Increased Formation of Early-Labeled Bilirubin in Rats with Iron Deficiency Anemia: Evidence for Ineffective Erythropoiesis

The production of early-labeled bilirubin and erythrocyte hemoglobin hemewas measured in rats with iron deficiency anemia, using glycine-2-¹⁴C asprecursor. The erythropoietic component of the early pigment fraction wassignificantly augmented and the formation of labeled hemoglobin depressed inthe anemic animals, findings characteristic of ineffective erythropoiesis. Bycontrast, the hepatic component of early-labeled bilirubin was substantiallyenlarged during the acute response to iron therapy. These experiments illustrate that overproduction of bilirubin may originate from both erythropoieticand hepatic sources of the early-labeled fraction of bile pigment, as well asfrom hemolysis of circulating red blood cells.

Submitted on November 14, 1968 Accepted on January 21, 1969     

Macrocytosis Resulting from Early Denucleation of Erythroid Precursors

In an attempt to elucidate the mechanism of production of macrocytosis inacute anemia, we studied changes in red cell volume and hemoglobin content, the RNA level of normoblasts and reticulocytes and RNA synthesis inreticulocytes of rabbits made anemic by blood loss or phenylhydrazine administration. The results were as follows:

(1) In severe phenylhydrazine anemia, red cell volume and hemoglobincontent per cell increased to twice the normal values.

(2) The RNA level of normoblasts decreases with the maturation of thecells and reaches a minimum at the orthochromatic stage. The decrease issimilar in the normal and anemic rabbits.

(3) The RNA level of reticulocytes in the bone marrow is higher in anemicthan in normal rabbits. In general, the RNA level of reticulocytes of anemicrabbits is comparable to that of the polychromatic normoblasts, while in normal rabbits this value is comparable to that of orthochromatic normoblasts.

(4) Autoradiographs of reticulocytes incubated with H³-uridine indicatethat the increased level of reticulocyte RNA of anemic rabbits is not due tonewly synthesized RNA.

From these results, we conclude that in an "emergency" situation of erythropoietic stimulation denucleation of normoblasts occurs at the polychromaticstage of red cell maturation, with skipping of the terminal cell division toorthochromatic cells and formation of macrocytic reticulocytes and red cells.

Submitted on August 28, 1963 Accepted on April 30, 1964     

Mitotic Indices of Human Bone Marrow Cells. III. Duration of Some Phases of Erythrocytic and Granulocytic Proliferation Computed from Mitotic Indices

1. Weighted average mitotic time of human bone marrow cells is estimated.Mitotic time is about 0.75 hours in red cell precursors and about 0.58 hoursin neutrophil precursors.

2. Estimated weighted average DNA-synthesis time is about 9 hours inred cell precursors and about 24 hours in neutrophil precursors.

3. Upper and lower limits for compartment transit times of proerythroblasts, basophilic normoblasts, polychromatic normoblasts, myeloblasts, promyelocytes and myelocytes are presented (table 2).

4. Total mass of nucleated red cell precursors is estimated at 3.5 x 10⁹/kg.and total mass of neutrophils and their precursors in the marrow at 8.8 x10⁹/kg.

Submitted on October 26, 1963 Accepted on January 24, 1964     

Inclusions of Hemoglobin in Erythroblasts and Erythrocytes of Thalassemia

Large, usually single inclusions, having the staining properties of Heinz-bodies and occurring spontaneously in thalassemic cells, are described. Theirfrequency is greatest in the normoblasts, either circulating ones or those foundin the bone marrow, and decreases sharply in more mature red cells. Splenectomy unmasks this inclusion phenomenon to a large extent. Some stainingand other properties of these inclusion bodies are described.

It is concluded that these inclusions represent precipitated hemoglobin,very likely uncombined -chains. The findings are discussed and related topresent-day knowledge on hemoglobin synthesis and erythrocyte turnover inthalassemia.

Submitted on July 23, 1962     

Erythropoietic (congenital) porphyria: A rare abnormality of the normoblasts

1. In 5 patients with erythropoietic (congenital) porphyria, unstained bonemarrow preparations were studied and photographed by fluorescence andabsorption microscopy. The same marrow slides were also studied after ordinarystaining.

2. Two morphologically different varieties of normoblasts were observed,which were designated as normal and abnormal cells. Normoblasts of theabnormal variety exhibited nuclear inclusion bodies containing hemoglobin.

