Reversible C3 Hypocomplementaemia in Megaloblastic Anaemia due to Vitamin B12 Deficiency

S ummary . Serum C3 and C4 levels have been determined in patients with Addisonian pernicious anaemia (PA) and megaloblastic anaemia due to vitamin B₁₂ deficiency from other causes, before and after treatment, in order to study the interaction between vitamin B₁₂ deficiency and Complement and the role of complement in the pathogenesis of the gastric lesion of PA. C₃ levels are significantly reduced in vitamin B₁₂ deficiency and return to normal on treatment; C₃ levels correlate with the degree of anaemia but not with serum vitamin B₁₂ levels at diagnosis. C₄ levels are normal. These observations suggest that the observed C₃ hypocomplementaenlia is not a consequence of immune mechanisms, but may be due to altered synthesis of C₃ complement component.     

Leucocyte Folate in Vitamin B12 and Folate Deficiency and in Leukaemia

Leucocyte folate concentrations were measured in 24 normal subjects, in 32 patients with subnormal serum folate concentrations and normal serum B₁₂ concentrations associated with chronic gastro-intestinal disease, in seven patients with leukaemia, and in 10 patients with untreated pernicious anaemia.
In the normal subjects, the leucocyte folate levels ranged from 60 to 123 ng./ml. of packed leucocytes. Among the patients with subnormal serum folate concentrations, leucocyte folate concentrations were normal in seven or eight patients with entirely normoblastic haemopoiesis, but were subnormal in all eight patients whose marrows showed normoblastic erythropoiesis and giant metamyelocytes, in all eight patients with obvious megaloblastic changes, and in all eight patients with overt megaloblastic anaemia due to folate deficiency.
Leucocyte folate was raised in six of the seven patients with leukaemia including patients with acute and chronic myeloid leukaemia, myelomonocytic leukaemia and chronic lymphatic leukaemia.
Of the 10 patients with untreated pernicious anaemia, three had raised, three had normal, and four subnormal leucocyte folate. The changes in leucocyte folate produced by B₁₂ therapy in four of the pernicious anaemia patients are described.     

Altered Erythrocyte Pyrimidine Activity in Vitamin B12 or Folate Deficiency

The net activity of orotidylic pyrophosphorylase and decarboxylase, sequential enzymes which catalyse the formation of uridine monophosphate from orotic acid in de novo pyrimidine biosynthesis, has been evaluated in erythrocytes of patients with folate or cobalamin deficiency. In patients with normoblastic haemopoiesis and normal cobalamin and folate status a direct relationship exists between the maturity of the peripheral blood erythrocyte population, as indicated by G6PD activity, and net orotidylic activity. In contrast in cobalamin or folate deficiency this co-ordinate relationship is not observed and net orotidylic activity is relatively reduced. Fractionation of erythrocytes by centrifugation demonstrates that this inordinately low orotidylic activity consistently occurs in the young erythrocyte population and is reversed by specific replacement therapy. In vitamin B12 or folate deficiency an impressive array of evidence now exists to implicate altered folate metabolism for the observed alterations in purine and pyrimidine metabolism (Das & Herbert, 1976). Of these changes the cornerstone defect resulting in megaloblastic maturation is impaired methylation of deoxyuridine monophosphate to thymidine monophosphate (Hoffbrand et al, 1976). In this context the reduced serum uridine plus uracil levels in patients with vitamin B12 deficiency (Parry & Blackmore, 1976) and the haematological response of these patients to orotic acid therapy (Rundles & Brewer, 1958) are not readily explicable. Since the conversion of orotic acid to uridine monophosphate depends on the integrity of the coupled activities of orotidylic pyrophosphorylase and decarboxylase, this study has quantitated this capacity in peripheral blood erythrocytes in patients with vitamin B12 or folate deficiency.     

Haemoglobin, Vitamin B12 and Folate Levels in the Elderly

S ummary . Community-based surveys of a representative sample of 533 subjects aged 65 yr and over were conducted in a mining valley and a seaside town in South Wales. Haemoglobin level, PCV, serum vitamin B₁₂, plasma and red-cell folate were measured and several simple tests of learing and memory were applied. The results give no evidence to suggest that anaemia is common and although low levels of vitamin B₁₂ and folate occurred no evidence of an associated impairment of health was detected.     

Effects of Dietary Methionine and Vitamin B12 Deficiency on Folate Metabolism

S ummary . Glutamate formimino transferase activity (GFA) and liver folic acid and vitamin B₁₂ levels were decreased in rats fed diets deficient in methionine and vitamin B₁₂. The addition of either methionine or vitamin B₁₂ resulted in a rise in GFA and in liver folate levels. However, L. casei serum folate was not affected by the dietary level of vitamin B₁₂ or methionine, but the S. faecalis serum folate decreased as the level of dietary methionine and vitamin B₁₂ was increased. The results of these studies do not appear to support the concept that vitamin B₁₂ deficiency results in a 'pile-up' of N⁵-methyl folate but suggests that it induces folate deficiency. Also the results suggest that the effect of vitamin B₁₂ on folate metabolism may be mediated via methionine metabolism.     

