Advances in the understanding of cobalamin assimilation and metabolism

The haematological and neurological consequences of cobalamin deficiency define the essential role of this vitamin in key metabolic reactions. The identification of cubilin-amnionless as the receptors for intestinal absorption of intrinsic factor-bound cobalamin and the plasma membrane receptor for cellular uptake of transcobalamin bound cobalamin have provided a clearer understanding of the absorption and cellular uptake of this vitamin. As the genes involved in the intracellular processing of cobalamins and genetic defects of these pathways are identified, the metabolic disposition of cobalamins and the proteins involved are being recognized. The synthesis of methylcobalamin and 5'-deoxyadenosylcobalamin, their utilization in conjunction with methionine synthase and methylmalonylCoA mutase, respectively, and the metabolic consequences of defects in these pathways could provide insights into the clinical presentation of cobalamin deficiency.     

New insights into carrier binding and epithelial uptake of the erythropoietic nutrients cobalamin and folate

PURPOSE OF REVIEW: In addition to malnutrition several genetic and acquired conditions may affect the homeostasis of cobalamin (vitamin B12) and folate, leading to megaloblastic anemia and other diseases. The present review describes new insight into protein handling of cobalamin and folate. RECENT FINDINGS: The recent solution of the three-dimensional structure of the cobalamin binder transcobalamin shows two separate domains enclosing the vitamin. This structure apparently also applies for the other homologous cobalamin binders, intrinsic factor and haptocorrin. Genetic studies of inherited cobalamin malabsorption and biochemical studies have now revealed that the functional receptor for uptake of intrinsic factor-vitamin cobalamin complexes also is a complex itself consisting of two different gene products, cubilin and amnionless. A role in folate uptake of megalin, an endocytic receptor for epithelial uptake of various proteins including transcobalamin, is now also indicated by the observation that megalin can mediate uptake of soluble folate receptor. SUMMARY: New data show the structure of cobalamin carriers and reveal novel proteins involved in the epithelial uptake of cobalamin and folate. Genetic abnormalities in three different genes encoding proteins in the epithelial uptake of cobalamin are now known to cause malabsorption of cobalamin and megaloblastic anemia.     

Food cobalamin malabsorption occurs frequently in patients with unexplained low serum cobalamin levels

Malabsorption of food-bound or protein-bound cobalamin with normal absorption of free cobalamin has been described in studies of patients with gastric dysfunction. We used the egg-yolk cobalamin absorption test to study 47 patients selected not because of known gastric disorders but because they had low serum cobalamin levels with normal Schilling test results. Their egg test results were significantly lower than in normal controls, while Schilling test results were normal. Twenty of the subjects had egg test excretion below 1.5%. No features distinguished them from the 27 who excreted more than 1.5% other than the presence of lower pepsinogen I:II ratios. Eight of 19 tested patients with food cobalamin malabsorption had no evidence of abnormal gastric status by blood tests and/or gastric analysis. Also noteworthy was the finding of food cobalamin malabsorption in 60% of tested patients who had neurologic, cerebral, or psychiatric abnormalities. Food cobalamin malabsorption appears to be associated frequently with otherwise unexplained low cobalamin levels. Low cobalamin levels in patients with normal Schilling test results cannot be dismissed as insignificant without also testing for food cobalamin malabsorption, whether or not the patients have known gastric dysfunction.     

Recognition of two intracellular cobalamin binding proteins and their identification as methylmalonyl-CoA mutase and methionine synthetase.

The granulocyte R-type cobalamin binding protein delivers cobalamin (Cbl) exclusively to hepatocytes, and transcobalamin II delivers Cbl to various mammalian cells. Both protein-Cbl complexes enter cells by pinocytosis, and the protein moieties are rapidly degraded in lysosomes. The liberated Cbl is subsequently bound to a high-molecular-weight intracellular cobalamin binding protein (ICB). The nature of ICB-Cbl is unknown but appears important because ICB-[57Co]Cbl is missing from cultured fibroblasts of a group of patients whose cells take up CN-[57Co]Cbl normally but do not convert it to either of its coenzyme forms. We have examined supernatants of sonicated rabbit livers and have found that 65% of the total endogenous Cbl elutes from Sephadex G-150 as ICB-Cbl and that this fraction also contains the two mammalian Cbl-dependent enzymes, methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase;EC 5.4.99.2) and methionine synthetase (tetrahydropteroylglutamate methyltransferase; 5-methyltetrahydropteroyl-L-glutamate:L-homocysteine-S-methyltransferase; EC 2.1.1.13). Gradient elution from DEAE-Sephadex reveals that 90--95% of the ICB--Cbl elutes with methylmalonyl-CoA mutase and 5--10% elutes with methionine synthetase. ICB--[57Co]Cbl first appears 2 hr after the intravenous injection of CN[57Co]Cbl bound to granulocyte R-type protein. This ICB-[57Co]Cbl is associated with either methylmalonyl-CoA mutase or methionine synthetase although the latter appears to be formed at a relatively faster rate. Our studies indicate that mammalian cells contain two ICBs, that these proteins are methylmalonyl-CoA mutase and methionine synthetase, and that the primary abnormality in the group of patients mentioned above lies at a step that is common to the formation of both Cbl coenzymes and that precedes the stable binding of Cbl to both methylmalonyl-CoA mutase and methionine synthetase.     