3. Red fluorescence, indicative of porphyrin, was found only in normoblastsbelonging to the abnormal cell variety. Normal normoblasts failed to exhibit redfluorescence.

4. The red fluorescence originated predominantly from the normoblasticnucleus. The fluorescence intensity of the cytoplasm was usually very low.Polychromatophilic erythrocytes also exhibited only weak fluorescence.

5. These findings indicate the coexistence of two different lines of normoblasts.The nuclei of cells belonging to the abnormal line probably form excessiveamounts of porphyrins and may release them into the plasma in the course ofcellular maturation. It is believed that these cells carry the abnormal traitrepresenting the "inborn error of metabolism."

6. A critical review of all cases of porphyria, reported as "congenital," hasshown that on the basis of the presented data, it was possible to establish thediagnosis of erythropoietic (congenital) porphyria beyond reasonable doubtin but 34 instances. Nineteen of the patients were female, fourteen were male.In a few families, more than one case was observed in the same generation, butin no instance was the disease recognized in subsequent generations.

7. Erythropoietic porphyria is a congenital disease, entirely distinct fromhepatic photosensitive ("cutanea tarda") porphyria. No evidence has beenfound of a genetic mixture of these two forms of porphyria.

Submitted on July 8, 1954 Accepted on November 3, 1954     

Congenital hemolytic jaundice; the pathogenesis of the hemolytic crisis

Six cases of congenital hemolytic jaundice with "hemolytic" crises are reported. It is demonstrated that during the development of the anemia an acuteaplastic condition is present in the erythropoietic tissue of the bone marrow withcomplete cessation of the formation of red cells. The reticulocytes disappear fromthe blood; jaundice, serum bilirubin and urobilinuria decrease to normal values;and the serum iron increases. This period is further characterized by leukopeniaand thrombocytopenia.

The spontaneous recovery is caused by a rapid regeneration of the erythropoietictissue resulting in a marked reticulocytosis in the peripheral blood, and there isalso leukocytosis, an increase in thrombocytes and a rapid fall of serum iron.

During the period of severe anemia an increase in the blood urea and uric acidoccurs.

Transfusion experiments revealed an average lifetime of approximately fifteendays for the red cells in congenital hemolytic jaundice, a fact which fully explainsthe development and symptoms of the crisis as a result of cessation in the formation of red cells.

The findings definitely contradict the theory that an acute increase in thehemolytic process is the reason for the crisis.

The crisis should be called aplastic and not hemolytic.

     

Chromatin Clumping in Mature Leukocytes: A Hitherto Unrecognized Abnormality

A 62-year-old man and a 73-year-old woman were found to have a nuclearabnormality characterized by exaggerated clumping of the chromatin in themature granulocytic and erythroid cells. The mature granulocytes showed lossof normal segmentation. The abnormality was not noted in other familymembers and appeared to be related to an atypical leukemic process. Thenuclear abnormality was shown to be associated with a defect in cellular production within the marrow. Granulocyte precursors and normoblasts incorporated ³H-TdR but did not divide. Electron microscopy suggested thedistribution of more than normal quantities of heterochromatin to matureleukocytes. Ferrokinetic studies disclosed ineffective erythropoiesis. The sharing of this abnormality by the myeloid and erythroid cells suggests a commonstem cell origin.

A functional defect of phagocytosis and cellular aggregation was demonstrated in the neutrophils, but bacterial killing was not affected. The nuclearanomaly described herein may be a pre-leukemic abnormality.

Since this paper was submitted for publication, we have treated two morepatients with an identical nuclear defect; an 82-year-old woman and a 79-year-old man. The woman succumbed of acute leukemic transformation, while theman is still alive.

     

The life span of red cells in the rat and the mouse as determined by labeling with DFP32 in vivo

The red cells of rats and mice were tagged in vivo by injection of diisopropylphosphorofluoridate (DFP), labeled with P³², and the disappearanceof radioactivity from the circulating red cells was determined. From the dataobtained, it is concluded that the disappearance of the red cells is linear withtime and that the red cells in both rats and mice have a true life span withoutmeasurable random destruction.

The life span of the erythrocytes was found to be 60.0 ± 3.2 (S.D.) daysin the rat and 40.7 ± 1.9 (S.D.) days in the mouse.

