A Comparison of the Behavior of 111In and 59Fe-labeled Transferrin on Incubation With Human and Rat Reticulocytes

The uptake by human and rat reticulocytesof ¹¹¹In and ⁵⁹Fe bound to transferrin hasbeen studied. The results indicate a significant difference between the behaviorof the two isotopes in both human and ratincubation mixtures. Reticulocyte uptakeof ¹¹¹In from human and rat serum was30% and 12% that of the ⁵⁹Fe after a30-min incubation. The process was temperature dependent, inhibited by sodiumarsenite, and related to the reticulocytepercentage of the cells in the reactionmixture. Washed reticulocytes, previouslyincubated for 30 min with either ⁵⁹Fe or¹¹¹In bound to serum were reincubated inunlabeled serum. Up to 85% of the ¹¹¹Inlabel and less than 10% of the ⁵⁹Fe on thereticulocytes were released on reincubation, indicating that, in contrast to ⁵⁹Fe,the majority of the ¹¹¹In label remainedmembrane bound. Specific binding of unlabeled In transferrin was demonstratedby its inhibitory effect on ⁵⁹Fe-transferrinuptake by rat reticulocytes. Both In- andFe-transferrin were found to have similarbinding affinities for the receptor sites.The ¹¹¹In remaining in lysates obtainedfrom washed reticulocytes after incubation with ¹¹¹In-labeled serum and reincubation in unlabeled serum did not appearto be associated with either the hemoglobin or heme molecules.

Submitted on October 2, 1973 Revised on October 28, 1973 Accepted on October 30, 1973     

The Metabolism of Transferrin-bound 111In and 59Fe in the Rat

The metabolism of transferrin-bound indium and iron were compared by in vivostudies in the rat. Rats were injected withserum transferrin labeled with ¹¹¹In and⁵⁹Fe, and, at intervals ranging from 20min to 5 days after injection, they werekilled. They were perfused through theportal vein with 200 ml of 0.9% saline,and the residual radioactivity, expressedas a percentage of the injected dose, wasmeasured in liver, spleen, kidney, muscle,washed red cells, red marrow, and femur.At 5 days, 76% of the injected ⁵⁹Fe wasrecovered in the red cell mass; only1%-2% of ¹¹¹In could then be recovered.Uptake and release of the ¹¹¹In label bythe femur was markedly less than that ofthe ⁵⁹Fe. Whereas 85% of the injected⁵⁹Fe could be recovered from the circulating red cells, liver, and spleen, only about15% of the injected ¹¹¹In could be so recovered. Approximately 35% of the injected ¹¹¹In was excreted, and 43% wasrecoverable from the carcass. The subcellular distribution of the two isotopes in theliver at timed intervals following intravenous injection was studied. While 35%of the ⁵⁹Fe activity in the homogenatewas associated with ferritin, only 4% ofthe ¹¹¹In could be so identified. The results indicate a significant difference between the metabolism of ¹¹¹In and ⁵⁹Fein the rat and make it unlikely that themetabolism of ^In in man bears muchsimilarity to that of iron.

Submitted on October 2, 1973 Revised on October 28, 1973 Accepted on October 30, 1973     

Hemoglobin Koln in a Black: Preand PostSplenectomy Red Cell Survival (DF32P and 51Cr) and the Pathogenesis of Hemoglobin Instability

A 17-yr-old black male with hemolysisand pigmenturia but no anemia was foundto have hemoglobin Köln (₂₂^{98 valmet}[FG5]). Splenectomy was done because ofcomplicating thrombocytopenia. Thrombokinetic studies with ⁵¹Cr tagged plateletssuggested hypersplenism, and after surgerythe platelet count returned to normal. Thered cell t ⁵¹Cr was more than doubled,but the red cell life span (DF³²P) wasmore modestly improved (30.6 47.2days). The "elution" of ⁵¹Cr from the redcells presplenectomy was 5.6%/day,whereas after surgery it was normal(1.9%/day), accounting for the disparitybetween the survival methods. Study of theisolated cyanferri derivative of hemoglobinKöln by ultracentrifugation at various saltconcentrations and various pH’s indicatedan increased tendency to dimer formationunder conditions where normal hemoglobin is a tetramer. This results from thesite and type of amino acid substitution andaccounts in part for its instability.

