Bone marrow cells from vitamin B12- and folate-deficient patients misincorporate uracil into DNA

Bone marrow cells from 15 patients with normal deoxyuridine (dU) suppression test results, 3 healthy subjects, and 11 patients with megaloblastic anemia caused by vitamin B12 or folate deficiency were examined for misincorporation of uracil into DNA. Cells were incubated with [5-3H] uridine for 2 hours and their DNA extracted. The DNA was hydrolyzed to deoxyribonucleosides with DNase 1, phosphodiesterase and alkaline phosphatase, and any dU present was separated from other deoxyribonucleosides by Aminex A6 chromatography. The quantity of dU/mg DNA and the radioactivity in the dU peak/mg DNA were then calculated. The results clearly showed that there was markedly increased uracil misincorporation into the DNA of vitamin B12- or folate-deficient marrow cells. Misincorporation of uracil into DNA may be an important biochemical lesion underlying both the megaloblastic change and the ineffectiveness of hematopoiesis in vitamin B12 and folate deficiency.     

Apoptosis in megaloblastic anemia occurs during DNA synthesis by a p53-independent, nucleoside-reversible mechanism

Deficiency of folate or vitamin B(12) (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts from p53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.     

Serum Uracil Levels in Acute Childhood Leukaemia

Serum ‘uracil + uridine’ expressed as uracil has been measured in 47 consecutive cases of acute leukaemia in children at the time of diagnosis, before treatment, and also both before and after therapy in a girl of 12 with Ph¹ positive chronic myeloid leukaemia in blastic transformation, and in an adult male age 31 with acute undifferentiated leukaemia. The results were compared with 97 previously reported normal subjects. The skew distribution of results in acute leukaemia rendered the mean value and standard deviation meaningless but the range was considerably wider than in the normal controls (range 7.2–77.1 μmol/l. in acute leukaemia; mean 15.7 μmol/l., range 5.7–40.5 μmol/l., SD 5.26 in controls). In 10 (21%) of the patients with acute leukaemia the level was more than 3 SDs and in five more than 8 SDs above the normal mean. There was a high degree of correlation between uracil concentration and total white cell count (r= 0.7894, P<0.001) and blast cell count (r= 0.7160, P<0.001) but no correlation was observed between serum uracil and the blood urea or the serum uric acid, vitamin B₁₂ or folate. The grossly elevated uracil levels, white cell and blast cell counts rapidly returned to normal or near normal after therapy. Retention of uracil by the kidney, increased DNA catabolism and the enzymatic deamination of cytosine are considered, and each in turn excluded, as a possible cause of the observed elevated uracil levels. It is submitted that the raised uracil level is a function of the circulating blast cell and that the results are consistent with the hypothesis that, at least in some cases of acute leukaemia, there may be an impairment in the amination of uracil to form cytosine akin to the impaired methylation of deoxyuridylate in the megaloblastic anaemias. The former would enhance the known mutagenic action of uracil, an action which is absent in the latter. The molecular mechanism, whereby this could give rise to a population of genetically abnormal cells incapable of normal differentiation, is discussed.     

Elevated serum S-adenosylhomocysteine in cobalamin-deficient megaloblastic anemia

Impaired methylation due to accumulation of S-adenosylhomocysteine (SAH) may contribute to the pathophysiology of cobalamin-deficient anemia. We assayed serum S-adenosylmethionine (SAM), SAH, total homocysteine (tHcy), and methylmalonic acid (MMA) in 15 subjects with cobalamin-deficient megaloblastic anemia and compared results with those of 19 subjects with anemia/pancytopenia due to other causes. Cobalamin-deficient subjects had a median hematocrit level of 20% and mean cell volume of 111.7 fL. The median serum cobalamin level was 37 pg/mL, MMA 3030 nmol/L, and tHcy 62.0 micromol/L. SAH was elevated in 13 of 15 subjects (median, 42 nmol/L) and the median SAM value was normal (103 nmol/L), but SAM/SAH ratio was low (2.5). The SAH was higher and SAM/SAH ratio was lower in cobalamin-deficient subjects compared with those with other anemias after excluding 4 patients with renal insufficiency. SAM concentrations were not low in cobalamin deficiency. Cobalamin injections corrected anemia, MMA, tHcy, SAM/SAH ratio, and SAH. Some hematologic variables were inversely correlated with SAH and cobalamin but not tHcy or MMA. In conclusion, serum SAH is elevated in cobalamin-deficient subjects with megaloblastic anemia and corrects with parenteral cobalamin therapy.     

