Local Cerebral Glucose Utilization in Two Models of B12 Deficiency

Local cerebral glucose utilization (LCGU), as measured by the 2-deoxy-D-[1-14C]glucose technique, reflects local cerebral functional activity. In an effort to elucidate mechanisms of the encephalopathy associated with deficiency of vitamin B12, LCGU was determined in two recently described models of effective B12 deficiency: exposure of rats to subanesthetic doses of nitrous oxide (N2O) and/or administration of 1-amino-cyclopentane-1-carboxylic acid (cycloleucine). Our results show that exposure of adult rats to N2O depresses LCGU selectively in cortical, auditory, and limbic structures, in association with a depression in whole-brain activities of the vitamin B12-dependent methyltetrahydrofolate-homocysteine methyl-transferase (EC 2.1.1.13, methionine synthetase). Cycloleucine has no discernible effect on LCGU in the adult rat and does not change the cerebral activity of methionine synthetase.     

Effects of Perinatal Vitamin B6 Deficiency on Dopaminergic Neurochemistry

Long-Evans dams were fed either a vitamin B6-deficient or a control diet from day 13-14 of gestation and throughout lactation. A control pair-fed group was also included because of differences in food intake between vitamin B6-deficient and control ad libitum dams. The progeny of vitamin B6-deficient dams had all the classic symptoms of B6 deficiency. These included weight loss, ataxia, tremor, and epileptic seizures. Concentrations of the neurotransmitter dopamine (DA), and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as D-2 dopamine receptor binding, 3,4-dihydroxyphenylalanine (DOPA) decarboxylase activity, and vitamin B6 levels were measured in the corpus striatum of progeny at 7, 14, and 18 days after birth. Striatal DA and HVA levels were significantly decreased in B6-deficient animals when compared to ad libitum or pair-fed controls. Daily injections of vitamin B6 to deprived animals from the 14th to 18th day after birth improved the abnormal movement and normalized the concentration of DA but not of HVA in corpus striatum. Striatal D-2 dopamine receptor binding using [3H]spiperone as ligand was significantly reduced in 18-day-old animals as compared to ad libitum and pair-fed controls. No significant differences were found at 14 days. The administration of vitamin B6 to deprived animals did not raise the level of D-2 receptor binding during the period of observation. Scatchard plots indicated that the differences in binding were due to changes in receptor number and not in KD. Corpus striatum DOPA decarboxylase activity with and without the addition of exogenous pyridoxal phosphate was significantly reduced in 14- and 18-day-old animals when compared to pair-fed controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synchronization of Division in Vitamin B12-Starved Euglena gracilis

SYNOPSIS Vitamin B₁₂ deficiency arrests cell division in Euglena gracilis. B₁₂ starvation for short periods made it possible to induce synchronous growth by addition of the vitamin. Culture conditions were established to optimize replenishment synchrony. The DNA content of E. gracilis in steady state culture and vitamin B₁₂ deficiency culture was measured by flow cytofluorometry and was consistent with colorimetric determinations. The cell volume and DNA distributions of E. gracilis in synchronous culture were analyzed and the sequential changes during the division cycle were computed. Synchronous culture permits more definitive studies of shifts in cell volume and DNA distributions, in which the biochemical events required for cell division are presumably synchronized.     

Lipid Composition of the Brain in the Vitamin B12-Deficient Fruit Bat (Rousettus aegyptiacus) with Neurological Impairment

Abstract: The Egyptian fruit bat Rousettus aegyptiacus develops severe vitamin B₁₂ deficiency when fed a diet of fresh peeled fruit and water. In a group of bats fed this diet, B₁₂ concentrations in the serum and brain were low, and neurological impairment, evidenced by deficient or absent hindlimb groping or grasping ability and climbing difficulties, was manifest. Control bats fed the identical diet supplemented with B₁₂ showed no such changes. Fatty acid analysis of whole brain homogenates showed a higher level of 20:4 in the deficient group. Phosphatidylcholine showed a marginally higher percentage of 18:3. The total percentage of branched chain fatty acids of phos-phatidylethanolamine was four times higher in deficient brains, comprising 2% of the total. Sphingomyelin showed a slightly higher percentage of 15:0, and a significantly lower percentage of long chain fatty acids C-24 and longer ( p < 0.01). The compositions of nonhydroxy fatty acids in cerebroside were unchanged. Examination of phospholipids showed that 8.9 ± 0.4% of total phosphorus was present as sphingomyelin in deficient bats, compared with 11.9 ± 1.2% in control animals (p < 0.05). There were no statistically significant differences in the concentrations of total brain lipid, protein, phospholipid, glycosphingolipid, cholesterol and plasmalogen between B₁₂-deficient and control bats.     