Submitted on January 8, 1973 Revised on April 30, 1973 Accepted on May 4, 1973     

Oxymetholone Treatment for the Anemia of Bone Marrow Failure

Oxymetholone was given to 28 adultswith chronic anemia from bone marrow disease. Changes in hematocritand red cell mass were correlated withserial assessments of erythropoietinand erythropoiesis. Erythropoietin excretion was enhanced more than five-fold over the level expected for thehematocrit in 70% of the patients. Only23% of the patients with an evaluabletreatment trial increased their red cellmass by at least 20%. In all responders, the T of ⁵⁹Fe disappearanceranged from 86-136 min and erythroniron turnover exceeded 0.25 mg/100ml blood/day. A decline in serum ironconcentration to the 50-100 µg/100 mlrange after 1 mo of oxymetholone wasfrequently associated with a subsequent response to therapy. Patientswith severe bone marrow failure, forwhom frequent red cell transfusionswere required, did not improve. Thefailure of other patients to respondwas attributed to complicating factorsthat either impaired maximal erythropoietin production or restricted ironsupply to the bone marrow. Hepatictoxicity was detected in less than10% of treated patients. Results support the use of oxymetholone in thetreatment of patients with moderatedegrees of bone marrow failure andsymptomatic anemia.

Submitted on October 6, 1971 Revised on April 18, 1972 Accepted on April 27, 1972     

Cytochemical and Radioautographic Identification of Cells Induced to Synthesize Hemoglobin

In order to facilitate the identification ofbone marrow cells in which hemoglobinsynthesis is initiated, erythropoiesis wasfirst suppressed in guinea pigs through theinduction of posthypoxic polycythemia,and then it was restimulated by bleedingand reexposure to hypoxia. Hemoglobinsynthesis was detected with ⁵⁵Fe incorporation on radioautographs, and itspresence was demonstrated in the lightmicroscope with the benzidine reactionand absorption of monochromatic light at 4046 Å. In the electron microscope,hemoglobin was detected in the cytoplasm by a general increase in electrondensity after treating the tissue withdiaminobenzidine (DAB) and OsO₄. Densitometric measurements were carried outon electronmicroscopic negatives, usingreticular cell cytoplasm as a base line. Innormal marrow, proerythroblasts were theearliest cells in which hemoglobin couldbe detected, but during the early phase oferythropoietic stimulation, hemoglobinwas demonstrated in transitional cellswith all the methods employed. Withoutthe specific demonstration of hemoglobin,these cells could not be recognized morphologically as erythroblasts nor couldthey be distinguished from the precursorsof bone marrow small lymphocytes.Transitional cells were numerous in themarrow at the time of stimulation, and40 hr later a small number of them werelabeled with ⁵⁵Fe and synthesized hemoglobin in detectable amounts. Proerythroblasts were absent at the time of the stimulus, and when they reappeared themajority were benzidine or DAB positiveand had incorporated ⁵⁵Fe. The findingssuggest that progenitor cells of erythroblasts are among the basophilic membersof the transitional cell population, anderythropoietic stimulation induces hemoglobin synthesis in them. The relationshipof these cells to the progenitors of otherhemopoietic cells, as well as to the pluripotent stem cell, is discussed.

Submitted on May 29, 1973 Revised on November 12, 1973 Accepted on November 15, 1973     

The Effect of Amelioration of Anemia on the Synthesis of Fetal Hemoglobin in Sickle Cell Anemia

Two adult patients with sickle cell anemia of blood group A and A₂Brespectively, received sufficient transfusions of group O blood to maintainnearly normal hemoglobin concentrations for 4 months or longer.

Serial samples of the erythrocytes of each recipient were obtained by agglutination with anti-A (and anti-B) serum. The proportion of Hb F in theagglutinated erythrocytes was determined. Early in the transfusion period, amarked rise in the proportion of Hb F was noted. This rise was attributed toprolonged survival of erythrocytes which contained larger proportions of Hb F.In the later part of the transfusion period, the proportion of fetal hemoglobindeclined to pre-transfusion levels or below. However, significant amounts offetal hemoglobin in the erythrocytes of each patient were demonstratedthroughout the period of study, and Fe⁵⁹ incorporation into Hb F in vivo wasdemonstrated in one patient after 4 months of transfusion therapy. Under theconditions of these studies, synthesis of Hb F continued despite prolongedcorrection of the anemia. A decline in the proportion of Hb F in the erythrocytes of one patient after 5 months of transfusions suggested that Hb F synthesis may ultimately be depressed by transfusions. It was suggested that theproportion of fetal hemoglobin observed in the erythrocytes might in certaindiseases reflect the degree of anemia present many months before.

