Erythrokinetics in pernicious anemia

Quantitative measurements of the erythropoietic activity of the marrow, ofcirculating red cell production and destruction have been made in patients withpernicious anemia in relapse and during response to vitamin B₁₂ therapy.

Total erythropoietic marrow activity as reflected by turnover of heme components proceeds at a rate of approximately 3 times normal. The delivery ofviable red cells to the circulating blood, however, does not increase above normal. This would indicate that the greater portion of marrow activity is ineffective in terms of blood production. This marrow dysfunction coupled with anincreased rate of cell destruction of approximately 3 times normal is responsiblefor the anemia. Total erythropoiesis is somewhat less, and effective erythropoiesisconsiderably less, than that which may be expected of the normal marrow underthe sustained stimulus of anemia.

The reticulocyte count is shown to be an unreliable index of blood productionin untreated pernicious anemia due to loss of reticulum from cytoplasma ofmany red cells before their delivery into circulation.

During the response to vitamin B₁₂ the ineffective erythropoiesis is convertedto effective erythropoiesis, whereas total erythropoiesis remains unchanged.The rate of blood production during recovery is 3 to 4 times normal.

Submitted on January 23, 1956 Accepted on May 3, 1956     

Pernicious anemia and related anemias treated with vitamin B12

Eleven cases treated with vitamin B₁₂ have been presented. Eight patients withpernicious anemia in relapse responded hematologically. Two patients with mildneurologic involvement were relieved by therapy with B₁₂ alone.

Consideration of the quantities of the crystalline vitamin required to promotemaximal erythropoiesis in pernicious anemia indicates that less than about 0.75µg. daily in doses at intervals of several days will not suffice to establish and maintain blood values as high as does adequate treatment with liver extract. Parenteraldaily doses of 1.0 µg. promoted good erythropoiesis in one patient, although itappears that the maximum rate of hemopoiesis may require the initial averagedaily dose of approximately 3.0 µg.

The reticulocyte count is an unreliable quantitative criterion of activity or adequacy of therapy.

It is suggested that hemopoietic factors in addition to PGA and B₁₂ may berequired by some patients to obtain maximal erythrocyte levels.

Vitamin B₁₂, as well as PGA, effects a reduction in the fecal urobilinogen output of patients with pernicious anemia. The significance of this finding is discussed.

No change in urinary excretion of pteroylglutamate or of porphyrin was detected in patients treated with vitamin B₁₂.

     

Pyridoxine-Responsive Anemia: Report of 2 Cases in Brothers and a Review of the Literature

The clinical and laboratory findings in two brothers with severe anemiaare presented. These findings were very similar in both cases. Evidence thatat least one of them suffered from a pyridoxine-responsive anemia is presented.It was assumed that the other had the same disorder since, in addition to thestriking similarity in the clinical and laboratory abnormalities, the latter’s anemia disappeared completely with the parenteral administration of vitamin Bcomplex, which provided him with 2 mg. of pyridoxine daily. These patientshave not relapsed 1.5 and 6 years after stopping the therapy.

A review of the reported cases with anemia responding to vitamin B₆ administration is presented. Common factors observed in some of these casesare: (1) severe anemia, microcytic and hypochromic in type, chiefly due to astriking maturation erythroblastic arrest at the basophilic stage with no defectin leukocytes or thrombocytes; (2) hyperferremia and hemosiderosis; (3) anabnormal tryptophan-loading test; and (4) complete or almost complete correction of all abnormalities with administration of vitamin B₆, even at smalldoses. These data were considered to suggest that the patients had a true deficiency in pyridoxine.

In other patients, no increased excretion of xanthurenic acid has been observed after a tryptophan-loading dose, and pyridoxine administration hasimproved the anemia only partially and has not reversed the serum iron parameters to normality. All of these other patients suffered from a familial hypochromic anemia not due to iron deficiency or from a "sidero-achrestic" or "refractory normoblastic" anemia.

The cause of the disorder in patients in the first group is unknown, but, byexclusion, increased requirement of vitamin B₆ seems to be the most likelypossibility.