Megaloblastic Anaemia of Infancy and Vitamin B12

The haematological status of 30 infants with a syndrome characterized by satisfactory growth, abnormal pigmentation, developmental retardation, hypotonia of muscles, hepatosplenomegaly, with or without tremors, has been studied. Anaemia of a moderate degree was seen in all. All but four had megaloblastic erythropoiesis. Serum vitamin B₁₂ levels were below 100 pg./ml. By increasing the infants' intake of vitamin B_12>, by raising the concentration of the vitamin in maternal milk, megaloblastic erythropoiesis could be corrected to normoblastic erythropoiesis. The other clinical manifestations of the syndrome also responded to the administration of vitamin B₁₂ to varying degrees.     

Role of Vitamin B12 in Folate Coenzyme Synthesis

Normal red cells in man were found to contain predominantly folate pentaglutamates with smaller amounts of tetra- and hexapolyglutamates. There was no change in the type of polyglutamate present in red cells from patients with vitamin B12 deficiency and primary folate deficiency. In contrast to the fall in red cell polyglutamate concentration in vitamin B12 deficiency, there was a marked fall in short-chain folates in early folate deficiency (treated non-anaemic epileptics) and a fall in both short chain and long chain polyglutamates in patients with severe folate deficiency and megaloblastic anaemia. These differences in folate distribution within cells exclude a primary failure to transport methylfolate into cells as the lesion in vitamin B12 deficiency. The failure of folate polyglutamate synthesis in ivtamin B12 deficiency arises either from a failure to provide the proper substrate for polyglutamate synthesis or to a direct requirement for vitamin B12 for polyglutamate synthesis.     

The Excretion of Methylmalonic Acid and Succinic Acid in Vitamin B12 and Folate Deficiency

The excretion of methylmalonic acid (MMA) and of succinic acid was measured in 18 control subjects and in 58 patients with vitamin B₁₂ and/or folate deficiency. The 18 control subjects excreted from 0.0 to 3·5 mg. MMA in 24 hours. MMA excretion was raised in 27 of 41 patients (Group I) with conditions associated primarily with B₁₂ deficiency. These 27 patients included 15 patients with Addisonian pernicious anaemia, two patients following total gastrectomy, two patients with anatomical lesions of the small intestine and eight patients following partial gastrectomy, whereas MMA excretion was normal in four patients with atrophic gastritis and in a further 10 patients following partial gastrectomy. In Group I, MMA excretion was raised in all but one of the patients with serum B₁₂ levels less than 100 pg./ml. due to uncomplicated B₁₂ deficiency, but was invariably normal in patients with borderline serum B₁₂ levels (from 100 to 160 pg./ml.). In the patients with serum B₁₂ levels less than 100 pg./ml., with associated iron and/or folate deficiency, the excretion of MMA tended to be lower than in patients with uncomplicated B₁₂ deficiency, and was often normal. MMA excretion was slightly raised in one of 17 patients with megaloblastic anaemia, primarily due to folate deficiency (Group II), although six of them had serum B₁₂ levels less than 100 pg./ml. The patient with a raised MMA excretion needed B₁₂ therapy for a full haematological remission. The 18 control subjects excreted from 2.0 to 12.5 mg. succinic acid in 24 hours. Succinic acid excretion was subnormal in nine and raised in five patients in Group I. These 14 patients all had serum B₁₂ levels less than 100 pg./ml. None of the Group II patients excreted subnormal amounts of succinic acid but three, including two patients with serum B₁₂ levels less than 100 pg./ml., excreted raised amounts of succinic acid.     

Reversal of Ineffective Erythropoiesis in Pernicious Anaemia following Vitamin B12 Therapy

Ineffective erythropoiesis was quantitated in a series of patients with pernicious anaemia at different times in relation to vitamin B₁₂ therapy by measuring the incorporation of [¹⁵N]δ aminolaevulinic acid and [¹⁵N]glycine into early labelled bilirubin. Prior to therapy ineffective erythropoiesis was grossly increased but this was reversed within 24 h of giving vitamin B₁₂, suggesting that most of the existing megaloblasts are enabled to mature into circulating red cells.     

