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.     

Nutrition in the Australian Aborigines—Effects of the fortification of white flour

Summary: The nutritional status of 66 part Aborigines was re-examined in 1974—with particular reference to blood levels of haemoglobin and vitamins—after white bread fortified with iron and the vitamins B₁ and PP (niacin) had been available for six and a half months to the population of Bourke, New South Wales. The results found in 1971 and 1974 are compared. A significant improvement from deficient to acceptable blood levels of vitamins B₁ and B₆ was found in 44% and 52% of the subjects respectively. This is attributed to the consumption of fortified bread since the levels of the other vitamins had remained either unchanged or worsened. The biochemical improvement in vitamin B₆ is attributed to the sparing effect of vitamin PP on vitamin B₆ requirement because the conversion of tryptophan to niacin is impaired in vitamin B₆ deficiency. Iron deficiency anaemia in children had decreased by 50% but this could have been due to many other factors besides the iron which had been added to the bread. Clinically there was a marked decrease in angular stomatitis and skin xerosis which could be related to the biochemical improvement of the two B-vitamins and a decrease in active trachoma and suppurative otitis media probably due to intensive treatment received since 1971. The results of this study and the extent of biochemical vitamin B₁ and B₆ deficiency found in other groups, indicate that fortification of bread may be of benefit to the community as a whole.     

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.     

Serum 'Uracil+Uridine'Levels in Pernicious Anaemia

S ummary . The serum 'uracil+ uridine'level, expressed as uracil, has been measured in 21 cases of vitamin B₁₂ deficiency, in which the serum folate was normal, and compared with the level in 97 normal subjects. The level in the vitamin B₁₂ deficient group (11.9 μmol/l.) was significantly lower than in the controls (15.7 μmol/l., P < 0.005). Nine of the former were complicated by systemic illness but the clinical and haematological features in the remaining 12 were consistent with the diagnosis of pernicious anaemia in relapse. The serum uracil level in this group was even lower (10.21 μmol/l., P < 0.01). This finding is unexpected in view of the generally accepted indirect role of vitamin B₁₂ in the methylation of deoxyuridine mono-phosphate to deoxythymidine monophosphate. Reasons are given for not accepting these results as reflecting the main biochemical lesion in vitamin B₁₂ deficiency. Although they do not give direct support to an impairment in the methylation of deoxyuridine monophosphate, they do not exclude it as they test only one possible metabolic pathway and moreover they could represent the result of more than one action of vitamin B₁₂ on uracil metabolism. They do show, however, that some aspect of uracil metabolism other than methylation is affected in vitamin B₁₂ deficiency in man.     

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.     

Vitamin B12 deficiency is the primary cause of megaloblastic anaemia in Zimbabwe

Summary. In a study of the pathogenesis and clinical features of megaloblastic anaemia in southern Africa, we evaluated 144 consecutive Zimbabwean patients with megaloblastic haemopoiesis. Vitamin B₁₂ deficiency was diagnosed in 86.1% of patients and was usually due to pernicious anaemia; isolated folate deficiency accounted for only 5/5% of cases. Anaemia was present in 95.8% of patients; the haemoglobin (Hb) was 6 g/dl in 63.9%. Neurological dysfunction was noted in 70.2% of vitamin B₁₂-deficient patients and was most striking in those with Hb values > 6 g/dl. Serum levels of methylmalonic acid, homocysteine, or both, were increased in 98.5% of patients.
Vitamin B₁₂ deficiency is the primary cause of megaloblastic anaemia in Zimbabwe and, contrary to textbook statements, is often due to pernicious anaemia. Isolated folate deficiency is less common. As reported in industrialized countries 75 years ago, anaemia is almost always present and often severe. Neurological dysfunction due to vitamin B₁₂ deficiency is most prominent in patients with mild to moderate anaemia.     

Uptake of Vitamin B12 by Phytohaemagglutinin-Transformed Lymphocytes

Phytohaemagglutinin (PHA)-transformed lymphocytes have been used as a model cell system to study the uptake of radioactive vitamin B₁₂ by haemopoietic cells. Both mature granulocytes and PHA-transformed lymphocytes took up more vitamin B₁₂ than mature, non-transformed lymphocytes. Uptake of vitamin B₁₂ by PHA-stimulated lymphocytes was greatest at 48–72 hr of culture, i.e. at about the time or just before the time of peak DNA synthesis.
Vitamin B₁₂ deficient lymphocytes took up significantly less vitamin B₁₂ than normal lymphocytes. Folate deficient lymphocytes took up an average of about 50% more vitamin B₁₂ than normal but the difference was not statistically significant for the numbers tested. Vitamin B₁₂ uptake by PHA-stimulated lymphocytes was related to their rate of RNA synthesis (measured by ³H-uridine uptake) and was closely related to active cytoplasmic protein synthesis since it was rapidly inhibited by puromycin and cycloheximide.     