Submitted on November 22, 1960 Accepted on February 14, 1961     

Fetal hemoglobin rescues ineffective erythropoiesis in sickle cell disease

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.     

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     

Experimental production of nutritional macrocytic anemia in swine. V. Hematologic manifestations of a combined deficiency of vitamin B12 and pteroylglutamic acid

A total of 20 swine were fed a diet adequate in all known respects except thatsoybean protein was substituted for casein, succinylsulfathiazole and a folic acidantagonist were added, and vitamin B₁₂ and pteroylglutamic acid were withheldfrom the vitamin supplement.

The animals developed macrocytic anemia, leukopenia and neutropenia, accompanied by erythroid hyperplasia of the bone marrow. Tue erythroblasts consisted mainly of immature macronormoblasts but a few atypical megaloblastswere also observed.

The anemia responded rapidly and completely to the administration of bothvitamin B₁₂ and pteroylglutamic acid. The administration of pteroylglutamicacid alone resulted in an immediate return of the blood and bone marrow towithin normal limits but after several months there was a partial hematologicrelapse in spite of continued therapy with this vitamin. The administration ofvitamin B₁₂ alone resulted in only partial remission of the anemia and the bonemarrow remained macronormoblastic although the megaloblasts tended to disappear.

Growth of the animals was stimulated by the administration of either vitaminbut the administration of both vitamins simultanseously resulted in the greatestrate of growth.

No manifestations of neurologic disturbances or of inscreased pigment excretionwere observed in the deficient swine.

Submitted on May 15, 1952 Accepted on July 22, 1952     

The Mechanism of the Development of Anemia in Untreated Chronic Lymphatic Leukemia

The following conclusions can be made concerning the cause of anemia inpatients with untreated chronic lymphatic leukemia excluding the rare casewith an autoimmune hemolytic anemia:

1. Early in the natural history of the disease there is a normal amount oferythroblastic tissue which produces red cells at a normal rate. The rate ofred cell destruction is also normal.

2. With progression of the disease the erythroblastic tissue is gradually replaced by lymphatic tissue, leading to a decrease in red cell production. Therate of red cell destruction is still normal.

3. Only late in the disease may decreased rate of red cell production be aggravated by an increased rate of red cell destruction.

Submitted on October 25, 1960 Accepted on December 9, 1960     

The Kinetics of Intravenously Injected Radioactive Vitamin B12: Studies on Normal Subjects and Patients with Chronic Myelocytic Leukemia and Pernicious Anemia

1. Studies of the fate of intravenously injected radioactive vitamin B₁₂have been performed in patients with normal, low and high serum concentrations of vitamin B₁₂.

2. Abnormal plasma disappearance curves were noted in chronic myelocytic leukemia, pernicious anemia in relapse and in remission, total gastrectomy and malabsorption syndrome.

3. In chronic myelocytic leukemia, the slow clearance of plasma radioactivity may be explained by the increased binding capacity of the plasmaproteins for vitamin B₁₂.

4. Plasma clearance of radioactivity is slower than normal in perniciousanemia, even in remission. The failure of the disappearance curve to returnto normal in pernicious anemia in complete remission suggests the existenceof a plasma "B₁₂-transferase," whose function is to transfer circulating B₁₂to the tissues. The disappearance curves suggest that the amount of such"B₁₂-transferase" is diminished in pernicious anemia, total gastrectomy andcertain Cases of malabsorption syndrome.

5. A relationship between a hypothetical "B₁₂-transferase" and intrinsicfactor is discussed.

     

Incidence of nucleoli in erythroblasts in patients suffering from refractory anemia of myelodysplastic syndrome

Nucleoli of erythroblasts have been studied in patients suffering from refractory anemia (RA) of myelodysplastic syndrome (MDS) and in control patients without a disturbed erythropoiesis in order to provide information on the incidence of nucleoli and micronucleoli in these cells. Nucleoli in erythroblasts were visualized by a simple cytochemical procedure for the demonstration of RNA which facilitated the visualization not only large nucleoli but also micronucleoli in advanced stages of the erythroblastic maturation. In control patients nucleoli were detected in all stages of erythroblastic development. In patients suffering from RA of MDS, a relatively large population of polychromatic and orthochromatic erythroblasts was characterized by a loss of nucleoli accompanied by the decreased incidence of micronucleoli characteristic of these cells. In contrast to control patients, in patients suffering from RA of MDS the number of nucleoli expressed by the values of the nucleolar coefficient of erythroblasts was smaller, particularly in both the early and terminal stages of erythroblastic development. Thus in patients with RA of MDS both the abnormal loss of nucleoli and decreased number of nucleoli in erythroblasts apparently represent and reflect a further abnormality of disturbed erythropoiesis.     