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     

Acquired increases of human erythrocyte purine enzymes.

The alterations of three erythrocyte purine enzymes were studied in 12 patients with diseases associated with reticulocytosis, two patients with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, seven patients with severe megaloblastic anemia, and 14 normal subjects. The specific activity of adenine phosphoribosyltransferase was positively correlated (r = 0.81) with the reticulocyte percentate in ten patients with a normal hypoxanthine-guanine phosphoribosyltransferase. Two apparent types of alterations of this enzyme were distinguished: (1) increased specific activity with a normal half life as in megaloblastic anemia, and (2) a prolonged half life with or without an elevation of specific activity as in the deficiency of hypoxanthine-guanine phosphoribosyltransferase. Hypoxanthine-guanine phosphoribosyltransferase and phosphoribosylpyrophosphate synthetase were increased in megaloblastic anemia, but were not correlated with the reticulocyte percentage and did not have a consistent change in the half life in the other disorders studied. The data show that acquired disorders associated with reticulocytosis may cause an elevation of the specific activity of purine enzymes in peripheral circulating erythrocytes. Therefore, these factors must be carefully considered in the interpretation of an elevated level of enzyme activity.     

Megaloblastic anemia in North Africa

We prospectively studied 478 patients with megaloblastic anemia living in Tunisia. Overall, 98% of patients had vitamin B12 deficiency. Pernicious anemia accounted for most of these cases, and median age at presentation was 45 years. Megaloblastic anemia occurred in 19 subjects under 15 years of age, and of these, nine had the Immerslund-Graesbeck syndrome.     

Characteristics of the levels of various nucleic acid precursors in the tissues of rabbits with various thyroid conditions

The content of the precursors and metabolites of nucleic acids in the brain and liver of normal rabbits, rabbits with hypothyrosis (thyroidectomy) and hyperthyrosis (100-150 Mg of L-thyroxine per 1 kg of body mass daily for 10-12 days) was determined by liquid chromatography with a high resolution. A decrease in the content of deoxyguanosine, thymine, hypoxanthine, cytosine, thymidine and an increase in the content of inosine, adenine and adenosine in the liver of animals with hypothyrosis were shown. In the brain of these rabbits the level of cytidine was lowered, and the levels of deoxyguanosine, thymine, inosine, hypoxanthine, adenine and uracil were on an increase. An increase in the levels of deoxyguanosine, thymine, adenine, thymidine and a decrease in the levels of cytidine, hypoxanthine, cytosine, adenine and uracil in the liver were noted in hyperthyrosis. In the brain of rabbits with hyperthyrosis an increase in the levels of deoxyguanosine, cytidine, inosine and adenine was combined with a decrease in the levels of thymine and hypoxanthine. These thyroid-dependent changes in the content of registered metabolites and precursors of nucleic acids in tissues suggest that the observed shifts illustrate one of many mechanisms of the regulation of cell metabolism by thyroid hormones.     

Persistence of neutrophil hypersegmentation during recovery from megaloblastic granulopoiesis.

We studied the rate of disappearance of neutrophil hypersegmentation in 23 patients with megaloblastic anemia during therapy with vitamin B12 and folic acid. In 14 patients with uncomplicated megaloblastic anemia, the neutrophil lobe average began to fall towards normal 11.4 +/- 0.3 (SEM) days after treatment was begun and became normal by 13.9 +/- 0.6 days. In nine patients with associated inflammatory disorders, the lobe average returned to normal more rapidly. In six initially neutropenic patients, a five-fold increase in total granulocyte count occurred by the eighth day and a seven-fold increment in the total number of circulating hypersegmented neutrophils. Granulocyte folate concentrations in five folate-deficient patients rose to normal levels within 4 to 7 days of starting therapy. Hypersegmentation thus persists for many days after correction of neutropenia and restoration of normal granulocyte folate levels have occurred. We conclude that cobalamin and folate are needed at two different steps in granulopoiesis.     