Coexistence of Pernicious Anaemia and Chronic Myeloid Leukaemia: An Experiment of Nature Involving Vitamin B12 Metabolism

S ummary . Chronic myeloid leukaemia (CML) and pernicious anaemia (PA) coexisted in a 51-yr-old man (who also had myasthenia gravis). His serum vitamin B₁₂ level was normal instead of the high value expected in CML, or the low value expected in PA. Short-term culture of his peripheral blood cells showed deranged DNA synthesis of the type observed in vitamin B₁₂ deficiency, indicating that his 'normal' serum vitamin B₁₂ level was not associated with normal amounts of vitamin B₁₂ available to his CML cells. A tracer dose of 0.45 μg of[⁵⁷ Co]vitamin B₁₂ disappeared abnormally slowly from his serum over 24 hr (as previously observed by others in patients with either CML or PA). A therapeutic injection of vitamin B₁₂ appeared to disappear abnormally slowly from his serum over a period of 2 yr. His serum showed a markedly elevated unsaturated vitamin B₁₂ binding capacity, with a less sharp increase in percentage of vitamin B₁₂ binding α as compared to β globulin than usually expected in CML.
Withholding therapeutic vitamin B₁₂ for 2 yr has been associated with stabilization of his WBC at the 40 000-50 ooo/μl range. Although this association could be chance rather than cause and effect, the patient does represent an 'experiment of nature' involving vitamin B₁₂ metabolism and does raise the question as to whether his leukaemia is retarded by his deficiency of vitamin B₁₂, which results from inadequate absorption of vitamin B₁₂ and possibly from elevated serum vitamin B₁₂ binding a globulin rendering his circulating vitamin B₁₂ metabolically inert.     

Solid-phase vitamin B12 assays using polyacrylamide-bound intrinsic factor and polyacrylamide-bound R-binder

S ummary . A new solid phase vitamin B₁₂ assay is described using intrinsic factor to measure microbiologically-available B₁₂ and R-binder to measure total B₁₂. The solid phase reagent consists of intrinsic factor coupled to polyacrylamide beads and salivary R-binder coupled to polyacrylamide beads. The assay is simple to perform and separates completely sera from controls and patients with megaloblastic anaemia due to B₁₂ deficiency.     

Methylmalonic Acid Excretion Studies

S ummary . Oral valine and isoleucine were equally effective in producing an increased urinary excretion of methylmalonic acid (MMA) in vitamin B₁₂ deficiency but oral MMA itself, methionine and leucine were unsatisfactory.
MMA excretion was increased in 16 out of 23 patients with vitamin B₁₂ deficiency after valine and/or isoleucine loading but remained normal in seven patients. These latter patients tended to have the highest serum vitamin B₁₂ levels and were the least anaemic. Thus, normal MMA excretion cannot be used as evidence to exclude vitamin B₁₂ deficiency. The excretion was within normal limits in patients with hepatic cirrhosis and in patients with folate deficiency.     

Correction of the DNA synthesis defect in vitamin B12 deficiency by tetrahydrofolate: evidence in favour of the methyl-folate trap hypothesis as the cause of megaloblastic anaemia in vitamin B12 deficiency

Summary. The critical disturbance of folate metabolism caused by vitamin B₁₂ deficiency which results in megalo- blastic anaemia remains controversial. Vitamin B₁₂ is required in the methionine synthase reaction in which homocysteine is converted to methionine and methyl tetrahydrofolate (methyl THF) to THF. The 'methyl-folate trap'hypothesis suggested that failure of demethylation of methyl THF with consequent deficiency of folate co-enzymes derived from THF is the crucial lesion caused by vitamin B₁₂ deficiency. A more recent theory suggested that reduced supply of methionine leads to reduced availability of 'activated formate'and hence of formyl THF and it is this defect that results in failure of folate co-enzyme synthesis. The present results, based on deoxyuridine suppression tests on 103 cases of megaloblastic anaemia, show that THF itself is equally capable of correcting the failure of thymidylate synthesis in vitamin B₁₂ deficiency as in folate deficiency. Although not as effective as formyl THF in correcting the dU blocking test in vitamin B₁₂ deficiency, this is equally so for the correction of the test by THF compared with formyl THF in folate deficiency. The results therefore favour the theory that it is in the supply of THF and not of 'active formate'or formyl THF that vitamin B₁₂ plays a critical role in folate metabolism.     