Experimental megaloblastic anemia in young guinea pigs

Megaloblastosis has been produced in young guinea pigs by feeding thempurified diets deficient its pteroylglutamic acid (PGA) or vitamin B₁₂ or both.The withholding of ascorbic acid from the diet did not produce megaloblastosisbut it greatly enhanced its development when PGA was also withheld. Spontaneous remission took place in several instances.

The possible role of growth of the animal and changes in the bacterial flora ofthe gastrointestinal tract has been discussed. Bacteriologic studies appeared toindicate a possible interrelationship between the occurrence of Escherichia coliin the stools of guinea pigs consuming deficient diets and the development ofmegaloblastosis. Diarrhea and infections are considered important in the pathogenesis of megaloblastic anemia but infections were not observed in animals ofthe present study. However, diarrhea was a prominent feature and appeared tobe a factor associated with megaloblastosis.

Anemia was usually present in association with megaloblastosis; however,megaloblastosis of a mild degree appearing in some of the animals fed the dietsdeficient in PGA or vitamin B₁₂ was not accompanied by anemia. When ascorbicacid was withheld in addition, then anemia always developed.

A dimorphic picture of macrocytosis and microcytosis was found most commonly in the peripheral blood of animals with megaloblastosis. This has beenexplained on the basis of regenerative macrocytosis or as a disturbance of thematuration of erythrocytes in such deficiencies.

Data obtained from peripheral blood are not a reliable indicator of megaloblastosis, nor is the clinical condition of the animal indicative of the pathologicstate.

One animal which had been fed a diet deficient in vitamin B₁₂ and PGA andwhich showed megaloblasts in its bone marrow was given injections of vitaminB₁₂. The megaloblastosis was corrected but the animal died on the sixteenth dayof treatment.

Two animals fed diets deficient in PGA and ascorbic acid were subsequentlygiven injections of PGA and ascorbic acid. The results demonstrated that administration of ascorbic acid alone did not interrupt the development of severemegaloblastosis in the continuing absence of PGA. When PGA was given, reticuocytosis then ensued and there was a reversal of megaloblastic erythropoiesis.

Submitted on March 31, 1955 Accepted on August 27, 1955     

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 transfusions of erythrocytes on untreated pernicious anemia

1. Three patients with untreated pernicious anemia were transfused withpacked erythrocytes to produce a normal hematocrit before they were treatedwith vitamin B₁₂. One patient with untreated pernicious anemia was transfusedwith 250 cc. plasma prior to therapy with vitamin B₁₂. No significant changeswere noted in this patient who received plasma.

2. Following erythrocyte transfusions the bone marrows of these patientswere considered to show the megaloblastic type of maturation with a reductionin percentage of the early megaloblasts (Stages I and II). The ME ratio of themarrows increased. A decrease in the absolute reticulocyte count occurred. Nochange occurred in the peripheral leukocyte and platelet counts and there wasno significant change in the clinical condition of the patients.

3. After therapy with vitamin B₁₂, at a time when the hematocrits were normal, the bone marrows became normoblastic, a small but definite reticulocytosisoccurred, the leukocyte and platelet counts returned to normal, and there wassubjective improvement in the condition of the patient.

4. It is concluded that in these three patients with untreated pernicious anemiamultiple transfusions of packed erythrocytes decreased erythropoiesis but didnot cause any basic change in the type of erythrocyte maturation.

Submitted on August 1, 1955 Accepted on November 17, 1955     

The effect of various metabolites on the growth of marrow cells in vitro

Marrow was cultured on chick plasma clots, glass surfaces and in suspensionand the influence of various metabolites and antimetabolites on the growth anddifferentiation of red cell precursors was studied.

