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

Abstract 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.

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

Blood ◽

10.1182/blood.v83.6.1656.bloodjournal8361656 ◽

1994 ◽

Vol 83 (6) ◽

pp. 1656-1661 ◽

Cited By ~ 2

Author(s):

SN Wickramasinghe ◽

S Fida

Keyword(s):

Bone Marrow ◽

Vitamin B12 ◽

Bone Marrow Cells ◽

Megaloblastic Anemia ◽

Folate Deficiency ◽

Test Results ◽

Dnase 1 ◽

Biochemical Lesion ◽

Suppression Test ◽

Marrow Cells

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.

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Could Cobalamin Deficiency Mimic a Clonal Aberration Cytogenetic? a Case Report

Blood ◽

10.1182/blood.v124.21.4882.4882 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4882-4882

Author(s):

Marcelo Bellesso ◽

Daniela Ferreira Dias ◽

Renato Torrescasana Centrone ◽

Laura Yolanda Chialanza Garcia ◽

Annelise Correa Wengerkievicz Lopes ◽

...

Keyword(s):

Bone Marrow ◽

Vitamin B12 ◽

Cytogenetic Analysis ◽

Bone Marrow Aspirate ◽

Bone Marrow Cells ◽

Megaloblastic Anemia ◽

Cobalamin Deficiency ◽

Normal Range ◽

Clonal Deletion ◽

Marrow Cells

Abstract A 32 years old female patient presented to our service complaining asthenia, apathy and loss of her power to work. It was observed, by laboratory test, pancytopenia (Hb: 6.0 g/dL; WBC: 2,100/mm³, platelets 95,000/mm³, Reticulocytes 0.8%) associated with marked elevation of LDH 1,800 U/L (normal range: 240 – 480U/L). Bone marrow aspirate demonstrated morphologic features of megaloblastic anemia. Moreover, low serum concentration of vitamin B12 < 150pg/mL (normal range: 200 – 95-pg/mL), Folic acid 15,9ng/mL (normal range: 3 – 17 ng/mL) confirmed Megaloblastic anemia by cobalamin deficiency. It was not evidenced gastritis. It was initiated the treatment with vitamin B12. However, in the next clinical attendance it was observed an unexpected cytogenetic result: 46,XX,del(9)(q13q22)[3]/76~78,XXX,+1,+3,+4,+9,+11,+12,+17,+20,+21[cp3]/46,XX[34]. It was interpreted that del(9)(q13q22)[3] could be a clonal cytogenetics aberration and the others data due to defects in synthesis of DNA by cobalamin deficiency. It was important, because in diagnosis of Megaloblastic anemia frequently it is not included cytogenetic analysis of bone marrow cells and a new cytogenetic analysis has become necessary due to this data. After two months, a complete hematological recovery was achieved and six months later, bone marrow aspirate and cytogenetic analisys were repeated. Normal morphologic bone marrow cells and normal cytogenetic: 46,XX[20] were evidenced. Therefore, it was really hard to conclude this case. First, cobalamin deficiency could promote this clonal deletion or, as a second hypothesis, a clonal cytogenetic with low proliferative rate was selected due to inefficient hematopoiesis, by vitamin B12 deficiency, and after the recovery of the hematopoiesis, this clonal was suppressed. Disclosures No relevant conflicts of interest to declare.

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5-Methyltetrahydrofolate related enzymes and DNA polymerase alpha activities in bone marrow cells from patients with vitamin B12 deficient megaloblastic anemia

Blood ◽

10.1182/blood.v59.4.832.bloodjournal594832 ◽

1982 ◽

Vol 59 (4) ◽

pp. 832-837

Author(s):

Y Kano ◽

S Sakamoto ◽

K Hida ◽

K Suda ◽

F Takaku

Keyword(s):

Bone Marrow ◽

Vitamin B12 ◽

Dna Polymerase ◽

Methylenetetrahydrofolate Reductase ◽

Bone Marrow Cells ◽

Megaloblastic Anemia ◽

Vitamin B12 Deficiency ◽

Dna Polymerase Alpha ◽

B12 Deficiency ◽

Marrow Cells

The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10- methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10- methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.

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5-Methyltetrahydrofolate related enzymes and DNA polymerase alpha activities in bone marrow cells from patients with vitamin B12 deficient megaloblastic anemia

Blood ◽

10.1182/blood.v59.4.832.832 ◽

1982 ◽

Vol 59 (4) ◽

pp. 832-837 ◽

Cited By ~ 8

Author(s):

Y Kano ◽

S Sakamoto ◽

K Hida ◽

K Suda ◽

F Takaku

Keyword(s):

Bone Marrow ◽

Vitamin B12 ◽

Dna Polymerase ◽

Methylenetetrahydrofolate Reductase ◽

Bone Marrow Cells ◽

Megaloblastic Anemia ◽

Vitamin B12 Deficiency ◽

Dna Polymerase Alpha ◽

B12 Deficiency ◽

Marrow Cells

Abstract The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10- methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10- methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.

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The effect of folate analogues and vitamin B12 on provision of thymine nucleotides for DNA synthesis in megaloblastic anemia

Blood ◽

10.1182/blood.v59.3.634.bloodjournal593634 ◽

1982 ◽

Vol 59 (3) ◽

pp. 634-640

Author(s):

MR Taheri ◽

RG Wickremasinghe ◽

BF Jackson ◽

AV Hoffbrand

Keyword(s):

Vitamin B12 ◽

De Novo ◽

Megaloblastic Anemia ◽

Vitamin B12 Deficiency ◽

Folate Deficiency ◽

Minor Role ◽

B12 Deficiency ◽

Thymidylate Synthesis ◽

Marrow Cells

The role of vitamin B12 in the folate dependent biosynthesis of thymidine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting ‘de novo’ thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)-thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6–3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6–3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, N6-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl-FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl- FH4.