Especially in the mouse the determination of the red cell life span withDFP³² is a simple procedure.

Submitted on November 20, 1957 Accepted on February 15, 1958     

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 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.

     

dgr-Aminolevulinic Acid Synthetase Activity in Human Plasma: Relation to Erythropoiesis and Evidence of Induction in Erythropoietic Porphyria

Measurement of -aminolevulinic acidsynthetase (ALA-S) activity in humanplasma has been carried out with samples from normal individuals, cases oferythropoietic porphyria (EP) and erythropoietic protoporphyria (EPP), and ofthe three principal forms of hepatic porphyria—acute intermittent porphyria,variegate porphyria, and porphyria cutanea tarda. The method of measurementdepends on formation of ¹⁴C-ALA whenthe plasma is incubated with ¹⁴C-succinicacid, succinyl-Co A synthetase, glycine,and other essential substances. The normal samples, as well as those from thehepatic porphyria cases, had small butsignificant activity of the same extent;those from the erythropoietic groupshowed consistently higher values, especially in the two cases of congenital type.A remarkably high value in one of thesecases in which there was outspoken erythropoiesis was believed to be related tothe presence of many fluorescing normoblasts in the peripheral blood. Followingmultiple transfusions these disappearedconcomitantly with striking reductionof the porphyria. The plasma ALA-Sactivity declined to 1.4% of the pretransfusion value. These results are consideredin respect to the question of induction ofALA-S in the developing red cells of thedisease, special attention being given tothe minor increase of ALA-S activity inthe plasma of a nonporphyric individualwhose peripheral blood contained largenumbers of circulating normoblasts.

Submitted on May 11, 1971 Revised on July 19, 1971 Accepted on July 20, 1971     

Studies on the pancytopenia of kala-azar

1. The peripheral blood changes in uncomplicated kala-azar are those ofpancytopenia; namely, anemia, leukopenia and thrombocytopenia. The red bloodcell morphology is normal and there is very little evidence of increased erythrogenicactivity. The leukopenia is due to a reduction in all types of cells, especiallyneutrophils.

2. When the disease is complicated by other infections the anemia is moresevere and anisocytosis, poikilocytosis, and polychromatophilia may appearand normoblasts may occasionally be seen in the peripheral blood. Leukocytosismay develop, the leukopenia may persist or the syndrome of agranulocytosis mayintervene.

3. As the duration of the disease increases, the spleen tends to become largerand the anemia, leukopenia and thrombocytopenia become progressively moresevere. Leukopenia generally appears first, followed by anemia and finally thrombocytopenia. The degree of leukopenia, anemia and thrombocytopenia followclosely the degree of splenic enlargement.

4. The bone marrow in kala-azar is hyperplastic and infiltrated by reticulo-endothelial cells. In spite of this there appears to be an abundance of blood formingtissue, especially erythropoietic tissue.

5. Differential cell studies on preparations of sternal marrow reveal a markedreduction in the polymorphonuclear neutrophils and eosinophils. Myeloblasts,promyelocytes, neutrophilic myelocytes and metamyelocytes are present in approximately normal proportions as are the lymphocytes and monocytes. Plasmacells are somewhat increased. Erythroid cells, especially polychromatic normoblasts are numerous and the leukocyte-erythroid ratio is altered, more than thenormal proportion of normoblasts being found. Megakaryocytes are present innormal or slightly reduced numbers. Staining abnormalities are noted in thesecells and there is a striking reduction in platelet production.

6. During effective anti-leishmanial therapy parasitized reticulo-endothelialcells disappear and the percentage of reticulo-endothelial cells gradually diminishes as the polymorphonuclear neutrophils increase. There is a significantincrease in the eosinophilic cells. Lymphocytes become more numerous and theplasma cells diminish in number. Nucleated red cells become less numerous andthe leukocyte-erythroid ratio returns to normal. At the same time the proportionof orthochromatic normoblasts to polychromatic normoblasts increases. Therelative number of megakaryocytes increases and platelet formation from themegakaryocytes is accelerated even beyond the normal. Huge groups of plateletsare frequently seen in the marrow smears. A rise in platelets in the peripheral bloodtakes place late, after there has been a significant rise in hemoglobin and leukocytes. The three cellular elements are restored to normal in the peripheral blood inthe same order as their reduction from normal.

