The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning.

Mutations in cobalamin or B12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5'-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ∼116-296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.

Paraoxonase and arylesterase activities in children with iron deficiency anemia and vitamin B12 deficiency anemia.

Paraoxonase-1 is an esterase enzyme and it has 3 types of activity, namely paraoxonase, arylesterase, and diazoxonase. It has been reported that paraoxonase-1 deficiency is related to increased susceptibility to development of atherosclerosis and cardiovascular disease. The aim of this study was to investigate serum paraoxonase and arylesterase activities in children with iron deficiency anemia and vitamin B(12) deficiency anemia. Thirty children with iron deficiency anemia, 30 children with vitamin B(12) deficiency anemia, and 40 healthy children aged 6 months to 6 years were enrolled in this study. Serum paraoxonase and arylesterase activities were measured with a spectrophotometer by using commercially available kits. Mean paraoxonase and arylesterase activities in vitamin B(12) deficiency anemia group (103 ± 73 and 102 ± 41 U/L, respectively) were significantly lower than mean activities of control group (188 ± 100 and 147 ± 34 U/L, respectively; P < .001 for both) and iron deficiency anemia group (165 ± 103 and 138 ± 39 U/L, respectively; P < .05, P < .001), whereas there were no significant differences between iron deficiency anemia and control groups (P > .05). Paraoxonase and arylesterase activities significantly increased after treatment with vitamin B(12) in vitamin B(12) deficiency anemia; however, there were no significant changes in the activities of these enzymes after iron treatment in iron deficiency anemia group. Important correlations were found between vitamin B(12) levels and both paraoxonase and arylesterase activities (r = .367, P < .001; r = .445, P < .001). Our results suggest that vitamin B(12) deficiency anemia causes important reductions in paraoxonase and arylesterase activities, and after vitamin B(12) therapy the activities of these enzymes returned to near-normal levels.

Maternal one-carbon metabolism, MTHFR and TCN2 genotypes and neural tube defects in India.

BACKGROUND: Neural tube defects (NTDs) are among the most common severe congenital malformations, representing a long-term public health burden in India. A deranged one-carbon metabolism and genes regulating this metabolism have been linked to NTDs. Vitamin B(12) deficiency is reported to be more prevalent than folate deficiency in the Indian population. We investigated the role of maternal nutritional and genetic markers related to one-carbon metabolism in the etiology of NTDs. METHODS: We conducted a multicenter case-control study to compare plasma folate, vitamin B(12) , homocysteine and holo-transcobalamin levels, and polymorphisms in methylenetetrahydrofolate reductase (MTHFR, 677C>T, 1298A>C, 1781G>A and 236+724A>G) and transcobalamin (TCN2, 776C>G) genes, in 318 women with NTD-affected offspring (cases) and 702 women with apparently healthy offspring (controls). The samples were collected at diagnosis in cases and at delivery in controls. RESULTS: We observed a significant association of high maternal plasma homocysteine concentrations with NTDs in the offspring (p = 0.026). There was no association of maternal folate or B(12) levels with NTDs (p > 0.05) but low maternal holo-transcobalamin predicted strong risk of NTDs in the offspring (p = 0.003). The commonly associated maternal polymorphism 677C>T in the MTHFR gene did not predict risk of NTDs in the offspring (p > 0.05) and 1298A>C and 1781G>A polymorphisms in MTHFR were protective (p = 0.024 and 0.0004 respectively). Maternal 776C>G polymorphism in TCN2 was strongly predictive of NTD in the offspring (p = 0.006). CONCLUSION: Our study has demonstrated a possible role for maternal B(12) deficiency in the etiology of NTDs in India over and above the well-established role of folate deficiency.

B12 in fetal development.

Vitamin B12 (cobalamin) is necessary for development of the fetus and child. Pregnant women who are vegetarian or vegan, have Crohn's or celiac disease, or have undergone gastric bypass surgery are at increased risk of B12 deficiency. Low serum levels of B12 have been linked to negative impacts in cognitive, motor, and growth outcomes. Low cobalamin levels also may be related to depression in adults. Some studies indicate that B12 supplementation may improve outcomes in children, although more research is needed in this area. Overall, the mechanisms of B12 action in development remain unclear. Further studies in this area to elucidate the pathways of cobalamin influence on development, as well as to prevent B12 deficiency in pregnant women and children are indicated.

Cobalamin deficiency results in an abnormal increase in L-methylmalonyl-co-enzyme-A mutase expression in rat liver and COS-7 cells.