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Incorporation of Radioactive Precursors into the Nucleic Acids of Bone Marrow Cells from Patients with Vitamin B12 or Folate Deficiency

Acta Haematologica ◽

10.1159/000207398 ◽

1980 ◽

Vol 63 (4) ◽

pp. 196-203 ◽

Cited By ~ 9

Author(s):

S.N. Wickramasinghe ◽

J.E. Sounders

Keyword(s):

Bone Marrow ◽

Nucleic Acids ◽

Bone Marrow Cells ◽

Folate Deficiency ◽

Vitamin B ◽

Marrow Cells

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The Value of Duodenal Biopsies in the Evaluation of Megaloblastic Anemia

Journal of Laboratory Physicians ◽

10.1055/s-0041-1730817 ◽

2021 ◽

Author(s):

Nasrin Nisha N. ◽

Sakthisankari Shanmugasundaram ◽

Kartikayan R. K.

Keyword(s):

Bone Marrow ◽

Vitamin B12 ◽

Megaloblastic Anemia ◽

Serum Vitamin ◽

Folate Deficiency ◽

Duodenal Biopsy ◽

Tropical Sprue ◽

Bone Marrow Biopsies ◽

Endoscopic Findings ◽

Duodenal Biopsies

Abstract Introduction Megaloblastic anemia is one of the common causes of anemia in India. Duodenal biopsies are routinely performed in the investigation of megaloblastic anemia. The present study was undertaken to analyze the value of duodenal biopsy in megaloblastic anemia and to correlate endoscopic findings with biopsy. As a secondary aim, the study has also analyzed the hematological profile and vitamin B12 and folate status of these patients. Materials and methods All the cases of megaloblastic anemia with bone marrow studies diagnosed at PSG Institute of Medical Sciences and Research during the two year period from January 2016 to December 2017 were retrieved. Clinical and laboratory findings (serum vitamin B12 and folate levels) and endoscopic findings were retrieved from hospital records of the patients. Duodenal biopsies of these patients reported in the histopathology department were retrieved and reviewed. Statistical analysis was done using SPSS software 20.0. Results There were 93 cases of megaloblastic anemia diagnosed on bone marrow biopsies. Tropical sprue was diagnosed in 49.5% of cases, followed by intraepithelial lymphocytosis (17.2%), peptic duodenitis (17.2%), and no significant pathology in 16% of cases. Pancytopenia was present in 54.8% of cases. Isolated vitamin B12 deficiency including low levels was present in 48.38% and folate deficiency was seen in 4.3% cases; 34.48% cases had both vitamin B12 and folate deficiency. Conclusion The incidence of tropical sprue in megaloblastic anemia is 49.5% in the study. Duodenal biopsy is valuable in the work up of megaloblastic anemia, irrespective of the endoscopic changes in identifying the etiology.

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Synthesis and secretion of the human vitamin B12-binding protein, transcobalamin II, by cultured skin fibroblasts and by bone marrow cells

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/0167-4889(85)90207-1 ◽

1985 ◽

Vol 845 (3) ◽

pp. 421-427 ◽

Cited By ~ 26

Author(s):

Marijke Fràter-Schröder ◽

Henk J. Porck ◽

Jale Erten ◽

Marianne R. Müller ◽

Beat Steinmann ◽

...

Keyword(s):

Bone Marrow ◽

Vitamin B12 ◽

Bone Marrow Cells ◽

Binding Protein ◽

Skin Fibroblasts ◽

Cultured Skin ◽

Marrow Cells ◽

Transcobalamin Ii

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Studies on the Uptake of Synthetic Conjugated Folates by Human Marrow Cells

Blood ◽

10.1182/blood.v42.1.141.141 ◽

1973 ◽

Vol 42 (1) ◽

pp. 141-146 ◽

Cited By ~ 15

Author(s):

A. V. Hoffbrand ◽

Edith Tripp ◽

Catherine M. Houlihan ◽

J. M. Scott

Keyword(s):

Bone Marrow ◽

Folic Acid ◽

Dna Synthesis ◽

Lactobacillus Casei ◽

Bone Marrow Cells ◽

Megaloblastic Anemia ◽

Chemotherapeutic Agents ◽

Hemopoietic Cells ◽

Marrow Cells

Abstract Pteroyltriglutamate shows little abiility (between 1% and 5%), compared to pteroylmonoglutamate (folic acid), to enter human marrow cells and to act as a coenzyme in intracellular DNA synthesis. This was shown by comparing the effectiveness of these two forms of the vitamin at stimulating folate-dependent pyrimidine incorporation into DNA in vitro in the bone marrow cells and lymphocytes of patients with megaloblastic anemia. It, therefore, appears that human hemopoietic cells (like Streptococcus fecalis rather than Lactobacillus casei) are unable to take up efficiently polyglutamyl forms of folate. The suggestion that polyglutamyl analogues of the antifolates might be more effective chemotherapeutic agents than corresponding monoglutamates, while biochemically possible, would appear to be precluded because of failure of transport of these compounds into human hemopoietic cells.

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