Effect of metal specific amino acid complexes and inorganic trace minerals on vitamin stability in premixes

Stability of vitamin activity in a swine premix containing metal specific amino acid complexes, inorganic trace minerals, or no trace minerals was evaluated over a 120-day storage period. Two vitamin-trace mineral premixes containing either metal specific amino acid complexes or inorganic trace mineral sources were formulated to contain 200% of NRC (1988) sow requirements for I, Cu, Zn, Mn, Fe, and Se based on a 5 g/kg dietary inclusion rate. A separate vitamin premix containing no trace minerals served as the control. The vitamin premix and the two vitamin-trace mineral premixes were formulated to contain the same level of vitamins. Vitamin levels exceeded NRC (1988) and were chosen to represent “typical” industry levels based on an informal survey of vitamin levels in commercial premixes in the U.S.A. Premixes were stored in an environmentally controlled feed storage room and samples were collected every month to determine vitamin activity. Minimal monthly vitamin stock losses in activity (0–1%) were observed for all vitamins except cyanocobalamin (2.8%) and choline (1.3%). Pantothenate, vitamin E, riboflavin, biotin and niacin were most resistant to destruction, while menadione, retinol, vitamin B-6, and thiamine were subject to the greatest loss of activity during the 120-day storage period. Use of metal specific amino acid complexes in vitamin-trace mineral premixes significantly reduced the loss of retinol, menadione, cyanocobalamin, thiamine, folates, vitamin B-6, and choline activity (P<0.05) compared to losses of vitamin activity in premixes containing inorganic trace minerals. Activity losses in retinol, cyanocobalamin, thiamine, and choline were similar between the vitamin premix and the vitamin-complexed trace mineral premix. Biotin activity was undetectable in the vitamin-complexed trace mineral premix due to unexplained analytical interference. Each vitamin was ranked according to relative vitamin assay cost, loss in vitamin activity per month, and susceptibility to multiple stress factors. This ranking was used to identify vitamins that could represent overall vitamin activity in a premix and could be assayed at a reasonable cost for a feed manufacturing quality control program. Retinol was identified as the best indicator vitamin, followed by thiamine, menadione, and cyanocobalamin. These results suggest that vitamin stability in swine vitamin-trace mineral premixes is improved when using metal specific amino acid complexes compared to inorganic trace mineral sources. More liberal safety margins for vitamins may be needed when formulating vitamin-trace mineral premixes using inorganic sources of trace minerals.     

Interactive effects of vitamins A and K3 on laying performance,egg quality,tibia attributes and antioxidative status of aged Roman Pink laying hens

Extending laying cycle is a tendency in hen breeding, but egg quality declines as laying hens age. The present study was conducted to investigate the interactive effects of vitamins A and K₃ on laying performance, egg and tibia quality, and antioxidative status of aged Roman Pink laying hens. In a 3 × 3 factorial arrangement, 1 080 87-week-old laying hens were allocated to nine groups with eight replicates in each group. Deficient, adequate and excess vitamins A (0, 7 000 and 14 000 IU/kg) and K₃ (0, 2.0 and 4.0 mg/kg) were supplemented into a basal diet with 1 320 IU/kg of vitamin A and 0.5 mg/kg of vitamin K₃. After 2 weeks of adaption to basal diet, hens were fed corresponding diets for 8 weeks. Vitamins A and K₃ did not significantly affect the laying performance. However, they showed interactive effects on yolk ratio at week 93 as well as tibia weight and diameter (P < 0.05), and hens fed deficient vitamins A and K₃ had the highest yolk ratio and tibia weight, but the lowest tibia diameter. Compared with deficient addition, adequate or excess vitamins A and K₃ increased yolk color at weeks 93 and 97 (P < 0.05). Compared with hens fed deficient or excess vitamins, hens fed adequate vitamins A and K₃ had higher eggshell strength at week 93 or 97 (P < 0.05). Increasing vitamin A elevated plasma total superoxide dismutase (T-SOD) activity and decreased hepatic glutathione peroxidase (GSH-Px) activity (P < 0.05). Excess vitamin K₃ increased hepatic T-SOD activity (P < 0.05). Vitamins A and K₃ exhibited interaction on the activities of antioxidative enzymes in eggshell gland (P < 0.05), and adequate or excess vitamins A and K₃ increased the activities of GSH-Px, T-SOD and catalase (CAT). Adequate and excess vitamin A up-regulated the mRNA expression of GSH-Px1, GSH-Px3 and SOD1 in eggshell gland (P < 0.05). Vitamins A and K₃ showed interactive effects on CAT mRNA expression in eggshell gland (P < 0.05) and hens fed adequate vitamins A and K₃ had the highest CAT mRNA levels. In conclusion, dietary addition of vitamins A and K₃ improved the eggshell quality and yolk color as well as antioxidative status in eggshell gland of aged laying hens. Adequate vitamins A and K₃ showed beneficial effects and excess levels did not exhibit superior effects.     