High serum cobalamin levels in the clinical setting--clinical associations and holo-transcobalamin changes

Whereas low cobalamin levels have been studied intensively, systematic information about high levels, especially in the clinical setting, is scarce. Therefore, a prospective comparison was done of 60 patients with high cobalamin levels and 75 with normal levels obtained by a hospital laboratory over a 2.5 month period. Associations with clinical disorders and laboratory test results were examined. Transcobalamin (TC) I and II were measured, especially the holoproteins (TC carrying circulating endogenous cobalamin) which were fractionated with microfine silica powder. High cobalamin levels (> 664 pmol/l; > 900 ng/l) occurred in 94 of 670 consecutive clinically requested assays (14%). The only independently significant associations with a high cobalamin level were renal failure among the clinical disorders (P=0.01), elevated serum creatinine (P=0.0001) and diminished albumin (P=0.0002) levels among laboratory tests. Both holo-TC I and holo-TC II levels were increased in renal failure (P=0.0001) but the increase was relatively greater in holo-TC II. The results indicate that high cobalamin levels are more frequent than low ones in clinical practice and appear to be associated often with renal failure. The elevation of both holo-TC II and holo-TC I suggests that several mechanisms are operative. The accumulation of holo-TC II suggests that cellular uptake of cobalamin by the abundant TC II receptors in the kidney may be impaired. The much better known association of high cobalamin levels with leucocytic disorders is rare, and no association was seen with liver disease.     

Climatically driven macroevolutionary patterns in the size of marine diatoms over the Cenozoic

Numerous taxonomic groups exhibit an evolutionary trajectory in cell or body size. The size structure of marine phytoplankton communities strongly affects food web structure and organic carbon export into the ocean interior, yet macroevolutionary patterns in the size structure of phytoplankton communities have not been previously investigated. We constructed a database of the size of the silica frustule of the dominant fossilized marine planktonic diatom species over the Cenozoic. We found that the minimum and maximum sizes of the diatom frustule have expanded in concert with increasing species diversity. In contrast, the mean area of the diatom frustule is highly correlated with oceanic temperature gradients inferred from the delta18O of foraminiferal calcite, consistent with the hypothesis that climatically induced changes in oceanic mixing have altered nutrient availability in the euphotic zone and driven macroevolutionary shifts in the size of marine pelagic diatoms through the Cenozoic.     

Spouses of demented patients with low cobalamin levels: a new risk group for cobalamin deficiency

Abstract: Low serum cobalamin levels are common in conditions such as dementia and often represent mild deficiency. We surveyed serum cobalamin levels prospectively in spouses and blood relatives of demented patients to determine if any familial predisposition exists for the low levels. Cobalamin status in most of the relatives found to have low levels was assessed further by means of blood counts, metabolic tests, neurologic evaluation, absorption studies and response to cobalamin therapy. Serum cobalamin levels in 36 spouses correlated with those of the 36 demented patients related to them (r = 0.46, p = 0.004). A significant association was not seen in 34 blood relatives of 34 demented patients (r = 0.27). Most importantly, 67% of the spouses of demented patients with low serum cobalamin had low values themselves, compared with only 3% of the spouses of patients with normal levels (p = 0.001). Detailed study of 4 of the 5 spouses (and 3 blood relatives) with low cobalamin levels showed no anemia in any case. Nevertheless, 4 of the subjects had metabolic evidence of deficiency and one had electrophysiological abnormalities; all these defects improved with cobalamin therapy. These observations identify a hitherto unsuspected group of people at high risk for cobalamin deficiency and suggest that spouses of demented patients with low cobalamin levels should also have their cobalamin levels measured. The increased frequency of low serum cobalamin levels in spouses of demented patients with low levels represents in most cases a true, mild cobalamin deficiency that responds to treatment.     