Haematological aspects of life with an ileostomy

S ummary . The haematological aspects of life with an ileostomy have been studied in 51 patients, of whom 39 had had ulcerative colitis and 12 had Crohn's dissease. The findings in these patients have been compared with those in 39 healthy volunteers who were matched for age and sex with the 39 patients who had had ulcerative colitis.
There was evidence of a mild degree of iron deficiency in the patients with an ileostomy. This was partly due to a pre-existing iron deficiency resulting from their preceding illness and operation, as the abnormality was less pronounced in the patients in whom the ileostomy had been established for more than 3 years. There was some evidence of excessive iron loss and a controlled trial of ferrous fumurate showed that the iron deficiency was largely corrected by this means. Circulating levels of vitamin B₁₂ were normal, but it is relevant that some of the Crohn's disease group were receiving parenteral supplements. The absorption of vitamin B₁₂ was low in the patients with Crohn's disease who had had an ileal resection of more than 17 cm. The absorption of vitamin B₁₂ in the patients who had had ulcerative colitis was increased and possible mechanisms are discussed. All but one of the patients had normal levels of plasma folate and in all the red cell blood folate was normal, which can be taken as an indication of a good dietary intake and adequate absorption.     

Effects of Chronic Alcohol Consumption on Enzyme Activities and Active Methionine Absorption in the Small Intestine of Pregnant Rats

The present study evaluates the effect of chronic alcohol intake on the intestinal transport of methionine during pregnancy. For this purpose, we have used an in vitro technique that allows measurement of the unidirectional influx of the amino acids across the brush-border membrane of the rat mid-jejunum, and the basolateral membrane enzyme Na⁺,K⁺-ATPase was also evaluated in the duodenum and jejunum. For chronic alcohol treatment, the rats were fed a liquid diet containing ethanol (36% of calories) or an isocaloric diet (pair-fed control) for 5 weeks before and during pregnancy. Animals were killed at 21 days of gestation. Results from the kinetic analysis revealed that chronic ethanol treatment reduces the maximum transport (J_m) of methionine uptake when compared with controls. Further experiments performed in the presence and absence of sodium have shown that ethanol selectively inhibited Na⁺-dependent methionine transport. At the same time, this treatment significantly reduced the levels of Na⁺,K⁺-ATPase in ethanol-fed rats compared with the controls. Alterations in methionine intestinal transport in pregnant alcohol-fed rats may contribute to the ethanol-induced fetal growth abnormalities.     

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.     

Inhibitory Effect of Eggs on Vitamin B12 Absorption: Description of a Simple Ovalbumin 57Co-Vitamin B12 Absorption Test

S ummary . Ovalbumin and egg yolks, mixed separately in vitro with radiocyanocobalamin (⁵⁷Co-vitamin B₁₂), were served to normal volunteers in a cooked form. Ovalbumin, and to a lesser degree, egg yolks were observed to inhibit vitamin B₁₂ absorption. This observation explains the rather poor assimilation of vitamin B₁₂ from eggs labelled in vivo with ⁵⁷Co-vitamin B₁₂.
The assimilation of vitamin B₁₂ from the ovalbumin ⁵⁷ Co-vitamin B₁₂ mixture was also measured in patients with structural alterations and functional disorders of the stomach. In contrast to the absorption test using crystalline radio-vitamin B₁₂, this simple food-vitamin B₁₂ absorption test successfully distinguished between the controls and subjects who had simple gastric achlorhydria and patients who had undergone a Billroth I or II operation or a successful vagotomy with pyloroplasty. Furthermore, this ovalbumin ⁵⁷Co-vitamin B₁₂ test successfully separated patients with pernicious anaemia from those with simple gastric achlorhydria. The ovalbumin ⁵⁷Co-vitamin B₁₂ test, in contrast to that using crystalline ⁵⁷Co-vitamin B₁₂, showed a positive correlation with the serum vitamin B₁₂ concentration. This ovalbumin⁵⁷ Co-vitamin B₁₂ test, being more physiologic and informative than the method using crystalline radio-vitamin B₁₂, is the first simple model for food-vitamin B₁₂ absorption to be proposed for research and for clinical purposes.     