Vitamin B₁₂ appeared to stimulate cell growth in cultures grown on clots and toconvert megaloblastic hematopoiesis to normoblastic in cultures grown on glass.

In suspension cultures of megaloblastic marrows in pernicious anemia serumsignificant cell increases were obtained with the addition of folinic acid, but notwith vitamin B₁₂. Both vitamin B₁₂ and folinic acid appeared to accelerate theconversion of megaloblasts to normoblasts in differential counts of stainedsmears of such cultures. No enhancing effect of intrinsic factor upon the activityof B₁₂ in suspension cultures of megaloblasts was observed. The effect of folinicacid could be blocked by colchicine and folinic acid could reverse the inhibitoryeffects of Amethopterin. These facts indicated that folinic acid acted in suspension cultures by stimulating mitosis.

Megaloblasts could be produced from normoblastic marrows by culturingthem in pernicious anemia serum, or in normal serum in the presence of Amethopterin.

Submitted on November 23, 1954 Accepted on February 14, 1955     

DIETARY FACTORS CONCERNED IN ERYTHROPOIESIS--Continued

Riboflavin is essential for normal erythropoiesis in rats, dogs, pigs, and monkeys.There is no evidence that this vitamin is required for normal erythropoiesis in man.The anemia in swine is normocytic.

Nicotinic acid deficiency is accompanied by a severe anemia in dogs. The type ofanemia produced is normochromic and may be either macrocytic or normocytic andis associated with a mild reticulocytosis. Limited observations indicate that thebone marrow is hypoplastic and that erythropoiesis stops at the erythroblasticlevel. An anemia due to a deficiency of this vitamin has not been demonstrated inother species nor in man.

Pyridoxine is essential for normal erythropoiesis in chicks, rats, dogs, and pigs.The anemia is microcytic and slightly hypochromic in type. Anisocytosis, microcytosis, polychromatophilia, and normoblasts can be seen in the blood smear.An irregular reticulocytosis is present. The bone marrow is hyperplastic and thereis an increase in the nucleated red blood cells. The anemia is accompanied byhemosiderosis of the tissues, an elevated serum iron level, and degeneration in thenervous system. There is no evidence of an increased rate of hemolysis. No relationship between pyridoxine and erythropoiesis has been demonstrated in man.

The "Lactobacillus casei group" includes the norite eluate factor, the L. caseifactor from liver, folic acid, the Streptococcus lactis R factor of Keesztesy et al., theyeast factor of Stokstad, the factor of Hutchings et al., vitamin M₁₁, xanthopterin,vitamin B_c, vitamin B_c conjugate, vitamins B₁₀ and B₁₁, and pyracin.

The L. casei factor from liver has been identified as pteroylglutamic acid. Theavailable evidence indicates that the norite eluate factor, folic acid, vitamin M,vitamin B_c, vitamin B₁₀, and vitamin B₁₁ are identical with pteroylglutamic acid.The Streptococcus lactic R factor of Keesztesy et al. may be pteroic acid. The yeastfactor of Stokstad is unidentified. The fermentation factor of Hutchings et al. hasbeen identified as pteroyltriglutamic acid. Vitamin B_c conjugate is now known tobe pteroylheptaglutamic acid. Thus the various members of this group are closelyrelated chemically and represent minor alterations of a basic structure. The corresponding deficiency syndromes are probably identical. In the rat the deficiency ismanifested by severe normocytic anemia, severe granulocytopenia, leukopenia,and thrombocytopenia. Nucleated red cells appear in the peripheral blood. Bonemarrow studies suggest a maturation arrest in the early stage of development of allthree of the cellular elements of the blood. The manifestations of the deficiency inthe chick are macrocytic anemia, leukopenia, and thrombocytopenia. Again immature red cells are present in the peripheral blood. In the monkey the manifestations of the deficiency are normocytic anemia, leukopenia, and thrombocytopenia.In human beings the synthetic L. casei factor from liver (pteroylglutamic acid) hasbeen shown to be effective in the treatment of various types of macrocytic anemiaincluding pernicious anemia and sprue. The relation of this substance to the antipernicious anemia substance in liver remains to be determined.