Submitted on February 26, 1964 Accepted on April 5, 1964     

Differentiation between Vitamin B12--deficient and Folic Acid--deficient Megaloblastic Anemias with C14--Histidine

Intermediary metabolism of the monocarbon pool and histidine in normalsubjects and patients with megaloblastic anemia was studied by continuousmeasurement of pulmonary excretion of C¹⁴O₂ and urinary excretion of C¹⁴after injection of L-histidine-2(ring)-C¹⁴. Cumulative pulmonary and renalexcretion of C¹⁴ for 1 month by two normal subjects approximates 45 percent of the amount injected. Within 4 months after injection of the doseused in this study, the resultant average tissue radiation decreases belowthe average natural terrestrial and cosmic radiation level.

Simultaneous determination of two parameters, (1) cumulative 1-hourpulmonary C¹⁴ excretion and (2) the time of occurrence of maximum C¹⁴O₂specific activity (T_max), may permit rapid and unequivocal differentiation between folic acid deficiency and vitamin B₁₂ deficiency in the pathogenesisof megaloblastic anemia. Folio acid deficiency results in marked diminutionof pulmonary C¹⁴ excretion (approximately 0.1 per cent of injection C¹⁴ in1 hour) and marked prolongation of C¹⁴O₂-specific activity T_max (approximately 3 hours), while both parameters are normal (approximately 1 percent and less than 1 hour, respectively) in patients with vitamin B₁₂ deficiencyand megaloblastic anemia.

Measurement during periods of reticulocyte response to either folio acidor vitamin B₁₂ demonstrate normal C¹⁴O₂-specific activity T_max but decreasedpulmonary C¹⁴ excretion. These observations suggest that prolongation ofC¹⁴O₂-specific activity T_max is a sensitive index of folic acid deficiency orblock and that if T_max is normal, pulmonary C¹⁴ excretion is a sensitiveindex of the relative partition of the active monocarbon pool between pathways for oxidation and pathways for nucleic acid synthesis.

This type of breath analysis seems to provide a quantitative dynamic representation of metabolic function which may be particularly useful in differentiating between the alterations of intermediary metabolism that occur inpatients with folic acid-deficient megaloblastic anemia and in patients withvitamin B₁₂-deficient megaloblastic anemia.

Submitted on August 24, 1962 Accepted on November 28, 1962     

The Use of 125I as a Membrane Protein Label for Erythrocyte Survival Studies

Radioactive iodide, incubated with erythrocytes in the presence of lactoperoxidaseand hydrogen peroxide, is covalentlybound to membrane proteins. Using thistechnique, rabbit erythrocytes were labeled with ¹²⁵I, reinjected into the donors,and erythrocyte survivals determined. Thevalues for the erythrocyte half-lives andthe shape of the decay curves were comparable to those reported using DF³²P. Following hemolysis of erythrocytes doublelabeled with ¹²⁵I and ⁵¹Cr, radioactivityfrom both these isotopes appeared mainlyin liver, lung, kidney, spleen, and urine.Enzymatic iodination provides a noneluting label of erythrocyte membraneproteins for the study of survival, sequestration and turnover of this red cellcomponent.

Submitted on June 16, 1973 Revised on September 13, 1973 Accepted on September 23, 1973     

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 life span of the red blood cell and the red blood cell volume in the chicken,pigeon and duck as estimated by the use of Na2Cr51O4, with observations on red cell turnover rate in the mammal,bird and reptile

Eight to 25 per cent of intravenously injected Na₂Cr⁵¹O₄ binds firmly witherythrocytes of the chicken, pigeon and duck. Calculation of the maximum lifespan of these avian red cells was made from the disappearance time of circulatingradioactivity. The maximum life span of the chicken erythrocyte was found to be35 days, of the pigeon erythrocyte 35-45 days, and the duck erythrocyte 42 days.Comparing the life span of avian erythrocytes with those of other animal species,the rate of red cell turnover in the mammals, birds, and reptile (turtle) was foundto correlate directly with basal heat production per kilogram body weight.