In Vitro DNA Synthesis by Bone Marrow Cells and PHA-stimulated Lymphocytes. Suppression by Nonradioactive Thymidine of the Incorporation of 3H-Deoxyuridine into DNA: Enhancement of Incorporation when Inadequate Vitamin B12 or Folate is Corrected

Previous studies demonstrated that excess deoxyuridine (dU) added to short-term bone marrow and PHA-stimulated lymphocyte cultures, blocks the incorporation of radioactive thymidine into DNA via the salvage pathway. In the current study, we investigated the effects of added thymidine (TdR) in varying concentrations (10⁻⁶to 1 μmol) on the incorporation of ³H-dU into thymine-DNA, i.e. we executed 'thymidine suppression tests.'Increasing concentrations of exogenous TdR caused progressive inhibition of ³H-dU incorporation into DNA, and decreasing ³H-dU incorporation was parallelled by increasing incorporation of added ¹⁴C-TdR. These findings demonstrate reciprocity of the salvage and the de novo pathways of thymine-DNA synthesis, presumably mediated by thymidine-triphosphate (dTTP), the common end product of both pathways, via feedback inhibition. In patients with folate and/or vitamin B₁₂ deficiency, the addition of appropriate vitamins to marrow and lymphocyte cultures enhanced the incorporation of ³H-dU into DNA. As predicted, this was not observed in normal subjects. The enhancing effect of these vitamins on in vitro incorporation of ³H-dU into DNA by deficient cell systems was similar to their correcting effect on abnormal dU suppression. These findings support the theoretical concept that the dU suppression test defines biochemical megaloblastosis due to deficiency of folate and vitamin B₁₂.     

The Content of Vitamin B12 in Adult and Foetal Tissue: A Comparative Study

S ummary . The vitamin B₁₂ content of 17 tissues in 28 adults and 17 foetuses was measured by a Euglena gracilis bioassay. The former were taken after sudden deaths, many accidental, and the latter after therapeutic abortions for mild psychiatric reasons. In the adult, the liver had the highest level of vitamin B₁₂ followed by the kidney, adrenal, pancreas and heart. The tissues of greatest vitamin B₁₂ concentration in the foetus were the liver, adrenal, kidney and lung. Foetal spleen, lung and colon were significantly higher than their adult counterparts, while foetal liver, kidney, heart and brain were significantly lower in comparison with the adult. Differences in vitamin B₁₂ levels may reflect adult and foetal differences in circulation, metabolic maturity or plasma vitamin B₁₂ transport.     

A Comparison of the Properties of Chicken Serum with Other Vitamin B12 Binding Proteins Used in Radioisotope Dilution Methods for Measuring Serum Vitamin B12 Concentrations

S ummary . The effects of pH, ionic strength, and vitamin B₁₂ concentration on the vitamin B₁₂ -binding capacities of chicken serum, normal human serum, chronic myelogenous leukaemia serum, and hog intrinsic factor concentrates are compared. The influence of pH on B₁₂-binding capacity was shown to be greatest at low pH and that of ionic strength was most evident below 0.1 ionic strength, particularly at acidic pH. The effect of B₁₂ concentration on B₁₂-binding capacity was shown to vary with different binders, the pH of binding, and the time of incubation.
It was possible to select conditions for carrying out the binding of B₁₂ to chicken serum so that the amount of B₁₂ bound was virtually unaffected by the kind of variations in pH, ionic strength, or concentration of B₁₂ that could occur in a radioisotope dilution assay of serum vitamin B₁₂. Under these conditions it was further demonstrated that the binding was unaffected by the presence of denatured human serum. In addition chicken serum had a very great binding capacity and affinity for B₁₂, was very stable on storage and was compatible with the use of protein-coated charcoal.
Some conclusions on the optimal use of B₁₂-binding protein in the radioisotope dilution assay of serum vitamin B₁₂ are made and the high potentiality of chicken serum in such a system is suggested.     

Preparation and Storage of Platelet Concentrates: I. FACTORS INFLUENCING THE HARVEST OF VIABLE PLATELETS FROM WHOLE BLOOD

Plasma and erythrocyte vitamin B₁₂ was measured in apparently healthy Indian lactovegetarians and compared with the healthy non-vegetarians from the same population. Comparison of the mean values for routine haematological parameters revealed no significant difference in various dietetic groups. The plasma vitamin B₁₂ levels were distinctly lower in the lactovegetarians than in the non-vegetarians. However, the mean erythrocyte vitamin B₁₂ levels in different dietetic groups showed no significant difference.