Antioxidant vitamins C, E and beta-carotene reduce DNA damage before as well as after gamma-ray irradiation of human lymphocytes in vitro

The protective effect of Vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in human lymphocytes in vitro was investigated. Cultured lymphocytes were exposed to increasing concentration of these vitamins either before or after irradiation with 2Gy of gamma-rays and DNA damage was estimated using micronucleus assay. A radioprotective effect was observed when antioxidant vitamins were added to cultured cells before as well after irradiation; the strongest effect was observed when they were added no later than 1h after irradiation. The radioprotective effect of vitamins also depended on their concentration; Vitamins C added at low concentration (1 microg/ml) before exposure of the cells to radiation prevented induction of micronuclei. Vitamin E at the concentration above 2 microg/ml decreased the level of radiation-induced micronuclei when compared to the cells irradiated without vitamin treatment. beta-Carotene was effective at all tested concentrations from 1 to 5 microg/ml and reduced the number of micronuclei in irradiated cells. The vitamins had no effect on radiation-induced cytotoxicity as measured by nuclear division index. The radioprotective action of antioxidant Vitamins C, E and beta-carotene was dependent upon their concentration as well as time and sequence of application.     

Vitamins as effectors of monensin production byStreptomyces cinnamonensis

Vitamins added to submergedStreptomyces cinnamonensis cultures stimulated the production of monensins. Vitamins B₂, B₃, B₅ and B₁₂ enhanced the production by about 50%, vitamins B₁ and B₆ by 100%. The addition of biotin in optimal concentration resulted in more than 3-fold increase in total production.     

PURIFICATION AND PROPERTIES OF CHOLINE ACETYLTRANSFERASE FROM THE NERVOUS SYSTEM OF DIFFERENT INVERTEBRATES

—Choline acetyltransferase has been purified from three invertebrate species, namely snail (Helix aspersa), cockroach (Periplaneta americana) and horse shoe crab (Limulus polyphemus.) All three enzymes followed a Theorell-Chance enzyme mechanism with a sequential addition of the substrates. All three enzymes were activated by sodium and potassium chloride and inhibited by high concentrations of magnesium or calcium chloride. The apparent K_m for choline and acetyl-CoA was for snail: K_mch= 370 μm ,K_mAcetyl-CoA= 51μm ; cockroach:K_mCh= 550 μm , K_mAcely-CoA= 16 μm horse shoe crab:K_mCn= 2700 μm K_mAcctyl-coA= 68 μm CoA inhibited the enzymes competitively with respect to acetyl-CoA and non-competitively with respect to choline. Acetylcholine inhibited the enzymes competitively with respect to choline and non-competitively with respect to acetyl-CoA. All the enzymes were inhibited strongly by 5,5′-dithiobis (2-nitrobenzoate), iodoacetate, acryloylcholine, chloracetylcholine and 3-bromacetonyltrimethyl-ammonium. The enzymes were only weakly inhibited by the styrylpyridine derivatives. The isoelectric points were 5.3 and 5.0 for the horse shoe crab and cockroach enzymes respectively. All three enzymes showed low affinity for a cation-exchanger (CM-Sephadex).     

Radioprotective effect of vitamins C and E

Albino rats were treated with aqueous vitamin C solution and vitamin E solution dissolved in olive oil at two concentrations, 100 and 300 mg/kg/day, for 6 months. Some of the animals were then subjected to whole-body irradiation. Chromosomal aberrations and mitotic activity in non-irradiated and irradiated groups were recorded. Both vitamins were found to be non-mutagenic. Vitamins C exerted a radioprotective effect but vitamin E was not radioprotective and it suppressed the radioprotection otherwise produced by olive oil.     