Using erythrocytes tagged with Na₂Cr⁵¹O₄ in vitro, the total red blood cellvolume was found to be 17-24 ml. per Kg. body weight in the rooster, 9-12 ml.per Kg. in the hen, 25-31 ml. per Kg. in the duck, and 31-34 ml. per Kg. in thepigeon. These values proved somewhat lower than those obtained from theindirect estimates of red cell volume, using plasma volume figures and peripherablood hematocrit.

Submitted on June 25, 1956 Accepted on August 18, 1956     

Iron in the Duodenal Mucosa of Normal, Iron-loaded and Iron-deficient Rats

Iron absorption studies with oral ⁵⁹FeSO₄were performed on 13 iron-depleted, 11iron-loaded, and 10 control rats, and nonheme iron was determined in both isolated epithelial cells and defoliated mucosa obtained from the duodenum. Meanabsorption by the control animals was18.5% of the dose. Both iron-depleted andiron-loaded groups showed significant differences in iron absorption (54.5% and2.2% respectively). Compared with thenormal controls, iron was decreased inthe epithelial cells of the iron-deficientgroup, whereas higher concentrationswere observed in the defoliated mucosaof iron-loaded animals. The latter observation was confirmed by the presence ofiron-laden macrophages seen in sectionsof the lamina propria of the iron-loadedrats.

Submitted on February 20, 1973 Revised on June 7, 1973 Accepted on June 13, 1973     

Radioactive vitamin B12 absorption studies: results of direct measurement of radioactivity in the blood

A new method for the determination of the absorption of vitamin B₁₂ has beendescribed using measurement of radioactivity in the blood or plasma after theingestion of physiologic test doses of Co⁶⁰ labeled vitamin B₁₂.

Although doses of 1.0 µc. (0.92 µg. vitamin B₁₂) gave higher counts, equallyreliable results were obtained with 0.5 µc. (0.46 µg. vitamin B₁₂). The radioactivitywas found in the plasma portion of the blood.

With this method it was possible to differentiate between all of nine patientswith pernicious anemia and 36 control subjects.

In non-pernicious anemia subjects and in pernicious anemia patients givenintrinsic factor, there was a relatively delayed rise in the blood or plasma radioactivity until a peak was reached in the 8 to 12 hour interval after the ingestionof the test dose. This absorption curve was quite different from the early rise observed by others after massive oral doses of vitamin B₁₂, indicating a differentmode of absorption.

Following the peak blood concentration the radioactivity gradually declinedand small amounts usually persisted for as long as one week, quite different fromthe rapid disappearance after parenteral administration previously reported.

This method appears valuable in the diagnosis of pernicious anemia and othervitamin B₁₂ malabsorptive states, in the evaluation of intrinsic factor activity,and in studies of various aspects of the metabolism of vitamin B₁₂.

Submitted on June 25, 1956 Accepted on August 12, 1956     

The Effect of Cold on Platelets. III. Adenine Nucleotide Metabolism After Brief Storage at Cold Temperature

The effect of cold on platelet adeninenucleotide (PAN) metabolism was studied.Spontaneous aggregation which occurswhen chilled platelet-rich plasma (PRP) issimultaneously warmed and stirred wasnot accompanied by the changes in adenine nucleotides associated with the release reaction. Connective tissue causedthe release of the same amount of ADPand conversion of equal amounts of ATPto IMP and hypoxanthine in cold-storedplatelets as it did in room temperaturestored platelets. However, cold did have animportant effect on PAN. In PRP stored atcold (0° C, 3° C) temperatures and warmedup to 37° C in the presence of ³H adenine,there was an increase in the conversion ofadenine to its metabolites and ultimatelyto hypoxanthine as compared to PRPstored at warmer temperatures. This effectcould not be prevented by ouabain,prostaglandin E₁, antibody to immunoglobulin M or adenosine.

Submitted on November 20, 1972 Revised on April 9, 1973 Accepted on April 19, 1973     

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.

