iron which is available for hemoglobin synthesiswhen the need arises. This iron is stored intracellularly in protein complex asferritin and hemosiderin. It would appear that wherever the cell is functionallyintact, such iron is available for general body needs.

Iron is transported by a globulin of the serum to and from the various tissuesof the body to satisfy their metabolism. Surplus iron carried by this iron-bindingprotein is deposited chiefly in the liver.

Storage iron may be increased in two ways. The first mechanism results fromthe inability of the body to excrete significant amounts of iron. Because of this,any decrease in circulating red cell iron (any anemia other than blood loss or irondeficiency anemia) is accompanied by a shift of iron to the tissue compartment.The total amount of body iron remains constant and is merely redistributed.

This is to be contrasted with the absolute increase in body iron and enlargediron stores which follow excessive iron absorption or parenteral iron administration. Enlarged iron stores in either instance may be evaluated by examination ofsternal marrow or determination of the serum iron and saturation of the ironbinding protein

In states of iron excess, differences in initial distribution are observed, dependingon the route of administration and type of iron compound employed. Iron absorbedfrom the gastro-intestinal tract and soluble iron salts injected in small amountsare transported by the iron-binding protein of the serum and stored predominantlyin the liver. Colloidal iron given intravenously is taken up by the reticulo-endothelial tissue. Erythrocytes appear to localize in greatest concentration in thespleen, while greater amounts of hemoglobin iron are found in the renal parenchyma. These latter differences in distribution reflect the capacity of various bodytissues to assimilate different iron compounds, which while present in the plasmaare not carried by the iron-binding protein.

Over a period of time an internal redistribution of iron from these various sitesoccurs through the serum iron compartment. The liver becomes progressivelyloaded with iron. When the capacity of the liver to store iron is exceeded, theserum iron increases and secondary tissue receptors begin to fill with iron. Thatiron in large amounts is toxic to tissues is suggested by the occurrence of fibrosisin the organs most heavily laden with iron. This sequence of events, whetherfollowing excessive iron absorption or parenteral iron administration is believedto be responsible for the clinical and pathologic picture of hemochromatosis.

     

Studies on the biosynthesis of heme in vitro by avian erythrocytes

1. A method, based on the uptake of radioiron into heme, is described for themeasurement of heme synthesis in a hemolysate of chicken erythrocytes.

2. The addition of glycine, -aminolevulinic acid, porphobilinogen or protoporphyrin 9 to the system augmented heme synthesis.

3. Citrate potentiated heme synthesis in the presence of glycine, but had noeffect when porphobilinogen was added. Succinate, in the presence of glycinedid not enhance heme synthesis.

4. The addition of coproporphyrin I or III, or uroporphyrin I or III, did notaugnment the uptake of radioiron into heme. The addition of mesoporphyrin 9or hematoporphyrin 9 enhanced the uptake of radioiron. These studies areiterpreted as suggesting that protoporphyrin, but not uroporphyrin or coproporphyrin, is a precursor of heme. For reasons discussed, further work will benecessary to determine if mesoporphyrin and hematoporphyrin are precursorsof protoheme.

5. The synthesis of heme was inhibited by the addition of malonate or lead.Evidence is presented that both of these substances affected heme synthesis atseveral different levels, particularly the formation of -aminolevulinic acid andthe incorporation of iron into protoporphyrin.

6. Plasma extracts from chickens, made anemic by bleeding or by the administration of phenylhydrazine, did not potentiate heme synthesis.

7. Normal, nonreticulated, human erythrocytes failed to synthesize heme inthe presence of glycine, -aminolevulinic acid, porphobilinogen on protoporphyrin 9.

Submitted on February 13, 1956 Accepted on April 15, 1956     

Evaluation of the Anemia of Rheumatoid Arthritis

Thirty-six patients with active rheumatoid arthritis in whom anemia wasslight were investigated, in part with radioiron and radiochromium, for abnormalities which might lead to anemia.

The primary defect appeared to be a pronounced reduction of abilityto absorb iron from the intestinal tract. There was an equally marked reduction in the serum concentration of iron which could not be attributedto the slight reduction in the iron-binding capacity of the serum. Despitethe reduction level of iron in the serum, the apparent turnover of iron wasso rapid that the total clearance per day was normal or moderately elevated.However, the fraction of the iron cleared from the blood and appearing inthe circulation in newly synthesized erythrocytes was slighfly depressed.Erythrocyte survival was normal or very slightly shortened; hemolysis seemsto play no important role. The fecal excretion of radioiron, not associatedwith erythrocytes, was greatly increased in two patients in whom there wasno evidence of intestinal bleeding. A moderate increase in plasma vohime wascommon.

Submitted on August 3, 1962 Accepted on November 3, 1962