In vivo evidence that cobalamin is absorbed by receptor-mediated endocytosis in the mouse

This study examines the mechanism of cobalamin absorption in the context of receptor-mediated endocytosis. Uptake, the amount of cobalamin that left the intestinal lumen, and transport, the component that was located in organs beyond the intestine, were measured after feeding saturating doses of [57Co]cobalamin to mice. Uptake from a 40-ng dose of [57Co]cobalamin at 1 h was 18.6 +/- 6.3 ng (mean +/- SD; n = 6). When the dose was given 1 h after 40 ng of unlabeled cobalamin, uptake was 18.5 +/- 1.5 ng, n = 6, indicating rapid clearance of the surface receptors. Transport of the initial and of a subsequent dose of cobalamin could not be detected for 2 h but the amounts and rates of transport were similar. Oral chloroquine and intraperitoneal cycloheximide reduced transport at 4 h to 4.3 +/- 1.6 ng, n = 8 and 5.6 +/- 2.4 ng, n = 7, respectively. Control values were 14.3 +/- 1.3 ng, n = 8. These results indicate that the transport of cobalamin involves a series of compartments, one of which may be lysosomal, and that there is a requirement for new protein synthesis.     

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

B(12)-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme.     

Effects of cobalamin, cobalamin analogues and cobalamin binding proteins on P388D1 mouse leukemic cells in culture.

Cobalamin-deficient P388D1 mouse leukemic cells were created by propagation in a cyanocobalamin-free medium in which the original fetal bovine serum was replaced by bovine serum albumin. These cobalamin-deficient cells gradually ceased to multiply when the medium contained 5-methyltetrahydrofolate. The growth of cells that had been cultured with this coenzyme was recovered following the addition of cyanocobalamin (CNCbl), at concentrations above 37 pM. In contrast to the effect of CNCbl, cobinamide, and cobalamin analogues prepared from hydroxy cobalamin by reaction with ascorbic acid, did not have a growth-inducing effect on these cells, nor did these analogues inhibit CNCbl-dependent growth. Transcobalamin II-cobalamin complex had a remarkably stimulating effect on cell growth. The growth inducing effect became apparent with a cobalamin concentration of only 0.37 pM. This was about 1/100th the level of free cobalamin required for cell growth. However, no growth-inducing effect was seen at an R protein-bound cobalamin concentration of 37 pM, indicating that once cobalamin has been bound to R protein, it loses its growth-promoting effect on these cells in culture.     

Low serum cobalamin levels in primary degenerative dementia. Do some patients harbor atypical cobalamin deficiency states?

Serum cobalamin (vitamin B12) levels were analyzed retrospectively in 17 patients with primary degenerative dementia and 11 with specific demonstrable causes of dementia (secondary dementia). The prevalence of low cobalamin levels was significantly increased in primary dementia (29% vs 0% in secondary dementia). Because typical findings of deficiency often seemed absent, we prospectively studied two other patients with primary dementia and low cobalamin levels. Neither of these two had megaloblastic anemia; one had a normal Schilling test while the other's was borderline. Despite this absence of the expected findings, the deoxyuridine suppression test gave biochemical evidence of cobalamin deficiency in both cases. Our survey of 28 patients thus established that low serum cobalamin levels are frequent in primary dementia. Our findings in the two prospectively studied cases (as well as in some of the patients in the survey) indicate that these levels are associated in at least some cases with an atypical deficiency state rather than with disorders such as pernicious anemia. Such atypical deficiency states cannot be identified by classic hematological criteria or by the Schilling test.     

High serum cobalamin levels in the clinical setting – clinical associations and holo‐transcobalamin changes

Whereas low cobalamin levels have been studied intensively, systematic information about high levels, especially in the clinical setting, is scarce. Therefore, a prospective comparison was done of 60 patients with high cobalamin levels and 75 with normal levels obtained by a hospital laboratory over a 2½ month period. Associations with clinical disorders and laboratory test results were examined. Transcobalamin (TC) I and II were measured, especially the holoproteins (TC carrying circulating endogenous cobalamin) which were fractionated with microfine silica powder. High cobalamin levels (> 664 pmol/l; > 900 ng/l) occurred in 94 of 670 consecutive clinically requested assays (14%). The only independently significant associations with a high cobalamin level were renal failure among the clinical disorders (P=0.01), elevated serum creatinine (P=0.0001) and diminished albumin (P=0.0002) levels among laboratory tests. Both holo‐TC I and holo‐TC II levels were increased in renal failure (P=0.0001) but the increase was relatively greater in holo‐TC II. The results indicate that high cobalamin levels are more frequent than low ones in clinical practice and appear to be associated often with renal failure. The elevation of both holo‐TC II and holo‐TC I suggests that several mechanisms are operative. The accumulation of holo‐TC II suggests that cellular uptake of cobalamin by the abundant TC II receptors in the kidney may be impaired. The much better known association of high cobalamin levels with leucocytic disorders is rare, and no association was seen with liver disease.     