     

Evidence for a Physiologic Role of Erythropoietin in Fetal Erythropoiesis

Administration of antibody prepared inrabbits against human urinary Ep to fetalsheep during the last third of the gestation period resulted in a suppression oferythropoiesis in the fetus. This wasevidenced by a sharp decrease in the numbers of circulating reticulocytes and a reduced rate of ⁵⁹Fe incorporation into RBC,spleen, and bone marrow, effects ascribable to a neutralization of endogenous Ep.In addition, there was a significant decrease in the numbers of nucleated erythroid cells in the bone marrows of thesefetuses. These results point to a physiologic role for Ep in fetal erythropoiesis.

Submitted on December 17, 1973 Revised on February 19, 1974 Accepted on February 20, 1974     

Variations in the Relationships Between Total 59Fe Uptake and the Uptakes Into Heme and Nonheme Fractions of Spleen and Bone Marrow in Irradiated Mice and Mouse Radiation Chimeras

A chemical method for the separation ofheme and nonheme iron-containing fractions in mouse tissues has been used tostudy the uptake of ⁵⁹Fe into both theheme and nonheme iron fractions inspleen and femoral bone marrow, in orderto investigate the possible relationshipsbetween the total uptake into spleen tissue and the rate of uptake into the hemefractions. In control mice, only aboutone third of the ⁵⁹Fe is directly associated with heme during the first dayafter administration, and this fraction decreases as radioactive red cells are released from the spleen into blood. Tendays after X-irradiation of mice, the proportion of ⁵⁹Fe in the spleen heme fraction 6 hr after intraperitoneal administration decreased from about 30 to 10%of the total splenic activity as the radiation dose was increased from 500 to 750rads. A similar alteration in the proportion of ⁵⁹ Fe in the spleen haem fractionoccurred in mouse radiation chimeras asthe dose of injected syngeneic bonemarrow cells was reduced from 5 x 10⁵to zero. Similar results were found infemoral bone marrow. These results indicate that in this system the rate of uptake into whole tissue is not a measureof the uptake into heme; it is necessary,therefore, to use the method involvingthe measurement of iron uptake intoheme, rather than the total tissue uptake,when estimating hemoglobin synthesisor erythropoiesis.

Submitted on February 15, 1971 Revised on April 5, 1971 Accepted on April 8, 1971     

The pathogenesis of postirradiation anemia

1. To assess the effects of irradiation on erythrocytes in vivo, one group ofrabbits was injected with radioiron-tagged erythrocytes immediately before, anda second group immediately after exposure to x-rays. A third control groupreceived the tagged erythrocytes but no irradiation. There was a drop in red cellmass in both irradiated groups as compared with the controls, but there was nodifference between the two irradiated groups, i.e., between the behavior ofirradiated and nonirradiated erythrocytes in irradiated hosts. These findings indicate that there is no direct effect of irradiation in the median lethal range onerythrocytes in vivo.

2. The degree of anemia was studied as a function of irradiation dose. It requires nearly lethal doses of x-rays to cause a conspicuous anemia. In the fatallyirradiated animals the drop in red cell mass and total circulating radioiron isprecipitous; in those nonfatally irradiated the drop is gradual and relativelyslight, even though the animals may have received the same dose.

3. Increase of specific organ activity of the liver and spleen of irradiated animals can be correlated with hemosiderin deposits and congestion. Deposition ofhemosiderin is a consequence of irradiation, its degree increasing with the effectiveness of the irradiation.

4. The erythrocyte mass of rabbits obtained by two direct isotopic technics(P³² and Fe⁵⁹) and one indirect technic (T-1824 dye) differ by constant ratios.The Fe⁵⁹/P³² ratio is 0.89; the Fe⁵⁹ values are regarded as correct. With the aid ofconversion factors either technic can be used. If an indirect technic is employedand the erythrocyte mass is estimated on the basis of plasma volume measurements, it is necessary to calculate first the average body hematocrit. This is doneby multiplying the large vessel hematocrit by a conversion factor, which is 0.83in normal rabbits.

Submitted on October 1, 1951 Accepted on December 22, 1951     

Hematologic responses in pernicious anemia to orotic acid

The oral administration of the pyrimidine precursor orotic acid in doses of3 to 6 Gm. daily to patients with pernicious anemia in relapse produced withsome regularity Partial remissions in the manifestations of vitamin B₁₂ deficiency. The best response occurred in a patient with postgastrectomy perniciousanemia. Little effect was seen in two undernourished patients who respondedwell, nevertheless, to B₁₂ therapy. Irregularity in the absorption of this poorlysoluble compound from the intestinal tract, possible changes en route, andother limiting nutritional factors may account for some variation in response.Preparations suitable for parenteral administration have not been developed.