Vitamin B12 Absorption by Inhalation

Cyanocobalamin in the form of an aerosol was inhaled by normal subjects, patients with pulmonary diffusion defects and patients with vitamin B₁₂ deficiency. In all groups there was a rapid increase in serum B₁₂ levels suggesting that absorption had occurred by diffusion through the lung alveoli.
Six patients with pernicious anaemia given daily inhalations showed complete clinical and haematological remission and a group of six patients with B ₂ deficiency were maintained in remission for 12 months. While effective, this method of administration of vitamin B₁₂ is not regarded as superior to that of injections and is therefore considered to have no therapeutic application. The possibility of inducing pulmonary damage is considered.     

Absorption of Vitamin B12 in Patients with Anaemia after Polya Partial Gastrectomy

The absorption of radioactive vitamin B₁₂ (cyanocobalamin) has been studied by the Schilling procedure in 27 patients with anaemia after Polya partial gastrectomy and in 10 patients with Addisonian pernicious anaemia.
In patients who have had Polya partial gastrectomy absorption of vitamin B₁₂ was often increased when the dose was given with a meal containing only a small quantity of the vitamin, and when the dose was given after an injection of histamine.
In the patients with pernicious anaemia histamine did not enhance absorption of vitamin B₁₂, and food did not alter the absorption of the vitamin when given with a small quantity of intrinsic factor.
Improved absorption of vitamin B₁₂ with food or after histamine is likely to be due to stimulation of a small residual secretion of intrinsic factor.
The results of the standard fasting Schilling test appeared to correlate better with the development of deficiency of vitamin B₁₂ than tests with food or after histamine.     

Observations on Folate Metabolism in Rats on a Vitamin B12 -Deficient Diet

Unless they were starved, rats fed on a vitamin B₁₂-deficient diet had significantly higher levels of folate activity for Lactobacillus casei in the plasma than controls receiving the same diet supplemented with cyanocobalamin. The folate activity in the livers of the deficient animals was significantly lower than in the controls, whether the rats were fed or starved. There was a significant linear correlation between the concentrations of folate activity and vitamin B₁₂ activity in liver.
No consistent differences were found between the rates of disappearance of intravenously-injected [5-¹⁴C]methyl tetrahydrofolate in vitamin B₁₂-deficient and -supplemented rats. The latter results do not support the 'methylfolate block' hypothesis.     

Cobalamin Dependent Methionine Synthesis and Methyl-Folate-Trap in Human Vitamin B12 Deficiency

The activity of methionine synthetase (MS) is important for the rapid growth of human haematopoietic cells and cultured lymphoblastoid cells. The MS reaction is the only known metabolic step in which both vitamin B₁₂ and folate are essential in a single enzyme reaction. In vitamin B₁₂ deficiency the MS activity in bone marrow cells is significantly lower than that in normal bone marrow. Free tetrahydrofolic acid (H₄PteGlu) is normally liberated from its metabolically inactive storage form, 5-methyl-H₄PteGlu (CH₃H₄PteGlu), in the cobalamindependent MS reaction. Thus, in vitamin B₁₂ deficiency H₄PteGlu is not available in sufficient concentration to maintain the de novo synthesis of thymidylate and purines, and accords with the methyl-folate-trap hypothesis. After treatment with amethopterin (Methotrexate^TM), the incorporation of ³Hdeoxyuridine into cellular DNA is reduced. In proliferating normal cells this effect of methotrexate can be prevented (and the cells rescued) with CH₃-H₄PteGlu or with CHO-H₄PteGlu (5-formyl-H₄PteGlu; Leucovorin^TM). On the other hand, in vitamin B₁₂ deficient bone marrow cells this so-called rescue-effect could only be achieved with CHO-H₄PteGlu and not with CH₃-H₄PteGlu. These observations also support the hypothesis of the methyl-folate-trap in vitamin B₁₂ deficiency. Decreased MS activity in vitamin B₁₂ deficiency seems to be the essential metabolic fault, which is responsible for secondary alterations of folate metabolism. Thus, measurement of MS activity may allow direct functional assessment of vitamin B₁₂ deficiency, at least with regard to DNA metabolism.     

Serum 'Uracil+Uridine'Levels Before and After Vitamin B12 Therapy in Pernicious Anaemia

S ummary . The serum 'uracil+uridine'levels, expressed as uracil, have been measured in 10 cases of pernicious anaemia both before and after treatment, and compared with the levels in 97 normal subjects. The mean pre-treatment value (8.82 μmol/l., range 6.0–12.0 μmol/l.) differed significantly from that of the normal controls (15.7 μmol/l., range 5.7–40.5 μmol/1., t = 8.8, P <0.001). This confirms the low serum uracil level previously reported in pernicious anaemia in relapse. The level rose progressively after treatment, reaching a maximum on the fourth day (mean 17.85 μmol/1., range 9.3–23.4 μmol/l.). This was not significantly different from the mean of the normal control group. The difference between the pre- and post-treatment levels was significant on days 3, 4 and 5 ( P <0.005, P <0.001 and P <0.005 respectively) and the rise preceded the reticulocyte response by 24 h. A further case was treated with physiological doses of vitamin B₁₂ (2 μg daily for 6 d) and a similar rise in the serum uracil level noted.
These results are not explained by any of the known functions of vitamin B₁₂. They are, however, similar to the changes in the serum methionine levels previously reported in pernicious anaemia. The latter were readily explained by the known action of vitamin B₁₂ on ' de novo 'methionine synthesis and it is suggested that the synthesis of uracil, like that of methionine, might be influenced by vitamin B₁₂ in man.     