Regulation of Cobalamin and Folate Metabolism by Methionine in Human Bone Marrow Cultures

In cobalamin deficiency folate metabolism is disturbed. In the liver this deranged metabolism can be overcome by methionine, however, methionine failed to overcome this abnormality in bone marrow cultures from cobalamin deficient patients. In cobalamin deficient E. coli mutant bacteria, methionine under different conditions could either inhibit or potentiate the growth of the organism. This study was therefore initiated to test the effect of methionine, under different conditions, on bone marrow cultures. The defective DNA synthesis in megaloblastic bone marrow due to cobalamin deficiency could be corrected by the in vitro addition of low 0.27 μmol (40 μg) but not high 6.7 μmol (1 mg) amounts of methionine. This was measured by the ability of deoxyuridine to suppress the ³H-thymidine incorporation into DNA. The effect of methionine in facilitating de novo DNA synthesis is probably due to the catalytic action of SAM which activates cobalamin dependent methyltransferase enzyme thus potentiating the effect of cobalamin. In contrast high concentrations of methionine may inhibit this enzyme.     

High prevalence of cobalamin deficiency in elderly outpatients.

OBJECTIVE: To measure the prevalence of cobalamin (vitamin B12) deficiency in geriatric outpatients as documented by both low serum cobalamin levels and elevations of serum methylmalonic acid and homocysteine and to determine the response to cobalamin treatment. DESIGN: Prospective study screening elderly subjects for cobalamin deficiency using radiodilution cobalamin assays as well as stable isotope dilution gas chromatography-mass spectrometry methylmalonic acid and homocysteine assays. In patients with serum cobalamin levels < or = 300 pg/mL, the response to cobalamin treatment in the group with levels of methylmalonic acid and/or homocysteine > 3 standard deviations (SD) above the mean for normals was compared with that of those without such elevations. SETTING: Outpatient geriatric clinics at the VA Medical Center and University Health Sciences Center, Denver, CO. PATIENTS: One-hundred and fifty-two consecutive outpatients, ages 65 to 99, were screened. Twenty-nine subjects with serum cobalamin levels < or = 300 pg/mL were prospectively evaluated and treated with cobalamin. MAIN OUTCOME MEASURES: Cobalamin, methylmalonic acid, homocysteine, complete blood counts, neurologic examination, and neuropsychological testing. RESULTS: The prevalence of cobalamin deficiency as defined by a serum cobalamin level < or = 300 pg/mL and levels of serum methylmalonic acid and/or homocysteine elevated to > 3 SD was 14.5% of the screened outpatients. A similar proportion of patients with low normal serum cobalamin levels (between 201 and 300 pg/mL) demonstrated elevated metabolites > 3 SD (56%) compared with patients with low serum cobalamin levels (< or = 200 pg/mL) (62%). Cobalamin therapy caused a marked fall or complete correction of the elevated methylmalonic acid and homocysteine levels in each patient who was treated prospectively. Results for complete blood count, lactate dehydrogenase, bilirubin, baseline neurologic score, and baseline neuropsychologic scores did not differ in the group of patients with elevated metabolites compared with those with normal metabolites. The mean red cell volume fell significantly in the patients with elevated metabolites after 6 months of cobalamin treatment. One patient with elevated metabolites had marked improvement in his neurologic abnormalities after 6 months of cobalamin treatment. CONCLUSION: There was a high (14.5%) prevalence of cobalamin deficiency as demonstrated by elevations in serum methylmalonic acid and homocysteine in addition to low or low normal serum cobalamin levels in elderly outpatients. The serum cobalamin level was insensitive for screening since similar numbers of patients with low normal serum cobalamin levels of 201-300 pg/mL compared with patients with low cobalamin levels (< or = 200 pg/mL) had markedly elevated metabolites which fell with cobalamin treatment. Additional studies will be required to define the full clinical benefit from treatment with Cbl in elderly subjects.     

Protein-bound cobalamin absorption declines in the elderly.