The early effects of orotic acid in pernicious anemia resembled those of smallamounts of B₁₂. Reticulocytosis appeared 7 to 14 days after the start of therapy.Gradual clinical and hematologic improvement followed. We have not beenable to evaluate the possible ability of this compound to reverse the neurologicmanifestations of pernicious anemia. Complete remissions in the disease werenever produced, however, by orotic acid, for at the height of improvement, redcell macrocytosis and some degree of megaloblastic cellular development persisted in the bone marrow. Patients maintained on orotic acid alone for 5 to 7months gradually relapsed with increasing anemia and lingual mucosal atrophy.Toxic effects of the chemical were not seen.¹⁰ In no instance did it producean effect in pernicious anemia like that of folic acid: a quick but suboptimalresponse at first, followed ultimately by relapse, with neurologic disease, lingual mucosal atrophy, and/or anemia with a hypocellular nonmegaloblasticbone marrow—all comparatively refractory at this stage to vitamin B₁₂ therapy.

Since orotic acid is known to function only as an intermediate in the synthesisof pyrimidines^{2, 9} (fig. 12), the hematopoietic effects of some precursors andderivatives of the compound were studied. None proved to be as active as orotic acid. Carbamyl aspartic acid given in doses of 3 Gm. daily was followedby a slight reticulocytosis in two patients. Aspartic acid given in doses up to15 to 20 Gm./day with 3 to 6 Gm. of orotic acid had little or no effect in twopatients. A concentrate of uridylic and cytidylic acids obtained from yeast,probably absorbed from the intestinal tract as nucleosides, showed some effectin one of the two patients to whom it was given. This same preparation was remarkably effective when given to a child with a congenital abnormality inpyrimidine biosynthesis who excreted large amounts of orotic acid in the urine.¹³

The parenteral administration of thymidine, 0.5 Gm. daily for six days, hadno hematopoietic effect in one patient, but a significant response occurred wheninosine was given with it concurrently. Sodium deoxyribonucleic acid (DNA)prepared from fish sperm by the hot alkaline extraction method produced amoderate reticulocytosis in one patient. A second crest followed the additionof orotic acid to her regimen. A less hydrolyzed DNA preparation given to another patient for one week had no effect, but there was apparently none eitherwhen orotic acid was given 6 Gm./day for the same length of time.

Vitamin B₁₂ and folic acid appear to have similar, overlapping or reciprocal,actions in different biologic systems. A comparison of the hematologic effectsof folic acid metabolites with that of orotic acid was accordingly undertaken.

Methionine reduces the vitamin B₁₂ requirement of bacteria, and the de novosynthesis of this amino acid is increased by cobalamin.^14-16 Methionine givento patients with pernicious anemia in relapse, however, seemed to depress hematopoiesis. It is of interest that the methyl group of methionine is not utilizedfor the biosynthesis of thymine in bacteria. The methyl group of thymine wasderived from glucose instead.¹⁷ The hematopoietic effect of serine has not yetbeen studied in patients with pernicious anemia in relapse.

Histidine, which is synthesized by folic acid containing enzymes, was givenwith the thought that it might become an "essential" amino acid under somecircumstances, or provide a general source of active formate or of transferableformimino groups usable in protein and/or nucleic acid synthesis,^16,18 A definite hematopoietic stimulus was obtained from DL-histidine in two Patientswith pernicious anemia in relapse. The response was quick and suboptimal,however, and did not potentiate that of orotic acid. One patient in partialhematologic remission after taking histidine for 12 weeks was then given folicacid. Additional benefit was not observed. Further studies of the effect of theseand other folic acid metabolites in pernicious anemia and in nutritional megaloblastic anemia are in progress.