Absorption and Transport of Vitamin B12

A review of recent advances is presented, mostly dealing with physiological mechanisms and the chemical and functional properties of the transport proteins. A new nomenclature is proposed for the R-proteins and transcobalamin II. Scepticism is expressed as to whether we are warranted in accepting the different R-proteins as separate entities.     

Forms of Vitamin B12 in Blood and Bone Marrow in Patients with Pernicious Anaemia

The biologically-active forms of vitamin B₁₂ in blood and bone marrow and changes induced in these by injections of cyanocobalamin have been measured in patients with pernicious anaemia. Bone marrow methylcobalamin was low before therapy, and increased 24 h after therapy. The largest portion of bone marrow vitamin B₁₂ was 5′deoxyadenosylcobalamin, and this increased more than did methylcobalamin during the 24 h after injection of cyanocobalamin. A single injection of roo μ of cyanocobalamin induced about 10 times the increase of the intracellular coenzyme forms of vitamin B₁₂ in bone marrow than followed injection of 100 μg. Plasma methylcobalamin was extremely low before therapy, and increased only moderately 24 h after therapy; the majority of plasma vitamin B₁₂ remaining as cyanocobalamin. In contrast, only a minority of intracellular bone marrow vitamin B₁₂ was cyanocobalamin 24 h after injection of cyanocobalamin. The degree of anaemia did not correlate with bone marrow methylcobalamin, nor did bone marrow cobalamin correlate significantly with cobalamin content of washed blood erythrocytes. Correlation was observed between intra-erythrocyte vitamin B₁₂ content and the degree of anaemia; the correlation being inverse with haemoglobin concentration in the peripheral blood. Inverse correlation also was observed between MCV and erythrocyte folate content. These studies suggest that megaloblastic maturation appears at different concentrations of bone marrow vitamin B₁₂ in different patients, presumably because in vitamin B₁₂ deficiency, eventual limitation of normoblastic maturation may be determined by factors such as folate metabolism, vitamin B₁₂ binders, and the affinity of vitamin B₁₂ dependent enzymes. Although the clinical response to 1000 μg of cyanocobalamin does not differ from that to 100 μg, the concentration of vitamin B₁₂ coenzymes in bone marrow cells was proportional to the cyanocobalamin injected.     

Vitamin B12 Binding Proteins in Liver Disease

Vitamin B₁₂ binding proteins were studied in patients with acute and chronic liver disease, and compared with vitamin B₁₂ binders in chronic myeloid leukemia. In acute viral hepatitis marked elevation of serum vitamin B₁₂ was common. Although the serum vitamin B₁₂ rose as high as that found in chronic myeloid leukaemia the unsaturated vitamin B₁₂ binding capacity (UBBC) was markedly elevated in the latter condition, whereas it was decreased in acute liver disease. In cirrhosis a moderate increase of serum vitamin B₁₂ and UBBC was common. Urinary vitamin B₁₂ excretion increased significantly only when the serum vitamin B₁₂ became markedly elevated in hepatitis. As in chronic myeloid leukaemia the alpha-globulin vitamin B₁₂-binder carried the bulk of the elevated serum vitamin B₁₂ in acute liver disease; the beta-globulin vitamin B₁₂-binder was decreased in serum but seemed to be the predominant binder in urine. This suggests that renal loss of beta-globulin binder is greater than renal loss of alpha-globulin binder. Only minor albumin binding of vitamin B₁₂ in liver disease was found in spite of marked elevation of the serum vitamin B₁₂ level. In cirrhosis the serum alpha-globulin binder was often increased and beta-globulin binder decreased. The cause and significance of these findings are discussed. The ability of serum vitamin B₁₂-binders of liver disease to ‘transfer’⁵⁷Co-B₁₂ to tissue was investigated in an in vitro rat liver homogenate system. In a limited study, vitamin B₁₂ uptake appeared to be within normal limits, except for suboptimal uptake from the beta-globulin binder in cirrhotic serum. Poor vitamin B₁₂ uptake from chronic myeloid leukaemia serum (in particular alpha-globulin binder) was confirmed.