Plasma alpha-tocopherol, retinol, cholesterol, and mineral concentrations in captive gorillas

Vitamins A, E, cholesterol, and mineral (calcium, copper, iron, magnesium, phosphorus, selenium, sodium, and zinc) concentrations were examined in the plasma of 74 captive lowland gorillas aged newborn to 41 years. Effects of age or sex on measured parameters were not significant. Plasma Mg and Ca concentrations were lower than reported captive gorilla means, whereas Na and P were higher. Since comparative gorilla values for certain blood components (vitamins E and A, copper, and selenium) are lacking, normal human values may provide the best available indicators for evaluating the plasma levels of these components in gorillas.     

HIGH AFFINITY CHOLINE UPTAKE: IONIC AND ENERGY REQUIREMENTS

Abstract— High affinity choline uptake into rat hippocampal synaptosomes was examined at 37°C when various ions were deleted from normal Kreb's-Ringer media. When sodium chloride was replaced by sucrose, lithium chloride, cesium chloride or rubidium chloride, choline uptake was markedly reduced. When the sodium concentrations of the Kreb's media were gradually reduced to zero, the uptake was gradually reduced in parallel. A kinetic analysis performed at low and normal sodium concentrations revealed changes in K_m and V^max values. When several non-chloride sodium salts were utilized, the uptake was reduced in all cases suggesting also a chloride-dependence in addition to the sodium-dependence. Omission of calcium chloride or magnesium sulfate from the media did not alter uptake. Sodium-dependent choline uptake was examined over a range of potassium concentrations (0–35 DIM). It was found that uptake was maximal between potassium concentrations of 0.35–4.8 mm but was reduced at both lower and higher potassium concentrations. The kinetics of uptake were examined under varying potassium concentrations, and at low potassium, only a change in V_max was observed while at high potassium concentrations, there were changes in both K^m and V^max values. Preincubation and incubation of synaptosomes with 0.1 m -ouabain, 0.1 mm -2,4-dinitrophenol and 1 mm -KCN caused a reduction in sodium-dependent uptake. When dextrose was omitted from the preincubation and incubation media there was also a reduction in sodium-dependent uptake. By contrast, the sodium-independent uptake was unaffected by the metabolic inhibitors or omission of dextrose, and had a very low Q₁₀. When various incubation temperatures were utilized in uptake experiments, the Q¹⁰ for the interval 37-27°C was 2.7 and the activation energy was 22.7 kcal/mol. Slightly different ionic dependences were observed when animals pretreated with pentobarbital of oentylenetetrazol were utilized as the source of synaptosomes.     

Adsorption of peroxidase on Celite 545 directly from ammonium sulfate fractionated white radish (Raphanus sativus) proteins

This paper demonstrates the direct immobilization of peroxidase from ammonium sulfate fractionated white radish proteins on an inorganic support, Celite 545. The adsorbed peroxidase was crosslinked by using glutaraldehyde. The activity yield for white radish peroxidase was adsorbed on Celite 545 was 70% and this activity was decreased and remained 60% of the initial activity after crosslinking by glutaraldehyde. The pH and temperature-optima for both soluble and immobilized peroxidase was at pH 5.5 and 40°C. Immobilized peroxidase retained higher stability against heat and water-miscible organic solvents. In the presence of 5.0 mM mercuric chloride, immobilized white radish peroxidase retained 41% of its initial activity while the free enzyme lost 93% activity. Soluble enzyme lost 61% of its initial activity while immobilized peroxidase retained 86% of the original activity when exposed to 0.02 mM sodium azide for 1 h. The K_m values were 0.056 and 0.07 mM for free and immobilized enzyme, respectively. Immobilized white radish peroxidase exhibited lower V_max as compared to the soluble enzyme. Immobilized peroxidase preparation showed better storage stability as compared to its soluble counterpart.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Gas chromatography of retinol and α-tocopherol without derivatization

An improved gas chromatographic method for the analysis of retinol and α-tocopherol in biological samples is described. The use of cold on-column injection in combination with wall coated open tubular column gas chromatography eliminates thermal decomposition of vitamin A and yields efficient separations of fat-soluble vitamins (A, D₂, D₃, and E) without derivatization. Peak tailing was judged to be minimal. Vitamins were quantified by flame ionization detection responses down to 3.5 ng injected, and their identifies were confirmed using gas chromatography-mass spectrometry. Extracts of biological samples were saponified, and sterols were removed using digitonin-impregnated celite chromatography before analysis by gas chromatography and gas chromatography-mass spectrometry. Recoveries of vitamins from a test diet ranged from 89 to 103%.     