The Role of Vitamin B12 Binding In the Uptake of the Vitamin By Euglena Gracilis

The uptake of [⁵⁷Co]B₁₃ (cyanocobalamin) by Euglena gracilis strain Z (ATCC 12716) occurred in 2 distinct phases-an initial rapid phase followed by a slower secondary phase. This secondary phase appeared after the saturation of the binding sites involved in the initial rapid phase and was energy-dependent and completely inhibited by 2,4-dinitrophenot, KCN and sodium azide. the subcellular localization of labeled cyanocobalamin taken up by the cell was mostly contained in the chloroplast fraction. the time course and the saturation kinetics of B₁₂ uptake by purified chloroplast fraction indicated that this fraction and the intact cell had a similar affinity for the vitamin B₁₂. This suggested that the chloroplasts contained the binding sites for vitamin ₁₂ and might regulate the uptake process in the intact cell. the kinetic properties of the overall ₁₂ uptake mechanism suggested that the initial phase represent the binding of vitamin ₁₂ to the available sites on the chloroplast. the secondary phase may represent the de novo synthesis of new binding sites.     

Vitamin B12 Production by the Gastro-intestinal Microflora of Baboons Fed either a Vitamin B12 Deficient Diet or a Diet Supplemented with Vitamin B12

The vitamin B₁₂-producing capacity of micro-organisms isolated from baboon faeces and gastric contents was measured using Lactobacillus leichmannii . The animals were fed either a diet deficient in or supplemented with vitamin B₁₂ (controls). Samples of gastric and small intestinal contents, obtained at laparotomy from two young vitamin B₁₂-deprived baboons, contained varying quantities of vitamin B₁₂. Many of the organisms isolated from these aspirates produced vitamin B₁₂ in vitro . The highest levels of vitamin B₁₂ were produced by anaerobic organisms. Gastric juice samples from vitamin B₁₂ deprived and control baboons contained similar types of organisms with like vitamin B₁₂-producing capacity. The vitamin B₁₂ content, pH and total bacterial counts of gastric juice samples aspirated after a 6 h fast from vitamin B₁₂ deprived baboons were not significantly different from those of the control animals. The pH values of gastric juice samples aspirated 18 h after feeding, however, were significantly lower than those of 6 h fasting samples in both groups. The mean vitamin B₁₂ levels in the total volumes of gastric juice aspirated after each fasting period were similar. The possible involvement of the gastrointestinal flora in the vitamin B₁₂ status of the baboon is discussed.     

Polymorphism of vitamin B12 [57Col binding proteins in rabbit serum

When rabbit serum labelled with vitamin B₁₂[⁵⁷Co] was subjected to starch gel electrophoresis and au;oradiography, three phenotypes of proteins capable of binding vitamin B₁₂ were observed. Family data revealed that these phenotypes (called TC-A, TC-AB and TC-B) are controlied by two codominant alleles (TC^A and TC^B), at an autosomic locus. Proteins capable of binding vitamin B₁₂ both in vivo and in vitro are commonly referred to as Transcobalamins and can be found in the serum of numerous animal species (for a review, see Glass, 1974; Allen, 1975; Stenman, 1975). Furthermore, Daiger et al. (1975a) have described seven different patterns of vitamin B₁₂ binding proteins which occur in human plasma and which are presumably controlled by four alleles. The present paper describes experiments in which both starch gel electrophoresis and autoradiography are used to identify three phenotypes of rabbit serum proteins responsible for binding vitamin B₁₂ in vitro. It was found that these three phenotypes are controlled by two allelic codominant genes, at an autosomic locus. Individual serum samples (30 μl), obtained from 385 White New Zealand rabbits varying in age from one month to three years, were incubated with 0.1 ng of vitamin B₁₂[⁵⁷Co] (specific activity: 180 μCi/μg; Lot 247; Radiochemical Centre, Amersham, England) at 37°C for 30 minutes. Starch gel electrophoresis and autoradiography were performed as described by Geldermann (1970) and Daiger et al. (1975b), respectively. Electrofocusing (pH range 3.5–9.5) was conducted in the 2117 Multiphor apparatus (LKB, Bromma, Sweden) according to the manufacturer's instructions. The resulting pH gradient was measured with a surface pH electrode (Ingold, Zürich, Switzerland).     

Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.     