The aim of the present study was to examine the effects of cobalamin (Cbl) on the activity and expression of L-methylmalonyl-CoA mutase (MCM) in rat liver and cultured COS-7 cells. The MCM holoenzyme activity was less than 5% of the total (holoenzyme+apoenzyme) activity in the liver although rats were fed a diet containing sufficient Cbl. When weanling rats were maintained on a Cbl-deficient diet, the holo-MCM activity became almost undetectable at the age of 10 weeks. In contrast, a marked increase in the total-MCM activity occurred under the Cbl-deficient conditions, and at the age of 20 weeks it was about 3-fold higher in the deficient rats than in the controls (108 (SD 14.5) v. 35 (SD 8.5) nmol/mg protein per min (n 5); P<0.05). Western blot analysis confirmed that the MCM protein level increased significantly in the Cbl-deficient rats. However, the MCM mRNA level, determined by real-time PCR, was rather decreased. When COS-7 cells were cultured in a medium in which 10% fetal bovine serum was the sole source of Cbl, holo-MCM activity was barely detected. The supplementation of Cbl resulted in a large increase in the holo-MCM activity in the cells, but the activity did not exceed 30% of the total-MCM activity even in the presence of Cbl at 10 micromol/l. In contrast, the total-MCM activity was significantly decreased by the Cbl supplementation, indicating that Cbl deficiency results in an increase in the MCM protein level in COS-7 cells as well as in rat liver.

Vitamin B12 deficiency results in the abnormal regulation of serine dehydratase and tyrosine aminotransferase activities correlated with impairment of the adenylyl cyclase system in rat liver.

The aim of the present study was to elucidate the mechanism of the vitamin B(12) deficiency-induced changes of the serine dehydratase (SDH) and tyrosine aminotransferase (TAT) activities in the rat liver. When rats were maintained on a vitamin B(12)-deficient diet, the activities of these two enzymes in the liver were significantly reduced compared with those in the B12-sufficient control rats (SDH 2.8 (sd 0.56) v. 17.5 (sd 6.22) nmol/mg protein per min (n 5); P < 0.05) (TAT 25.2 (sd 5.22) v. 41.3 (sd 8.11) nmol/mg protein per min (n 5); P < 0.05). In the B(12)-deficient rats, the level of SDH induction in response to the administration of glucagon and dexamethasone was significantly lower than in the B(12)-sufficient controls. Dexamethasone induced a significant increase in TAT activity in the primary culture of the hepatocytes prepared from the deficient rats, as well as in the cells from the control rats. However, a further increase in TAT activity was not observed in the hepatocytes from the deficient rats, in contrast to the cells from the controls, when glucagon was added simultaneously with dexamethasone. The glucagon-stimulated production of cAMP was significantly reduced in the hepatocytes from the deficient rats relative to the cells from the control rats. Furthermore, the glucagon-stimulated adenylyl cyclase activity in the liver was significantly lower in the deficient rats than in the controls. These results suggest that vitamin B(12) deficiency results in decreases in SDH and TAT activities correlated with the impairment of the glucagon signal transduction through the activation of the adenylyl cyclase system in the liver.

Cobalamin (Cbl) C/D deficiency: clinical, neurophysiological and neuroradiologic findings in 14 cases.

The early onset type of cobalamin (Cbl) C/D deficiency is characterised by feeding difficulties, failure to thrive, hypotonia, seizures, microcephaly and developmental delay. It has an unfavourable outcome, often with early death and significant neurological impairment in survivors. While clinical and biochemical features of Cbl C/D deficiency are well known, only a few isolated case reports are available concerning neurophysiological and neuroimaging findings. We carried out clinical, biochemical, neurophysiological and neuroradiologic investigations in 14 cases with early-onset of the Cbl CID defect. Mental retardation was identified in most of the cases. A variable degree of supratentorial white matter atrophy was detected in 11 cases by MR imaging and tetraventricular hydrocephalus was present in the remaining 3 patients. Waking EEG showed a clear prevalence of epileptiform abnormalities, possibly related to the high incidence of seizures in these cases. Increased latency of evoked responses and/or prolongation of central conduction time were the most significant neurophysiological abnormalities. The selective white matter involvement, shown both by neuroradiologic and neurophysiological studies, seems to be the most consistent finding of Cbl C/D deficiency and may be related to a reduced supply of methyl groups, possibly caused by the dysfunction in the methyl-transfer pathway.

Targeted disruption of the methionine synthase gene in mice.

Alterations in homocysteine, methionine, folate, and/or B12 homeostasis have been associated with neural tube defects, cardiovascular disease, and cancer. Methionine synthase, one of only two mammalian enzymes known to require vitamin B12 as a cofactor, lies at the intersection of these metabolic pathways. This enzyme catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Human patients with methionine synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypomethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. To better study the pathophysiology of methionine synthase deficiency, we utilized gene-targeting technology to inactivate the methionine synthase gene in mice. On average, heterozygous knockout mice from an outbred background have slightly elevated plasma homocysteine and methionine compared to wild-type mice but seem to be otherwise indistinguishable. Homozygous knockout embryos survive through implantation but die soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in methionine synthase. Whether any human patients with methionine synthase deficiency have a complete absence of enzyme activity is unclear. These results demonstrate the importance of this enzyme for early development in mice and suggest either that methionine synthase-deficient patients have residual methionine synthase activity or that humans have a compensatory mechanism that is absent in mice.

