The anemia of the Di Guglielmo syndrome

The physiopathology of the anemia of the Di Guglielmo syndrome (erythremic myelosis) was studied in 11 patients with the acute and chronic varieties of the disease. Ferrokinetic studies were performed in three additionalpatients.

1. The anemia was normochromic and macrocytic; in contrast to the meancorpuscular volume, which was elevated, the mean corpuscular hemoglobinwas often normal. In several patients the mean corpuscular hemoglobin concentration was slightly lower than normal, suggesting slight hypochromia.

2. Reticulocytes were often increased but bore no relationship to the degree of the anemia nor to the shortening of the red cell life span. The reticulocyte count is an unreliable index of blood production in this disease.

3. The degree of erythroblastemia was highly variable. No direct correlation existed between the degree of erythroblastemia and the acuteness of thedisease, nor was there any relationship between the degree of erythroblastemiaand either the degree of anemia or the degree of erythrocytic destruction.

4. The bone marrow showed striking erythroblastic hyperplasia. This wasusually of the megaloblastic type. Primitive erythroblasts (erythrogones) wereconspicuous. The erythroblastic hyperplasia was out of proportion to the relatively minor reticulocytosis or the relatively slight diminution in red cellsurvival.

5. The nucleated red cells of the marrow showed variable numbers ofmegaloblasts and megaloblastoid forms, suggesting the presence of a vitaminB₁₂ deficiency (pernicious anemia). However, the vitamin B₁₂ concentrationof the serum was elevated, and there was no response to the administrationof vitamin B₁₂ or folic acid.

6. Varying numbers of erythroblasts in the bone marrow and in the peripheral blood showed periodic acid-Schiff (PAS)-positive granules in the cytoplasm. No chemical abnormalities of hemoglobin could be detected either bythe method of paper electrophoresis or by the alkali denaturation test.

7. Diminished red cell survival was present in most cases, but it was of arelatively slight degree. It was due to an "intracorpuscular" defect of the redcells.

8. The often great increase in fecal urobilinogen output as compared witha relatively minor rate of red cell destruction suggests "heme pigment diversion" or increased destruction of precursor red cells, as in pernicious anemia,where the same phenomenon has been observed.

9. The great increase in the number of erythroid cells in the bone marrowand the increased rate of iron turnover as compared with the relatively minorincrease in red cell destruction and iron utilization point to an "ineffective"type of erythropoiesis. The high degree of "ineffective erythropoiesis" seenin this disease may be characteristic of the neoplastic proliferation of the redcell series.

10. In conclusion, the anemia of the Di Guglielmo syndrome is due to acombined disturbance: (1) an "ineffective" type of erythropoiesis of markeddegree, perhaps due to an acquired (neoplastic) defect in the uptake or utilization of B₁₂ by the erythroblasts and (2) increased hemolysis resulting fromthe increased destruction of defective red cells.

Submitted on June 11, 1958 Accepted on August 13, 1958     

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     

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     

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.

     

Erythrokinetics. IV. The plasma iron turnover as a measure of erythropoiesis

Experimental and clinical studies have been performed to define more clearlythe significance of the plasma iron turnover.

It has been shown that the plasma iron turnover is not affected by the rate ofred cell destruction and to only a limited extent by increased body stores. Itdoes, however, reflect the degree of erythroid marrow activity and is a sensitiveindicator for measuring changes in such activity.

A series of 85 studies in normal subjects and in patients with various hematologic disorders were carried out to define the range of response with anemia andto assess the clinical value of the plasma iron turnover as an index of erythropoiesis. In states of marrow hyperfunction it is increased from 3 to 6 times normaland may be depressed to approximately half normal with marrow hypofunction.The plasma iron turnover is increased with marrow dyspoiesis. This increase is ameasure of total erythropoiesis and does not indicate the production of viablered cells.

Submitted on July 30, 1956 Accepted on November 4, 1956     

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.

     

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.

