Combined dietary folate, vitamin B-12, and vitamin B-6 intake influences plasma docosahexaenoic acid concentration in rats.

BACKGROUND: Folate, vitamin B-12, and vitamin B-6 are essential nutritional components in one-carbon metabolism and are required for methylation capacity. The availability of these vitamins may therefore modify methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) by PE-N-methyltransferase (PEMT) in the liver. It has been suggested that PC synthesis by PEMT plays an important role in the transport of polyunsaturated fatty acids (PUFAs) like docosahexaenoic acid (DHA) from the liver to plasma and possibly other tissues. We hypothesized that if B-vitamin supplementation enhances PEMT activity, then supplementation could also increase the concentration of plasma levels of PUFAs such as DHA. To test this hypothesis, we determined the effect of varying the combined dietary intake of these three B-vitamins on plasma DHA concentration in rats. METHODS: In a first experiment, plasma DHA and plasma homocysteine concentrations were measured in rats that had consumed a B-vitamin-poor diet for 4 weeks after which they were either continued on the B-vitamin-poor diet or switched to a B-vitamin-enriched diet for another 4 weeks. In a second experiment, plasma DHA and plasma homocysteine concentrations were measured in rats after feeding them one of four diets with varying levels of B-vitamins for 4 weeks. The diets provided 0% (poor), 100% (normal), 400% (enriched), and 1600% (high) of the laboratory rodent requirements for each of the three B-vitamins. RESULTS: Plasma DHA concentration was higher in rats fed the B-vitamin-enriched diet than in rats that were continued on the B-vitamin-poor diet (P = 0.005; experiment A). Varying dietary B-vitamin intake from deficient to supra-physiologic resulted in a non-linear dose-dependent trend for increasing plasma DHA (P = 0.027; experiment B). Plasma DHA was lowest in rats consuming the B-vitamin-poor diet (P > 0.05 vs. normal, P < 0.05 vs. enriched and high) and highest in rats consuming the B-vitamin-high diet (P < 0.05 vs. poor and normal, P > 0.05 vs. enriched). B-vitamin deficiency significantly increased plasma total homocysteine but increasing intake above normal did not significantly reduce it. Nevertheless, in both experiments plasma DHA was inversely correlated with plasma total homocysteine. CONCLUSION: These data demonstrate that dietary folate, vitamin B-12, and vitamin B-6 intake can influence plasma concentration of DHA.

Plasma choline concentration varies with different dietary levels of vitamins B6, B12 and folic acid in rats maintained on choline-adequate diets.

Choline is an important component of the human diet and is required for the endogenous synthesis of choline-containing phospholipids, acetylcholine and betaine. Choline can also be synthesised de novo by the sequential methylation of phosphatidylethanolamine to phosphatidylcholine. Vitamins B6, B12 and folate can enhance methylation capacity and therefore could influence choline availability not only by increasing endogenous choline synthesis but also by reducing choline utilisation. In the present experiment, we determined whether combined supplementation of these B vitamins affects plasma choline concentration in a rat model of mild B vitamin deficiency which shows moderate increases in plasma homocysteine. To this end, we measured plasma choline and homocysteine concentrations in rats that had consumed a B vitamin-poor diet for 4 weeks after which they were either continued on the B vitamin-poor diet or switched to a B vitamin-enriched diet for another 4 weeks. Both diets contained recommended amounts of choline. Rats receiving the B vitamin-enriched diet showed higher plasma choline and lower plasma homocysteine concentrations as compared to rats that were continued on the B vitamin-poor diet. These data underline the interdependence between dietary B vitamins and plasma choline concentration, possibly via the combined effects of the three B vitamins on methylation capacity.

Synaptic proteins and phospholipids are increased in gerbil brain by administering uridine plus docosahexaenoic acid orally.

The synthesis of brain phosphatidylcholine may utilize three circulating precursors: choline; a pyrimidine (e.g., uridine, converted via UTP to brain CTP); and a PUFA (e.g., docosahexaenoic acid); phosphatidylethanolamine may utilize two of these, a pyrimidine and a PUFA. We observe that consuming these precursors can substantially increase membrane phosphatide and synaptic protein levels in gerbil brains. (Pyrimidine metabolism in gerbils, but not rats, resembles that in humans.) Animals received, daily for 4 weeks, a diet containing choline chloride and UMP (a uridine source) and/or DHA by gavage. Brain phosphatidylcholine rose by 13-22% with uridine and choline alone, or DHA alone, or by 45% with the combination, phosphatidylethanolamine and the other phosphatides increasing by 39-74%. Smaller elevations occurred after 1-3 weeks. The combination also increased the vesicular protein Synapsin-1 by 41%, the postsynaptic protein PSD-95 by 38% and the neurite neurofibrillar proteins NF-70 and NF-M by up to 102% and 48%, respectively. However, it had no effect on the cytoskeletal protein beta-tubulin. Hence, the quantity of synaptic membrane probably increased. The precursors act by enhancing the substrate saturation of enzymes that initiate their incorporation into phosphatidylcholine and phosphatidylethanolamine and by UTP-mediated activation of P2Y receptors. Alzheimer's disease brains contain fewer and smaller synapses and reduced levels of synaptic proteins, membrane phosphatides, choline and DHA. The three phosphatide precursors might thus be useful in treating this disease.

Cytidine and uridine increase striatal CDP-choline levels without decreasing acetylcholine synthesis or release.