7. Evidence is presented which contradicts the view that the pancytopenia isdue to a crowding out of the bone marrow by reticulo-endothelial cells.

8. Certain similarities between the hematologic changes in this disease andthose accompanying the hypersplenic syndromes are noted.

     

Quantitative Recovery of 14C-labeled Carbon Monoxide (14CO) From Viable Heme-labeled Red Blood Cells in the Rat

Long-term recovery of ¹⁴C-labeled carbon monoxide (¹⁴CO) from labeled,transfused red blood cells (RBC) wasstudied in buffalo rats. Donor RBC(labeled with ¹⁴C-2-glycine) weretransfused into host rats, and the ¹⁴COformed from degradation of labeledhemoglobin heme was collected overthe next 110+ days. The heme-equivalent ¹⁴CO recovery in 13 animalsaveraged 102.1 ± 2.1% (mean ± SE)of activity in hemoglobin heme ofdonor RBC. This confirms that hemeof circulating RBC destroyed by random hemolysis and senescence isquantitatively converted to CO.

Submitted on December 14, 1971 Revised on February 23, 1972 Accepted on February 26, 1972     

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     

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     

Hemoglobin Hope: A Beta Chain Variant

A new hemoglobin, hemoglobin Hope, with a beta chain abnormality hasbeen found in three generations of a St. Louis Negro family. The abnormalhemoglobin in the heterozygous state caused neither clinical stigmata norabnormality in the red blood cells. Hemoglobin Hope was detected by agargel electrophoresis at pH 6.2, but could not be differentiated from hemoglobinA by starch block electrophoresis at pH 8.6. Also, it could not be separatedfrom hemoglobin A by paper, or starch gel electrophoresis employing a rangeof buffers from pH 6.2 to 8.6. Amino acid analysis showed that aspartic acidwas substituted for glycine at position 136 of the beta chain. HemoglobinHope may be formulated as ₂^A₂^{136 gly-asp}.

Submitted on March 30, 1964 Accepted on September 30, 1964     

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     

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     

Role of bilirubin overproduction in revealing Gilbert's syndrome: is dyserythropoiesis an important factor?

Gilbert's syndrome was diagnosed in 37 patients with unconjugated hyperbilirubinaemia without overt haemolysis or structural liver abnormality, who had a marked reduction in hepatic bilirubin UDP-glucuronosyltransferase activity (B-GTA) (as compared with that of 23 normal subjects). No significant correlation existed in these patients between serum bilirubin level and the values of B-GTA, thus suggesting that factors other than a low B-GTA must influence the degree of hyperbilirubinaemia in Gilbert's syndrome. Studies of 51Cr erythrocyte survival and 59Fe kinetics in 10 unselected patients demonstrated slight haemolysis in eight, whereas mild ineffective erythropoiesis was suggested in all from a low 24-hour incorporation of radioactive iron into circulating red cells. This overproduction of bilirubin resulting from mild haemolysis and perhaps dyserythropoiesis might reflect only an extreme degree of the normal situation. It certainly contributes to the hyperbilirubinaemia of Gilbert's syndrome and may play a major role in the manifestation of this condition.     

Congenital Erythropoietic Porphyria. II. The Effects of Induced Polycythemia

Polycythemia and anemia were inducedin a patient with congenital erythropoietic porphyria as a possible means ofaltering erythropoiesis and its attendantporphyrin production. Maintenance ofhematocrits at 50 per cent and 60 percent for periods of 2 weeks decreasedboth erythropoiesis and porphyrin excretion to about one-half of their initiallevels. Conversely with a hematocrit of25, erythropoiesis and porphyrin production were increased to about twice thebasal level. Examination of the marrowshowed fluorescence in all late normoblasts and indicated that the patient hasonly one red cell population. During induced polycythemia, a shift in marrowerythroid population to relatively matureforms, apparently related to delayedenucleation, was accompanied by a retention of the reticulocyte pool andfluorescent cells within the marrow. During anemia, the occurrence of relativelymore immature erythroid cells was accompanied by a shift of the reticulocytepool and fluorescent cells into the circulating blood. During polycythemia, increases were observed in the relativenumber of nuclear vacuoles peculiar tothis disease, in the retention of radioironin the marrow and in the relative amountof stercobilin excretion. In anemia theseparameters decreased towards normal.

Submitted on June 30, 1969 Revised on March 16, 1970 Accepted on April 1, 1970