The extrinsic factor of Castle is still unidentified. It now seems reasonable thatit is related in some way to pteroylglutamic acid. It is unlikely that it is identicalsince the synthetic L. casei factor is effective even in the absence of normal gastricjuice. A deficiency of the extrinsic factor in man results in an anemia which isidentical with pernicious anemia and the bone marrow is cytologically indistinguishable. An accompanying neutropenia and thrombocytopenia are also frequently seen. The anemia responds rapidly to the parenteral administration ofhighly purified antipernicious anemia liver extracts and to pteroylglutamic (folic)acid. Achlorhydria is generally not present. Macrocytic anemia of nutritional originoccurring in the tropics varies from this anemia in one important aspect. It fails torespond to highly purified liver extracts. This strongly suggests that the factorresponsible for the deficiency is distinct from that of the extrinsic factor of Castle.A deficiency of this factor has been produced in monkeys and the deficiency syndrome consists of a macrocytic anemia with a megaloblastic bone marrow. Theanemia fails to respond to highly purified liver extracts which are effective in thetreatment of pernicious anemia but does respond to crude liver extracts and tomarmite, an autolyzed yeast extract. The relation between this factor and the L.casei factor has not been investigated.

The role of ascorbic acid in erythropoiesis is not clear. Although the scorbuticstate in both guinea pigs and human beings is frequently accompanied by anemiait is questionable whether the anemia is due specifically to a deficiency of ascorbicacid. Much of the animal experimentation is inconclusive because pure ascorbicacid supplements were not used. Further work in animals is needed. In man it hasbeen both asserted and denied that synthetic ascorbic acid is effective in relievingthe anemia. It would seem, however, that there are some scorbutic patients whorespond specifically to pure ascorbic acid. The anemia accompanying scurvy hasbeen reported as macrocytic, normocytic, and microcytic. An induced, uncomplicated ascorbic acid deficiency in a human being did not result in anemia.

Pantothenic acid deficiency results in a normocytic anemia of moderate degree inpigs in about two-thirds of the animals. There is evidence which suggests that adeficiency of this vitamin in rats may result in anemia, granulocytopenia, and bonemarrow hypoplasia. Not all animals show these changes and pantothenic acid,although completely preventive, does not exert a curative action in all animals.There seems to be a relation between pantothenic acid deficiency and a deficiencyof the L. casei factor in the rat.

Choline deficiency in dogs results in a severe anemia. In many animals this changeis irreversible. This may be explained by the irreversible liver damage which ispresent.

Biotin is necessary for the production of hemoglobin values greater than 14grams per cent in dogs maintained on a highly purified ration. There is no evidencethat biotin has an effect on erythropoiesis in other species.

In addition to the factors described above it has been shown that monkeys,pigeons, and guinea pigs require at least one more additional factor for normalerythropoiesis.

There is no evidence that thiamine, p-aminobenzoic acid, and inositol are concerned in erythropoiesis in any species.

Considering the relative size of the globin fraction of the hemoglobin moleculeit is understandable that a deficiency of protein results in anemia. This has beendemonstrated in rats and dogs. It has been pointed out that because of a markedreduction in the total blood volume only when the total circulating hemoglobinis determined and adjusted to a unit of surface can the true severity of the anemiabe appreciated. Equine globin contains all ten of the "essential" amino acids andat least nine "nonessential" amino acids. Human globin has not been so extensivelystudied. It would be expected that a deficiency of any one of the "essential" aminoacids would give rise to anemia. Actually, specific deficiencies of tryptophan,lysine, phenylalanine, and isoleucine have been produced in the rat and anemiadeveloped in each instance. The morphological characteristics of these anemiashave not been carefully investigated. The anemia due to tryptophan deficiency inthe rat has been stated to be normocytic and normochromic. An anemia probablydue to a lack of tryptophan has been produced in pigs. This anemia is normocytic,normochromic, and accompanied by a hypoplastic or normoplastic bone marrowand a normal level of iron in the serum. No increase of hemosiderin in the tissueshas been noted. Whether the anemia produced in rats by feeding deaminized caseinis due to a toxic substance rather than a deficiency of lysine is unsettled althoughlarge amounts of lysine prevent its development. Evidence that glycine is utilizedin the synthesis of the pyrrole rings of protoporphyrin has been obtained by labeling this amino acid with N¹⁵ and feeding the labeled compound to rats. Pyrroleshave also been synthesized in vitro from glycine. Similar evidence is available toindicate that acetic acid, or a derivative of it, is utilized for porphyrin synthesis.