团头鲂幼鱼对不同浓度胆碱的利用率及7种常见饲料原料中胆碱生物学效价的评定

研究常见饲料原料中胆碱的生物学效价,为渔用配合饲料配制提供基础数据。试验选取团头鲂平均体重（3.50.1）g 990尾,以酪蛋白、明胶等原料配制4个氯化胆碱添加水平（0、1030、1230、1430 mg/kg）的纯合饲料,并在1030 mg/kg的基础上分别添加7种常见饲料原料（鱼粉、豆粕、菜粕、棉粕、次粉、麸皮、米糠）,使得各组配合饲料中胆碱含量为1230 mg/kg,共11组,每组3重复,饲养8周。根据肝脏胆碱沉积量和试验鱼胆碱摄食量,评定团头鲂幼鱼对不同浓度的胆碱利用率和这7种原料中胆碱的生物学效价。结果表明:在4个纯和饲料中,肝脏胆碱沉积量和增重率随着饲料胆碱水平从0到1230 mg/kg饲料的增加而显著上升（P0.05）,并在饲料胆碱水平从1230到1430 mg/kg饲料的增加而差异不显著（P0.05）;在同一胆碱水平（1230 mg/kg）的条件下,原料组的增长率均高于对照组;团头鲂幼鱼对鱼粉、豆粕、菜粕、棉粕、次粉、麸皮和米糠的胆碱生物学效价分别为87.42%、112.54%、76.84%、98.00%、95.91%、43.88%、91.5%。分析可知,团头鲂生产饲料中尚需要额外添加氯化胆碱方能满足其对胆碱的需要,实际添加量与饲料所使用的原料有关。       

Quantification of fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry

Sensitive quantification method for fat-soluble vitamins in human breast milk by liquid chromatography-tandem mass spectrometry was developed. Vitamins A, D and E were extracted from 10.0 mL of breast milk after saponifying by basic condition. Vitamin K derivatives were extracted from 3.0 mL of breast milk after lipase treatment. The corresponding stable isotope-labeled compounds were used as internal standards. For the determination of vitamin D compounds, derivatization with a Cookson-type reagent was performed. All fat-soluble vitamins were determined by liquid chromatography-tandem mass spectrometry in the positive ion mode. The detection limits of all analytes were 1-250 pg per 50 microL. The recoveries of fat-soluble vitamins were 91-105%. Inter-assay CV values of each vitamin were 1.9-11.9%. The mean concentrations of retinol, vitamin D3, 25-hydroxyvitamin D3, alpha-tocopherol, phylloquinone and menaquinone-4 were 0.455 microg/mL, 0.088 ng/mL, 0.081 ng/mL, 5.087 microg/mL, 3.771 ng/mL, and 1.795 ng/mL, respectively (n=82). This method makes possible to determine fat-soluble vitamins with a wide range of polarities in human breast milk. The assay may be useful for large-scale studies.     

Factors affecting the transport of β-amino acids in rat renal brush-border membrane vesicles. The role of external chloride