A modified protein-bound cobalamin absorption test was used to study dietary cobalamin absorption in healthy adults of different age groups and patients with isolated low serum concentrations of cobalamin. Dietary cobalamin absorption was significantly reduced in healthy adults aged 55-75 years compared with young adults, with a further reduction in those older than 75 years. No difference was detected between dietary cobalamin absorption in patients with isolated low serum cobalamin and controls of a similar age group. Cobalamin malabsorption was associated with elevated serum gastrin. The diagnostic value of this protein-bound cobalamin absorption test in the elderly was limited by the frequent finding of reduced absorption in healthy elderly people with normal serum cobalamin concentrations. The performance of such tests should be evaluated in different age groups before application in diagnosis.     

Diagnosis of cobalamin deficiency: II. Relative sensitivities of serum cobalamin, methylmalonic acid, and total homocysteine concentrations

The serum cobalamin level has been generally considered to be essentially 100% sensitive in the detection of the clinical disorders caused by cobalamin deficiency. We tested this hypothesis in two groups of patients. In patients with pernicious anemia or previous gastrectomy who received less than monthly maintenance therapy, early hematologic relapse was associated with elevation of the serum methylmalonic acid, total homocysteine, or both metabolites in 95% of instances, although the serum cobalamin was low in only 69%. In the absence of hematologic relapse, the methylmalonic acid was abnormal more than twice as frequently as the serum cobalamin. We also reviewed the records of 419 consecutive patients with recognized clinically significant cobalamin deficiency. Twelve patients were identified in whom deficiency was clearly present although the serum cobalamin was greater than 200 pg/ml. Anemia was usually absent or mild, but 5 had prominent neurological involvement that subsequently responded to cobalamin. Both the serum methylmalonic acid and total homocysteine were increased in each patient. The serum cobalamin was normal in 9 (5.2%) of 173 patients with recognized cobalamin deficiency seen in the last 5 years. Antibiotic treatment lowered the serum methylmalonic acid but not the total homocysteine level in two cobalamin-deficient patients, suggesting that propionic acid generated by the anaerobic gut flora may be a precursor of methylmalonic acid in deficient patients. We conclude that the serum cobalamin is normal in a significant minority of patients with cobalamin deficiency and that the measurement of serum metabolite concentrations facilitates the identification of such patients.     

Acquired and inherited disorders of cobalamin and folate in children

Cobalamin deficiency in the newborn usually results from cobalamin deficiency in the mother. Megaloblastic anaemia, pancytopenia and failure to thrive can be present, accompanied by neurological deficits if the diagnosis is delayed. Most cases of spina bifida and other neural tube defects result from maternal folate and/or cobalamin insufficiency in the periconceptual period. Polymorphisms in a number of genes involved in folate and cobalamin metabolism exacerbate the risk. Inborn errors of cobalamin metabolism affect its absorption, (intrinsic factor deficiency, Imerslund‐Gräsbeck syndrome) and transport (transcobalamin deficiency) as well as its intracellular metabolism affecting adenosylcobalamin synthesis (cblA and cblB), methionine synthase function (cblE and cblG) or both (cblC, cblD and cblF). Inborn errors of folate metabolism include congenital folate malabsorption, severe methylenetetrahydrofolate reductase deficiency and formiminotransferase deficiency. The identification of disease‐causing mutations in specific genes has improved our ability to diagnose many of these conditions, both before and after birth.     

Malabsorption of protein-bound cobalamin but not unbound cobalamin during cimetidine administration

The suppressive effects of cimetidine on acid, pepsin, and intrinsic factor secretion have been well documented; however, the effect of cimetidine on cobalamin absorption has not been assessed. The absorption of both unbound [⁵⁷Co] cyanocobalamin and protein-bound [⁵⁷Co] cyanocobalamin was evaluated in 12 patients with duodenal ulcer disease during and after discontinuation of cimetidine therapy. Cimetidine administration did not lead to malabsorption of unbounds cobalamin but caused malabsorption of protein-bound cobalamin (0.22±0.08%, [mean±1sem] versus 2.3±0.10% in control subjects,P<0.01). This malabsorption was reversible upon discontinuation of cimetidine. Patients on cimetidine therapy malabsorb protein-bound cobalamin and, during long-term treatment, are at risk for developing cobalamin deficiency. This malabsorption of protein-bound cobalamin is not detectable by the usual tests of cobalamin absorption which employ unbound cobalamin.During the performance of these investigations Dr. King was the recipient of a Research Associate Award and Dr. Toskes, a Clinical Investigation Award, from the Veteran Administration.Supported in part by grant RR-82 and training grant AM07209 from the National Institutes of Health.Presented in part at the Annual Meeting of the American Gastroenterological Association in Las Vegas, Nevada, May 1978.