Three mineral elements, iron, copper, and cobalt, have been shown to be essentialfor normal erythropoiesis in at least one species each. Iron is probably required forerythropoiesis in all mammals. A deficiency results, at least in the chronic stages,in a microcytic hypochromic anemia and is accompanied by a normoblastic, hyperplastic bone marrow and a low serum iron level, an increased amount of protoporphyrin in the erythrocytes, and an elevated serum copper level. Nucleated red bloodcells are occasionally seen in the peripheral blood and the reticulocytes are increased.

The fundamental concepts of iron metabolism have changed greatly in recentyears. These may be summarized. Iron is absorbed chiefly in the duodenum. In manit is absorbed principally as ferrous iron. Dogs absorb both valence forms wellalthough some animals absorb the ferrous form more readily than the ferric form.Rats absorb both forms equally well. The absorption of iron is also dependent uponthe concentration of the iron in the intestine, upon the solubility of the iron salt,and in the human being at least upon the presence of reducing substances in the dietas well as the reducing action of the gastric hydrochloric acid. In addition to thesefactors the need of the body for iron may determine, to a certain degree, the amountabsorbed. This is known as the "selective absorption" theory. Recently it has beensuggested that apoferritin acts as a receptor compound in the mucosal cell. As theconcentration of the plasma iron falls, ferrous iron is removed from the mucosal cellresulting in a diminution of ferritin in the mucosa. When the ferritin has diminishedto a point where the cell is no longer saturated with respect to ferrous iron, moreiron is absorbed into the mucosal cell. Once absorbed the iron is transported in theplasma to the tissues where it is stored to a great extent as ferritin, a protein-ironcomplex. The iron is then used over and over again for hemoglobin synthesis. Ironis excreted from the body in only insignificant quantities. This theory requiressubstantiation.

Copper has been shown to be essential for normal erythropoiesis in chickens,mice, rats, rabbits, dogs, pigs, sheep, cattle, and infants. A deficiency of this mineral in rats is manifested by a microcytic hypochromic anemia and a moderatereticulocytosis. A condition due to a deficiency of copper, known as "enzooticataxia," occurs in sheep in Western Australia. Anemia may be severe. In younglambs it is microcytic and hypochromic and is accompanied by demyelinization ofthe nervous system and hemosiderosis of the tissues. In adult sheep the anemia isslightly macrocytic and hypochromic. Blood smears reveal anisocytosis, poikilocytosis, Howell-Jolly bodies, normoblasts, numerous macrocytes, stippling, andpolychromatophilia. Similar blood changes have been reported in copper-deficientcattle in Western Australia. In nutritional anemia in infants the rate of erythropoiesis is accelerated when copper is given in addition to iron. In adults supplementalcopper therapy may be of value in a few cases. Such cases, if they occur, are rare.Most cases will respond if adequate doses of iron are given. This does not necessarilyindicate that copper is not needed for erythropoiesis or that it is not a dietary essential but rather that the quantities needed are so small that sufficient copper is present in the body stores in adult life, in the diet, or as a contaminant in the iron usedtherapeutically to supply the needs. No case of uncomplicated copper deficiency hasbeen reported in man. The manner in which copper is related to the formation of redcells is not understood.