Lactobacillus reuteri CRL 1098 prevents side effects produced by a nutritional vitamin B12 deficiency

Aims:  To evaluate the efficiency of the vitamin B_12-producing Lactobacillus reuteri CRL1098 strain in preventing the symptoms caused by a nutritional cobalamin-deficient diet in pregnant female mice and their weaned offspring.
Methods and Results:  Pregnant female mice were divided into three groups: animals fed with a B₁₂-deficient diet (DD), animals fed with DD plus L. reuteri CRL1098 and animals fed with a B₁₂-sufficient diet. The animals received the different feedings from the end of gestation up to weaning. At the end of the trials, they and their corresponding offspring were bled to determine haematological, immunological and histological parameters. The administration of the pseudovitamin B₁₂-producing strain prevented the symptoms observed in female and weaned young animals fed with a nutritional B₁₂-deficient diet.
Conclusions:  Our data suggest that the pseudovitamin B₁₂ produced by L. reuteri CRL1098 is biologically active and effective in preventing the pathologies caused by the nutritional deficiency of B₁₂ both in pregnant mice and their offspring.
Significance and Impact of the Study:  The ability of L. reuteri CRL1098 to prevent a nutritional vitamin deficiency was demonstrated for the first time. The addition of a GRAS micro-organism to complement the B₁₂ content in deficient foods is an interesting biotechnological alternative.     

Existence of Three Subtypes of Bradykinin B2 Receptors in Guinea Pig

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin B12 transport in Escherichia coli: energy coupling between membranes

Cells of Escherichia coli possess high-affinity active transport systems of vitamin B12 and iron-siderophore complexes. Specific outer-membrane proteins carry out the energy-dependent transport across the outer membrane, in conjunction with the TonB coupling protein. Mutagenesis experiments have identified a conserved region near the amino-terminus of the outer-membrane transporters that is necessary for energy-coupled transport. The ability of extragenic suppressor mutations in tonB to correct the transport defect indicates that TonB couples the proton-motive force to the outer-membrane proteins by direct contact.     

Rescue by vitamin B12 of Escherichia coli cells treated with colicins A and E allows measurement of the kinetics of colicin binding on BtuB

Abstract Sensitivity of Escherichia coli bacteria to colicins A and E1 was significantly increased by overproduction of the BtuB receptor protein. The amount of vitamin B₁₂ needed before colicins A and E1 treatment to protect cells against killing was found to be a function of the number of BtuB molecules present at the cell surface. Cells treated by colicins A and E were rescued from killing by addition of vitamin B₁₂ shortly after colicin treatment. The rate of reversal by vitamin B₁₂ may correspond to the kinetics of irreversible binding to BtuB of the various colicins.     

High Doses of Vitamin B6 in the Rat Are Associated with Inhibition of Hepatic Tryptophan Metabolism and Increased Uptake of Tryptophan into the Brain

Abstract: Acute administration of vitamin B₆ to rats (10 mg/kg body weight) led to reduced urinary excretion of N ¹-methyl nicotinamide and methyl pyridone carboxamide, indicating inhibition of the oxidative metabolism of tryptophan. There was a considerable reduction in the production of ¹⁴CO₂ from [ ring -2-¹⁴C]tryptophan, and a significant inhibition of hepatic tryptophan oxygenase when measured in liver homogenates, together with an increase in the concentration of tryptophan in plasma. There was an increase in both the concentration of tryptophan in the brain and the uptake into the brain of peripherally administered [³H]tryptophan, accompanied by a small increase in the rate of synthesis of 5-hydroxy-tryptamine in the brain. It is suggested that this increase in the uptake of tryptophan into the brain following a relatively large dose of vitamin B₆ may explain the beneficial action of the vitamin in some cases of depressive illness.     

Serum Folate and Vitamin B12 Levels in Children from Mozambique

In order to investigate the behaviour of biochemical parameters in children from Mozambique, we have determined the serum levels of folic acid and vitamin B12, two well known markers of nutritional anemia.     

Cloning, sequencing and expression in Escherichia coli of the gene encoding component S of the coenzyme B12-dependent glutamate mutase from Clostridium cochlearium

Abstract Adenosylcobalamin (coenzyme B₁₂) dependent glutamate mutase catalyzes the carbon skeleton rearrangement of ( S )-glutamate to (2S,3 S )-methylaspartate. This is the first step of the fermentation of glutamate by the strict anaerobic bacterium Clostridium cochlearium . The enzyme consists of the two protein components E and S. The gene encoding component S ( glmS ) was cloned in Escherichia coli and its nucleotide sequence was determined. The nucleotide sequence and the deduced amino acid sequence showed very strong identities to the sequence of the glmS (also called mutS ) gene (80%) and to component S (82%) from the related C. tetanomorphum , respectively. Cell-free extracts of E. coli carrying the glmS gene showed glutamate mutase activity which was strictly dependent on the addition of coenzyme B₁₂ and component E purified from C. cochlearium . Enzyme activity of the recombinant protein was achieved up to 2200 nkat/g wet cells wich is due to a ten-fold overexpression compared with the activities determined in cell-free extracts of C. cochlearium . This is the first report of overexpression of an active component of glutamate mutase. A rapid purification procedure consisting only of ammonium sulfate precipitation and a gel filtration step was developed to obtain large amounts of pure component S in a short time.