AIMS: Treatments that increase acetylcholine release from brain slices decrease the synthesis of phosphatidylcholine by, and its levels in, the slices. We examined whether adding cytidine or uridine to the slice medium, which increases the utilization of choline to form phospholipids, also decreases acetylcholine levels and release. METHODS: We incubated rat brain slices with or without cytidine or uridine (both 25-400 microM), and with or without choline (20-40 microM), and measured the spontaneous and potassium-evoked release of acetylcholine. RESULTS: Striatal slices stimulated for 2 h released 2650+/-365 pmol of acetylcholine per mg protein when incubated without choline, or 4600+/-450 pmol/mg protein acetylcholine when incubated with choline (20 microM). Adding cytidine or uridine (both 25-400 microM) to the media failed to affect acetylcholine release whether or not choline was also added, even though the pyrimidines (400 microM) did enhance choline;s utilization to form CDP-choline by 89 or 61%, respectively. The pyrimidines also had no effect on acetylcholine release from hippocampal and cortical slices. Cytidine or uridine also failed to affect acetylcholine levels in striatal slices, nor choline transport into striatal synaptosomes. CONCLUSION: These data show that cytidine and uridine can stimulate brain phosphatide synthesis without diminishing acetylcholine synthesis or release.

Oral uridine-5'-monophosphate (UMP) increases brain CDP-choline levels in gerbils.

We examined the biochemical pathways whereby oral uridine-5'-monophosphate (UMP) increases membrane phosphatide synthesis in brains of gerbils. We previously showed that supplementing PC12 cells with uridine caused concentration-related increases in CDP-choline levels, and that this effect was mediated by elevations in intracellular uridine triphosphate (UTP) and cytidine triphosphate (CTP). In the present study, adult gerbils received UMP (1 mmol/kg), a constituent of human breast milk and infant formulas, by gavage, and plasma samples and brains were collected for assay between 5 min and 8 h thereafter. Thirty minutes after gavage, plasma uridine levels were increased from 6.6 +/- 0.58 to 32.7 +/- 1.85 microM (P < 0.001), and brain uridine from 22.6 +/- 2.9 to 89.1 +/- 8.82 pmol/mg tissue (P < 0.001). UMP also significantly increased plasma and brain cytidine levels; however, both basally and following UMP, these levels were much lower than those of uridine. Brain UTP, CTP, and CDP-choline were all elevated 15 min after UMP (from 254 +/- 31.9 to 417 +/- 50.2, [P < 0.05]; 56.8 +/- 1.8 to 71.7 +/- 1.8, [P < 0.001]; and 11.3 +/- 0.5 to 16.4 +/- 1, [P < 0.001] pmol/mg tissue, respectively), returning to basal levels after 20 and 30 min. The smallest UMP dose that significantly increased brain CDP-choline was 0.05 mmol/kg. These results show that oral UMP, a uridine source, enhances the synthesis of CDP-choline, the immediate precursor of PC, in gerbil brain.

A Drosophila temperature-sensitive seizure mutant in phosphoglycerate kinase disrupts ATP generation and alters synaptic function.

A novel paralytic mutant, nubian, was identified in a behavioral screen for conditional temperature-sensitive seizure mutants in Drosophila melanogaster. nubian mutants display reduced lifespan, abnormal motor behavior, altered synaptic structure, and defective neurotransmitter release. The nubian mutant disrupts phosphoglycerate kinase (PGK), an enzyme required for ATP generation in the terminal stage of the glycolytic pathway. Consistent with altered ATP generation in nubian animals, brain extracts show a threefold reduction in resting ATP levels compared with controls. Microarray analysis of nubian mutants reveals altered transcription of genes implicated in glucose and lipid metabolism. Disruption of ATP generation in nubian animals is accompanied by temperature-dependent defects in neuronal activity, with initial seizure activity, followed by an activity-dependent loss of synaptic transmission. nubian mutants also display structural defects at the synapse, with larger varicosity size but normal varicosity number, indicating that these synaptic parameters are regulated independently. Both exocytotic (NSF) and endocytotic (dynamin) ATPase/GTPase activity are required for normal synaptic transmission. Biochemical and physiological analyses indicate that synaptic defects in nubian animals are secondary to defective endocytosis, suggesting that endocytotic pathways may be generally more sensitive to altered ATP levels than those used for exocytosis. Alterations in ATP metabolism likely disrupt similar pathways in humans, because PGK deficiency is associated with mental retardation, seizures, and exercise intolerance. Given the behavioral similarities between disruptions of PGK function in Drosophila and humans, the analysis of nubian animals may reveal conserved neuronal responses associated with altered ATP generation within the brain.

Stimulation of CDP-choline synthesis by uridine or cytidine in PC12 rat pheochromocytoma cells.

Oral administration of CDP-choline to rats raises plasma and brain cytidine levels and increases brain levels of phosphatidylcholine (PC). In contrast, in humans oral CDP-choline increases plasma levels of uridine. To determine whether uridine can also enhance PC synthesis, we developed an assay for CDP-choline, an immediate and rate-limiting precursor in PC synthesis, and measured this intermediate in clonal PC12 rat pheochromocytoma cells incubated with various concentrations of uridine or cytidine. Addition of uridine (50-100 microM) to the incubation medium caused significant elevations in UTP, CT, USAP and CDP-choline levels in PC12 cells. Uridine had no effect on the synthesis of diacylglycerol (DAG) or the activity of the phosphotransferase which catalyzes the synthesis of PC from DAG and CDP-choline. Hence uridine treatment was unlikely to inhibit the conversion of endogenous CDP-choline to PC. These results suggest the possibility that uridine may also enhance PC synthesis in intact brain.