The role of cobalt in erythropoiesis is unique. A deficiency results in anemia. Theadministration of small amounts to normal animals produces a polycythemia,whereas the administration of large amounts depresses erythropoiesis. The enzooticoccurrence of cobalt deficiency in sheep and cattle has been reported from variousregions of the world. Anemia is present and is oftentimes severe. The anemia iseither normocytic or microcytic and usually hypochromic. Blood smears revealanisocytosis and poikilocytosis. There is a hypoplasia of erythrogenic tissue in thebone marrow, hemosiderosis of the tissues and a reduction in reticulocytes in theblood. An experimental anemia due to cobalt deficiency has not been produced ineither rats or dogs. There is no substantial or convincing evidence that cobalt isneeded by human beings for normal erythropoiesis. The administration of smallamounts of cobalt to normal rats, dogs, guinea pigs, frogs, mice, rabbits, chickens,pigs, and ducks produces a marked polycythemia which is accompanied by areticulocytosis, hyperplasia of the bone marrow, and an increased erythropoieticactivity in the spleen and liver. Larger doses of cobalt inhibit erythropoiesis. Themetabolism of cobalt is unlike that of iron. The excretion of cobalt from the bodyonce it is absorbed is exceedingly rapid and is principally through the kidneys.

In conclusion, certain vitamins, namely, riboflavin, nicotinic acid, pyridoxine,"folic acid," and the extrinsic factor, have been shown to be essential for normalerythropoiesis in at least one species each. It has been claimed that ascorbic acid,pantothenic acid, choline, and biotin play a role in erythropoiesis but these claimsneed substantiation. There is no substantial evidence that thiamine or inositol isconcerned in red cell formation. The significance of p-aminobenzoic acid has yet tobe determined. Protein is essential for normal red blood cell formation. The globinfraction of the hemoglobin molecule contains all ten of the "essential" amino acidsas well as many of the "nonessential" ones. The stroma of the red cells also contains amino acids. It is logical, therefore, to assume that in the absence of any oneof the so-called essential amino acids hemoglobin formation cannot take placenormally. Actually specific deficiencies of tryptophan, lysine, phenylalanine, andisoleucine have been produced in the rat and anemia has developed in each instance.There is evidence to show that glycine and acetic acid, or a derivative of it, areutilized in the synthesis of the pyrrole rings of protoporphyrin. Three mineralelements, iron, copper, and cobalt, have been shown to be essential for normalerythropoiesis.

Note: I gratefully acknowledge my indebtedness to Dr. Maxwell M. Wintrobe for his kind advice and aidin the preparation of this review as well as for the liberal use of his extensive reprint file.

     

Urinary excretion of vitamin B12

1. Twelve patients, 6 with pernicious anemia and 6 without pernicious anemiaas controls, were studied as to urinary excretion of vitamin B₁₂.^*

2. Microbiologic tube assay using Lactobacillus leichmanii A.T.C.C. # 4797was the method of B₁₂ assay.

3. Dosages used were 42.2, 63.3, 84.4, 211.0 µg given both in ascending anddescending order at daily intervals.

4. Amounts of 84.4 to 211.0 µg of vitamin B₁₂ parenterally resulted invariablyin the excretion of 53 to 68 per cent of the injected vitamin B₁₂ within 18 hours.The percentage of excretion rose remarkably as the dosage increased.

5. An amount of 42.2 µg of vitamin B₁₂ parenterally resulted in relativelylittle excretion when this was the initial dose, but the excretion was appreciablewhen these doses followed the injection of the larger doses. "Saturation" of thebody apparently occurs. In general more vitamimi B₁₂ is excreted in the smallerdoses if these follow the larger doses.

6. No significant differences could be detected between the pernicious anemiaand nonpernicious anemia patients.

7. A possible explanation of these results is proposed.

Submitted on July 18, 1951 Accepted on October 1, 1951     

Experimental research on the proliferative and differentiative activity of the erythroblast in chronic erythremia,the chronic form of Di Guglielmo''s disease

Using the method of culture in vitro, the authors have studied the proliferative and differentiative activities of the erythroblastic tissue in chronic erythremic myelosis.

The results of the research allowed the authors to reach the following conclusions:

1. the erythroblastic cells of chronic erythremic myelosis reveal indisputabledifferentiative activity in vitro;

2. the rate of maturation of the erythremic erythroblasts is slower than normal;

3. the proliferation of the erythroblastic tissue in chronic erythremic myelosisis normal;

4. in the genesis of the erythroblastic hyperplasia in chronic erythremic myelosis, the most evident factor is a defect of maturation of the erythroblasts.