Neutrophil myeloperoxidase measurement uncovers masked megaloblastic anemia

We report the observation of a high neutrophil myeloperoxidase activity (MPXI) in patients with megaloblastic anemia. MPXI is rapidly measured as part of an automated complete blood count (Technicon H*1, Technicon Instruments Corp, Tarrytown NY). We describe the range of MPXI levels in healthy and patient populations and in 10 cases of megaloblastic anemia, including five having elevated mean cell volume (MCV) and five without macrocytosis. Regardless of the MCV, our megaloblastic patients had hypersegmented neutrophils and elevated MPXI levels without visible alteration of granule content. MPXI measurement may be particularly useful in identifying cases of "masked megaloblastic anemia" where the MCV is below 100 fL. The advantage of the MPXI over other methods of uncovering masked megaloblastic anemia is its simplicity when performed as part of a routine complete blood count on an automated hematology instrument.     

Anemia in alcoholics

In order to develop a diagnostic approach to the common problem of anemia associated with alcoholism, 121 chronic alcoholics admitted to a general medical service with a low hematocrit were evaluated. Multiple contributing causes of anemia were present in most patients. Megaloblastic marrow change was found in 33.9% of patients, sideroblastic change in 23.1%, absent iron stores in 13.2%, aggregated macrophage iron in 81.0%, and acute blood loss in 24.8%. The MCV was of little value in predicting the presence of megaloblastic change unless markedly elevated (greater than 110 fl). In 15 of 41 patients with megaloblastic marrow morphology (36.6%) the MCV was normal or low. Among 40 patients with MCV values between 100 and 110 fl, megaloblastic change was not present in the bone marrow smears of 24 (60.0%). Neutrophil hypersegmentation was 95% specific but only 78% sensitive for megaloblastic change; in contrast, the presence of macroovalocytosis was 90% sensitive but only 68% specific. Serum lactic dehydrogenase, plasma folate, and erythrocyte folate levels had such low sensitivities and specificities for megaloblastic change as to be of little predictive value. Hematologic responses to folic acid were often inadequate in patients with megaloblastic morphologic changes, apparently because of associated acute and chronic illness. Our findings are consistent with the hypothesis that 2 mechanisms account for the development of megaloblastic hematopoiesis in alcoholics: induction of folate deficiency and a direct toxic effect of alcohol on erythroid precursors independent of folate depletion, as reflected by the presence of normal plasma and erythrocyte folate levels in several patients with megaloblastic change. In no patient was sideroblastic change the sole apparent cause of anemia. Megaloblastic hematopoiesis and aggregated macrophage iron frequently accompanied sideroblastic change. Examination of the blood smear revealed siderocytes in one-third of patients with sideroblastic marrows and dimorphic erythrocyte morphology in the majority. Dimorphic blood smears, however, were neither sensitive nor specific for sideroblastic change. Serum iron concentrations were usually not elevated in the group with sideroblastic abnormalities. In predicting marrow iron stores, serum iron and iron-binding capacity concentrations were often non-diagnostic or misleading. Serum ferritin levels less than 100 ng/ml, however, showed 100% sensitivity and 95% specificity for absent marrow iron stores despite the frequent presence of abnormal liver function. On the basis of our findings, practical guidelines have been formulated for the evaluation and therapy of anemia in alcohol     

Serum transferrin receptor in the megaloblastic anemia of cobalamin deficiency.

In order to further study the relation between transferrin receptor and erythropoiesis we examined serum receptor levels in megaloblastic anemia, which is the classic example of ineffective erythropoiesis. We studied 33 patients with unequivocal cobalamin deficiency, only 22 of whom were anemic. High serum transferrin receptor levels were found in 12 patients, all of whom were anemic and had high lactate dehydrogenase (LDH) levels; in contrast, only 10 of the 21 patients with normal receptor levels were anemic. Receptor correlated most strongly with LDH (r = 0.573, p < 0.001) and, inversely, with hemoglobin values (r = -0.560, p < 0.001); it also correlated with ferritin and total bilirubin levels, but not with cobalamin, MCV or erythropoietin. No association was found with the hemolytic component of megaloblastic anemia, represented indirectly by haptoglobin levels. Changes induced by cobalamin therapy were also examined in 13 patients. Transferrin receptors rose in all 6 patients who initially had high levels and in 2 of 3 patients who had borderline levels, but not in the 4 patients with initially normal levels. The receptor levels began to rise within 1-3 days, peaked at about 2 weeks and returned to normal at about the 5th wk. The findings indicate that serum transferrin receptor levels reflect the severity of the megaloblastic anemia. The elevated receptor levels rise further with cobalamin therapy, however, as effective erythropoiesis replaces ineffective erythropoiesis, and these persist until the increased erythropoiesis returns to normal.     