Biochemical studies in the past have given no indication that vitamin B₁₂ isconcerned with pyrimidine biosynthesis. The latter process, of considerablecurrent interest also in reference to the development of pyrimidine antimetabolites, is outlined in figure 12.^19-23 Carbamyl phosphate and aspartic acid areconverted by known enzymatic reactions to orotic acid. The latter is rapidlyconverted to pyrimidine nucleotides, derivatives, and to the pyrimidine moietiesof nucleic acids. Hurlbert and Potter injected small amounts of orotic acidintraperitoneally into rats.¹⁹ About one-third of the chemical was immediatelyexcreted unchanged and another third immediately taken up by the liver. Orotic acid was quickly converted in the liver quantitatively to acid-solublemetabolites, particularly uridine-5'-phosphate, derivatives of this compound,and cytidine-5'-phosphate. The uridine phosphate pool appeared to be theimmediate metabolic precursor of the uracil of the ribonucleic acid (RNA) inthe nucleus and a major source of the pyrimidines of the cytoplasmic RNA. Alarge contribution to liver DNA pyrimidine was observed in tissue regeneratingafter partial hepatectomy.²⁴ Thymidine is formed in vitro from uracil deoxyriboside by a reaction inhibited by Aminopterin.²⁵

Vitamin B₁₂ may facilitate nucleoside and nucleic acid synthesis by differentmechanisms in different biologic systems. In bacteria there is evidence thatit promotes the synthesis of methionine, nucleosides and/or deoxyribose, andpossibly activates protein sulfhydryl groups.¹⁴ In animals it may promote methyl group neogenesis, but there is increasing doubt that it has anything to dowith transmethylation.^16,26 The indications are that vitamin B₁₂ has differentfunction(s) than that of folic acid, which is concerned in the synthesis, transferand/or incorporation of formate, formimino and hydroxymethyl groups.

The vitamin B₁₂ requirement of the human adult on a weight basis is 10 to 15times less than that of animals,²⁶ but a disease unique to man, pernicious anemia, results from its lack. Evidence relating the deficiency in pernicious anemiato pyrimidines was first obtained by Vilter and associates who reported incomplete hematologic remissions in patients to whom they gave 15 to 30 Gm. ofthymine or uracil daily.^{11, 12} Thymine was effective in two of their patients whohad relapsed while taking folic acid. Uracil had no effect in a patient withmegaloblastic anemia of pregnancy, but who did respond to thymine.

Nieweg, et al., studied the relationship of vitamin B₁₂ to folic acid in themegaloblastic anemias. They cited evidence to support the idea that in thehuman, vitamin B₁₂ was particularly concerned with pyrimidine formationand RNA-protein synthesis.²⁷

The degree of remission that can be produced in patients with perniciousanemia in relapse by the administration of orotic acid suggests, too, that onemajor consequence of vitamin B₁₂ deficiency in the human is a defect in pyrimidine biosynthesis and/or incorporation. Other processes, such as purine ringformation, may also be affected. The mechanism by which orotic acid inducespartial remissions in pernicious anemia is unknown. It could serve merely asa metabolite which when supplied from exogenous sources would circumventa block in its synthesis or in that of a precursor. Increasing the supply of oroticacid could possibly overcome by mass action a defect in the synthetic pathwayat a later stage. In view of demonstrated feed-back regulatory mechanisms inpyrimidine synthesis,²⁸ however, there are too many ways by which orotic acidcould influence metabolism in the presence of vitamin B₁₂ deficiency to justifyfurther speculation.

Submitted on October 25, 1955 Revised on October 7, 1957 Accepted on October 7, 1957     

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     

A New Abnormal Hemoglobin O Padova, agr 30 (B11) Glu rarr Lys, and a Dyserythropoietic Anemia With Erythroblastic Multinuclearity Coexisting in the Same Patient

A patient with a not previously describedabnormal hemoglobin (^{30Glu Lys}) anddyserythropoietic anemia with erythroblastic multinuclearity is reported. Theheat stability and the functional propertiesof the new abnormal hemoglobin, namedhemoglobin O Padova, are normal, although the replacement lies in the ₁₁ interchain contact. The hemolytic condition,which was much improved by splenectomy, therefore appears to be linked tothe hereditary erythroblastic multinuclearity similar to hereditary erythroblasticmultinuclearity with positive acidifiedserum test (HEMPAS). In addition to theleading features observed in publishedcases of this entity, our case exhibitedsome immunologic peculiarities.

Submitted on November 8, 1973 Accepted on June 11, 1974