Submitted on July 11, 1955 Accepted on March 23, 1956     

Free Serum Vitamin B12 Level in Certain Hematologic Disorders

The variation of the free fraction of serum vitamin B₁₂ level was studied incertain hematologic disorders. In normals, the mean ratio of free to totalvitamin B₁₂ was 10.2 per cent. In leukemias—chronic myeloid, acute myeloidand acute lymphatic—the mean value of free vitamin B₁₂ was higher thannormal. In chronic myeloid and acute lymphatic leukemias, the free vitaminB₁₂ level did not on an average exceed 20 per cent of the total. In acute myeloid leukemia, the free fraction was relatively more increased, constituting31.1 per cent of the total on an average. In aplastic anemia, the mean valuesof free vitamin B₁₂ and the ratio (31.2 per cent) of free to total vitamin B₁₂were higher than normal. The patterns presented by acute myeloid leukemiaand aplastic anemia appeared to be more or less similar.

     

Observations on the mechanism of intranasal absorption of vitamin B12 in pernicious anemia

Satisfactory clinical and hematologic response was obtained from the singleapplication to the nasal mucosa of 200 and 150 micrograms of crystalline vitaminB₁₂ in two patients with pernicious anemia.

Thirty-two patients with pernicious anemia in relapse and 55 in remissionhave been controlled satisfactorily by intranasal B₁₂ either in saline solution,lactose powder, or as crystals. Absorption of the vitamin was probably directand did not require binding with "intrinsic substance." Chronic disease of thenose did not contraindicate this modality of therapy; local irritation of themucous membranes by the crystalline B₁₂ or the vehicles employed has notbeen observed.

Submitted on December 15, 1954 Accepted on June 22, 1955     

Cyanocobalamin, Ascorbic Acid and Pteroylglutamates in Normal and Megaloblastic Bone Marrow

The B₁₂ activity as estimated by Lactobacillus leichmannii, the folic-acid-like activity by Streptococcus faecalis (F.A.A.) and the ascorbic acid concentration have been determined in the blood and buffy coat of bone marrowof normal subjects, 10 patients with pernicious anemia in relapse, a group ofpatients with non-Addisonian megaloblastic anemia and some patients withiron deficiency.

A correlation between the serum B₁₂ and the plasma ascorbic acid andtheir respective levels in bone marrow was observed. The marrow and serumB₁₂ levels in prenicious anemia were abnormally low, but they did not differfrom a group of 5 patients with hypochromic normoblastic anemia who hadboth low serum and marrow levels. The concentration of F.A.A. in the marrowof patients with pernicious anemia was reduced, but it was felt that this wasmore likely a manifestation of the megaloblastic anemia rather than a causativefactor.

One of six patients with megaloblastic anemia of pregnancy had no detectabledeficiency, while the other five had reduced B₁₂, folic acid and ascorbic acidconcentrations. The possible therapeutic implications are discussed.

There was a significant reduction in the bone marrow concentration ofascorbic acid in all patients with megaloblastic anemia.

Submitted on March 26, 1959 Accepted on October 9, 1959     

Visual impairment due to optic neuropathy in pernicious anemia: report of case and review of the literature

1. A man with optic nerve involvement and pernicious anemia regainednormal vision after treatment with vitamin B₁₂.

2. In a search of the literature we found 28 cases with both perniciousanemia and optic neuropathy. The hematologic, neurologic and ophthalmologicfindings were analyzed in these cases. In each case, the diagnosis of perniciousanemia was established. Optic atrophy associated with pernicious anemiamay be part of the pathologic process of pernicious anemia. If the patient istreated early with vitamin B₁₂ or liver extract, optic nerve function returns.

Sixteen other cases with optic atrophy and possible pernicious anemia inthe literature had inadequate information to substantiate the diagnosis.

3. The use of a cobalt⁶⁰-labelled vitamin B₁₂ absorption test may be helpfulin optic nerve disorders of obscure etiology.

Submitted on June 17, 1958 Accepted on August 7, 1958