     

A modified urinary excretion test for measuring oral cobalt60 labeled vitamin B12 absorption and its application in certain disease states

1. Factors relating to the urinary excretion test for oral vitamin B₁₂ absorptionwere studied. A modified procedure is presented giving a high percentage ofurinary recovery of radioactivity in the normal.

2. Values in the pernicious anemia range were found in the presence of acuteinfection and severe renal insufficiency. Other limitations in the interpretationof the test are discussed.

Submitted on July 31, 1956 Accepted on October 7, 1956     

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     

Serum vitamin B12 in leukemias and malignant lymphomas

High serum vitamin B₁₂ levels were found in chronic myeloid leukemia and inacute leukemia with myeloid differentiation. Following x-ray therapy and repeated blood transfusions, a drop of serum vitamin B₁₂ was found.

In chronic lymphatic leukemia, undifferentiated stem cell leukemia, Hodgkin’s disease, and multiple myeloma, the serum vitamin B₁₂ concentrations werenormal.

In polycythemia with marked leukocytosis the serum B₁₂ was normal.

In myelosclerosis high B₁₂ values may be found.

The serum vitamin B₁₂ in chronic myeloid leukemia is in a bound form andthe binding capacity for added B₁₂ is increased.

Serum vitamin B₁₂ determination may be of some value in differentiatingvarious types of leukemia and other myelo-proliferative disorders.

Submitted on October 14, 1956 Accepted on February 24, 1957     

The effect of irradiation on the plasma erythropoietic stimulating factor

1) Protein-free plasma extracts from rabbits made anemic by the administration of phenylhydrazine immediately following total body x-irradiation arecapable of stimulating erythropoiesis in the normal rat as demonstrated by erythrocytosis, reticulocytosis, and increased marrow erythropoietic activity.

2) Plasma extracts from rabbits made anemic by total body x-irradiationalone contain an erythropoietic stimulating factor.

3) These data would indicate that the stimulating factor is not produced byhemopoietic or other radiosensitive tissue and its formation is not dependentupon a regenerative marrow.

Submitted on May 23, 1956 Accepted on June 29, 1956     

Pernicious anemia without gastric atrophy and in the presence of free hydrochloric acid; report of a case

A case is described of Addisonian pernicious anemia in a girl aged 16 years,associated with a normal gastric mucosa and free hydrochloric acid in the stomach. Partial, but significant deficiency of intrinsic factor activity was demonstrated in her gastric juice by the use of Co⁵⁶-labeled vitamin B₁₂.

Submitted on September 1, 1956 Accepted on November 26, 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 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.

     

Studies on urinary excretion of vitamin B12CO60 in pernicious anemia for determining effective dosage of intrinsic factor concentrates

Studies were made in pernicious anemia patients on the urinary excretion ofB₁₂Co⁶⁰ after a small oral dose followed by a large parenteral injection of nonradioactive vitamin B₁₂.

(1) Increasing doses of intrinsic factor concentrates give increasing excretionsof radioactivity at low doses; little additional increase at moderate doses; andat times a subsequent diminution at excessive doses. Data on 34 tests of aparticular intrinsic factor concentrate in 18 pernicious anemia patients tend tosupport an excretion proportional to the logarithm of intrinsic factor dosage atlow to moderate levels, but do not exclude the possibility of a linear approachto a plateau.

(2) Assay by hematologic response was compared with the urinary excretiontests in 13 pernicious anemia patients. This data shows a relation between thetwo tests though the correlation is far from complete.

(3) Methods are outlined for testing all intrinsic factor preparations with thesame amount of tracer vitamin B₁₂ as will be incorporated commercially. Moresensitive comparative tests of similar intrinsic factor preparations may be madeusing smaller amounts of B₁₂Co⁶⁰.

(4) The literature is reviewed to determine which variations in technic mightlead to the most reliable quantitation of intrinsic factor activity.

Submitted on August 8, 1955 Accepted on January 12, 1956     

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     

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     

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

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

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

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

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

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

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

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

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     

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