Diabetic acido-ketosis revealing thiamine-responsive megaloblastic anemia

Thiamine-responsive megaloblastic anemia (TRMA) is a rare autosomal recessive disorder characterized by megaloblastic anemia, diabetes mellitus and progressive sensorineural deafness. We report the cases of two infants, aged 4 and 5 months, hospitalized for diabetic ketoacidosis requiring insulin therapy. Laboratory tests revealed megaloblasic anemia, thrombocytopenia and normal thiamine level. Neurosensorial investigations showed bilateral deafness and ophthalmic involvement. Treatment with oral thiamine normalized hematological disorders and controlled diabetes; however, thiamine therapy had no impact on neurosensorial disorders.     

Arsenic intoxication as a cause of megaloblastic anemia

We have described a case of chronic arsenic intoxication associated with pancytopenia and megaloblastic erythropoiesis. The patient had the typical laboratory manifestations of effective erythorpoiesis due to a megaloblastic process, including macroovalocytes, mild pancytopenia, low reticulocyte index, increased marrow cellularity with erythroid hyperplasia, and morphologic evidence of megaloblastic maturation in the marrow. The patient's serum folate and vitamin B12 were normal, and the anemia regressed without therapy. Our case suggests that the combination of megaloblastosis with normoblastic or megaloblastic karyorrhexis,should raise the suspicion of arsenic intoxication in the mind of the observer. In addition, arsenic should be added to the list of agents causing a reversible megaloblastic anemia.     

Coexistence of Megaloblastic Anemia and Iron Deficiency Anemia in a Young Woman with Chronic Lymphocytic Thyroiditis

Pernicious anemia is a megaloblastic anemia caused by vitamin B12 deficiency, and is the end-stage of autoimmune gastritis that typically affects persons older than 60 years. It is the most common cause of vitamin B12 deficiency. Pernicious anemia can also be diagnosed concurrently with other autoimmune diseases. We report the occurrence of megaloblastic anemia in a 22-year-old woman with chronic autoimmune thyroiditis for 10.5 years. Recently, she presented with microcytic anemia, and iron deficiency anemia was diagnosed initially. After administration of ferrous sulfate, macrocytic anemia was revealed and vitamin B12 deficiency was detected. Pernicious anemia was highly suspected because of the endoscopic finding of atrophic gastritis, and high titer of antigastric parietal cell antibody, as well as elevated serum gastrin level. After intramuscular injections of hydroxycobalamine 100 microg daily for 10 days, and monthly later, her blood counts returned to normal.     

A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil

Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G.C --> A.U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and Pfu) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea.     

Thymidylate synthetase activity in bone marrow cells in pernicious anemia.

The tritium release assay for the demonstration of thymidylate synthetase activity has been applied to the measurement of enzyme activity in the bone marrow of four patients with pernicious anemia and nine normal subjects. On the average, an approximately ninefold increase in enzyme activity was observed in patients with pernicious anemia. In the absence of 5, 10-methylene-tetrahydrofolate, enzyme activity was reduced in both normal and in pernicious anemia cells. Addition of 5, 10-methylene-tetrahydrofolate to the assay medium resulted in a far greater activation of thymidylate synthetase activity in megaloblastic bone marrow cells than in the cells of control subjects.     

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.     

An N-Glycosidase from Escherichia coli That Releases Free Uracil from DNA Containing Deaminated Cytosine Residues

An enzyme that liberates uracil from single-stranded and double-stranded DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers in the absence of Mg(++) has been purified 30-fold from cell extracts of E. coli. The enzyme does not release uracil from deoxyuridine, dUMP, uridine, or RNA, nor does it liberate the normally occurring pyrimidine bases, cytosine and thymine, from DNA. The enzymatic cleavage of N-glycosidic bonds in DNA occurs without concomitant cleavage of phosphodiester bonds, resulting in the formation of free uracil and DNA strands of unaltered chain length that contain apyrimidinic sites as reaction products. The enzyme may be active in DNA repair, converting deaminated dCMP residues to an easily repairable form.