Extremely low activity of methionine synthase in vitamin B-12-deficient rats may be related to effects on coenzyme stabilization rather than to changes in coenzyme induction.

Severely vitamin B-12 (B-12)-deficient rats were produced by feeding a B-12-deficient diet. The status of B-12 deficiency was confirmed by an increase in urinary methylmalonate excretion and decreases in liver B-12 concentrations and cobalamin-dependent methionine synthase activity. Rat liver methionine synthase existed almost exclusively as the holoenzyme. In B-12-deficient rats, the level of methionine synthase protein was lower, although the mRNA level was not significantly different from that of control rats. When methylcobalamin, the coenzyme for methionine synthase, was administered to the B-12-deficient rats, growth, liver B-12 concentrations and urinary excretion of methylmalonate were reversed although not always to control (B-12-sufficient) levels in a short period. During this recovery process, methionine synthase activity and its protein level increased, whereas the mRNA level was unaffected. We reported previously that rat apomethionine synthase is very unstable and is stabilized by forming a complex with methylcobalamin. Thus, the extremely low activity of methionine synthase in B-12-deficient rats may be related to effects on "coenzyme stabilization" (stabilization of the enzyme by cobalamin binding) rather than to changes in "coenzyme induction."

Advantages of serum pepsinogen A combined with gastrin or pepsinogen C as first-line analytes in the evaluation of suspected cobalamin deficiency: a study in patients previously not subjected to gastrointestinal surgery.

OBJECTIVES: Since there is a significant overlap in serum cobalamin concentrations between healthy and cobalamin-deficient individuals, we wanted to compare two different principles for use as supplementary tests to serum cobalamin concentration in patients with suspected cobalamin malabsorption and deficiency. DESIGN: Clinical study of consecutive patients. SETTING: The catchment area of Sahlgrenska University Hospital, Göteborg. SUBJECTS: A total of 112 patients with suspected cobalamin deficiency who had not previously undergone gastrointestinal surgery. INTERVENTIONS: Gastroduodenoscopy with biopsies taken from the gastric body and the duodenum, Schilling test, and measurement of serum methylmalonic acid (MMA), total homocysteine (Hcy), pepsinogens A and C, and gastrin. MAIN OUTCOME MEASURES: Number of patients with gastric body atrophy identified with the combination of MMA and Hcy, and pepsinogen A combined with pepsinogen C or gastrin. RESULTS: About 95% of the patients with severe gastric body atrophy had abnormal concentrations of serum pepsinogen A and/or gastrin or pepsinogen A/C ratio, whereas 65% had abnormal metabolite concentrations. Serum pepsinogen A combined with pepsinogen C identified 100%, and combined with gastrin 88%, of the patients with gastric body atrophy and elevated metabolite tests, and 67 and 75%, respectively, of those who had not yet developed elevated metabolite tests. CONCLUSIONS: Pepsinogen A, combined with pepsinogen C or gastrin, should be the first option in evaluating patients with suspected cobalamin deficiency who have not previously undergone gastrointestinal surgery.

Elevated deoxyuridine triphosphate nucleotidohydrolase (dUTPase) activity in the cobalamin-deficient megaloblastic bone marrow cells.

The biochemical basis underlying the pathogenesis of megaloblastic anemia due to vitamin B12 deficiency was examined. Megaloblastic bone marrow cells show a marked imbalance in the deoxynucleoside triphosphate pools. If cellular dUTP concentration is elevated due to metabolic block in a similar way to dCTP, ensuing uracil misincorporation into DNA would result in deranged DNA composition and impaired cell function. Therefore, dUTP-degrading activity was measured in blood cells from various diseases. dUTPase (dUTP nucleotidohydrolase) activity of bone marrow cells was, on average, 4.4 times higher in 8 patients with untreated megaloblastic anemia than that of other normoblastic conditions. Such elevated dUTPase may help lower cellular dUTP levels in megaloblastic anemia. However, it is not clear whether the observed increase of dUTPase in megaloblastic cells is sufficient to suppress uracil misincorporation below the harmful level.

[Monoamine oxidase activity in rat liver in vitamin B12 deficiency]. / Monoaminooksidaznaia aktivnost' v pecheni krys pri nedostatochnosti vitamina B12.

Changes in the activity of monoamine oxidase (MAO-B) in liver homogenates and mitochondria from normal and vitamin B12-deficient rats have been studied in various functional states of the mitochondria. Preincubation of liver preparation at 32 degrees C increased the MAO affinity for benzylamine in samples of normal (but not vitamin B12-deficient rats). Succinate added to the incubation medium decreased Km and increased Vmax in both normal and vitamin B12-deficient animals. A correlation was found between the decline of MAO-B activity under vitamin B12 deficiency, deceleration of succinate oxidation and reduction of the transmembrane potential.

Cobalt-vitamin B12 deficiency and the activity of methylmalonyl CoA mutase and methionine synthase in cattle.

Cobalt deficiency was induced in cattle by feeding two groups of animals either a basal diet that was very low in Co (12.9-17.6 micrograms Co per kg), or the same diet supplemented with cobalt, for a total of 64 weeks. Vitamin B12 deficiency was induced, as judged by hepatic concentrations of vitamin B12 and plasma concentrations of MMA. However, the activity of holo-methylmalonyl CoA mutase was significantly reduced only in brain. This was reflected in very minor alterations in the tissue concentrations of branched chain- and odd numbered-fatty acids. The activity of holo-methionine synthase was significantly reduced in liver and brain, but there were no consequent alterations in the concentrations of phosphatidyl choline and phosphatidyl ethanolamine. This study confirms that cattle are less susceptible to the effects of cobalt deficiency than sheep, and concludes that prolonged cobalt deficiency had little significant effect on tissue metabolism.

Cobalt-vitamin B-12 deficiency decreases methionine synthase activity and phospholipid methylation in sheep.

Two groups of lambs were fed either a Co-deficient or a Co-sufficient whole barley-based diet for 28 wk to induce a severe Co-vitamin B-12 deficiency. Holo and apo methionine synthase activities were significantly lower in the liver, kidney and spinal cord of Co-deficient animals compared with controls. Neither form of this enzyme in the brain was affected by Co deficiency. The ratio of the tissue concentrations of S-adenosyl methionine to S-adenosyl homocysteine was significantly lower only in the liver of Co-deficient animals, suggesting that the activity of hepatic SAM-dependent methyltransferase enzymes would be impaired. Measurements of tissue concentrations of phosphatidyl choline and phosphatidyl ethanolamine revealed lower concentrations of phosphatidyl choline and a lower phosphatidyl choline:phosphatidyl ethanolamine ratio in both liver and brain of the Co-deficient animals. The latter finding occurred in the absence of changes in either methionine synthase activity or the methylation ratio and may result from impaired availability of hepatic phosphatidyl choline for transport into the brain.

Vitamin B-12 deficiency increases the specific activities of rat liver NADH- and NADPH-linked aquacobalamin reductase isozymes involved in coenzyme synthesis.

Rat liver contains both NADH- and NADPH-linked aquacobalamin reductases, which are involved in the synthesis of the vitamin B-12 coenzymes and are distributed in both the mitochondrial and microsomal membranes. To clarify the physiological roles of these hepatic enzymes, vitamin B-12-deficient rats were used to study the effect of the deficiency on the enzyme activities. Male rats fed a vitamin B-12-deficient diet for 11 wk developed a severe vitamin B-12 deficiency with a high urinary methylmalonate excretion (214.3 +/- 115.2 mumol/d) and approximately 96% lower hepatic vitamin B-12 content. Tissues of the vitamin B-12-deficient rats were assayed for NADH- and NADPH-linked aquacobalamin reductase activities. The specific activities of both enzymes in homogenates of liver, kidney or upper intestine were shown to be three- to 20-fold greater in the vitamin-deficient rats than in the control rats. In liver, the vitamin deficiency specifically elevated the specific activities of the mitochondrial NADH-linked and microsomal NADPH-linked enzymes. These are likely the isozymes involved in vitamin B-12 coenzyme synthesis.

Vitamin B12-induced reduction of platelet monoamine oxidase activity in patients with dementia and pernicious anaemia.

Platelet monoamine oxidase (MAO) activity has previously been shown to be increased in patients with senile dementia of Alzheimer type (SDAT) and in patients with megaloblastic anaemia. Moreover, low serum B12 levels were found to be 4-5 times more frequent in SDAT compared with an unselected population of similar age. In the present investigation, platelet MAO activity was estimated in 14 SDAT patients with relatively low serum B12 levels and in 4 patients with pernicious anaemia. Before B12 therapy, platelet MAO activity was significantly increased in both patient groups compared with a control group. After B12 therapy, platelet MAO activity was significantly reduced in both patient groups to apparently normal levels. The present results show that B12 status is a controlling factor of platelet MAO activity and confirm that a significant connection exists between vitamin B12 deficiency and primary degenerative dementia disorders, such as SDAT.