The effect of a variety of ions and other solutes on the accumulation of the β-amino acid, taurine, was examined in rat renal brush-border membrane vesicles. Initial taurine uptake (15 and 30 s) is sodium-dependent with a typical overshoot. This Na⁺ effect was confirmed by exchange diffusion and gramicidin inhibition of taurine uptake. External K⁺ or Li⁺ do not increase taurine accumulation more than Na⁺-free mannitol, except that the combination of external K⁺ and Na¹ in the presence of nigericin enhances uptake. Of all anions tested, including more permeant (SCN⁻ and NO₃⁻) or less permeant (SO₄²⁻), chloride supported taurine accumulation to a significantly greater degree. Preloading vesicles with choline chloride reduced taurine uptake, suggesting that external Cl⁻ stimulates uptake. Since this choline effect could be related to volume change, due to the slow diffusion of choline into vesicles, brush-border membrane vesicles were pre-incubated with LiCl, LiNO₃ and LiSO₄. Internal LiCl, regardless of the final Na⁺ anion mixture, reduced initial rate (15 and 60 s) and peak (360 s) taurine uptake. Internal LiNO₃ or LiSO₄ with external NaCl resulted in similar or higher values of uptake at 15, 60 and 360 s, indicating a role for external Cl⁻ in taurine uptake in addition to Na⁺ effect. Although uptake by vesicles is greatest at pH 8.0 and inhibited at acidic pH values (pH less than 7.0), an externally directed H⁺ gradient does not influence uptake. Similarly, amiloride, an inhibitor of the Na⁺/H⁺ antiporter, had no influence on taurine accumulation over a wide variety of concentrations or at low Na⁺ concentrations. Taurine uptake is blocked only by other β-amino acids and in a competitive fashion. d-glucose and p-aminohippurate at high concentrations (> 10⁻³ M) reduce taurine uptake, possibly by competing for sodium ions, although gramicidin added in the presence of d-glucose inhibits taurine uptake even further. These studies more clearly define the nature of the renal β-amino acid transport system in brush-border vesicles and indicate a role for external Cl in this uptake system.     

In Vitro Effects of Arachidonic and L-Glutamic Acids on the High-Affinity Choline Transport in Rat Hippocampus

A second messenger role for arachidonic acid (AA) in the regulation of the high-affinity choline uptake (HACU) was suggested. It was repotted that micromolar concentrations of AA applied in vitro decreased the HACU values and increased the specific binding of [³H]hemicholinium-3 ([³H]HCh-3). It was published that L-glutamic acid (GA) applied in vivo produced a fall in the HACU values. In addition, GA liberates free AA. In this study, an ability of GA to influence in vitro the activity of presynaptic cholinergic nerve terminals via its effect on the release of AA is investigated in hippocampal synaptosomes of young Wistar rats. Millimolar concentrations of GA decrease both the high- and low-affinity choline uptake, the specific as well as nonspecific binding of [³H]HCh-3 and the activity of Na⁺,K⁺-ATPase. Kinetic analysis (Lineweaver-Burk and Scatchard plots) reveals a change in V_max and B_max, but not in K_M and K_D. It appears very likely that under normal conditions GA applied in vitro is not able to change markedly the choline transport via its effect on the release of AA. Results confirm the hypothesis about an indirect inhibitory role for glutamatergic receptors on cholinergic cells.     

The effect of salt and temperature on the conformational changes of P1LEA‐22, a repeat unit of plant Late Embryogenesis Abundant proteins

The effect of choline chloride on the conformational dynamics of the 11‐mer repeat unit P1LEA‐22 of group 3 Late Embryogenesis Abundant (G3LEA) proteins was studied. Circular dichroism data of aqueous solutions of P1LEA‐22 revealed that the peptide favors a polyproline II (PPII) helix structure at low temperature, with increasing temperature promoting a gain of unstructured conformations. Furthermore, increases in sample FeCl₃ or choline chloride concentrations causes a gain in PPII helical structure at low temperature. The potential role of PPII structure in intrinsically disordered and G3LEA proteins is discussed, including its ability to easily access other secondary structural conformations such as α‐helix and β‐sheet, which have been observed for dehydrated G3LEA proteins. The observed effect of FeCl₃ and choline chloride salts on P1LEA‐22 suggests favorable cation interactions with the PPII helix, supporting ion sequestration as a G3LEA protein function. As choline chloride is suggested to improve salt tolerance and protect cell membrane in plants at low temperature, our results support adoption of the PPII structure as a possible damage‐preventing measure of Late Embryogenesis Abundant proteins.     

Homologous metabolic and gene activating routes for vitamins E and K

Vitamins E and K share structurally related side chains and are degraded to similar final products. For vitamin E the mechanism has been elucidated as initial ω-hydroxylation and subsequent β-oxidation. For vitamin K the same mechanism can be suggested analogously. ω-Hydroxylation of vitamin E is catalyzed by cytochrome P450 enzymes, which often are induced by their substrates themselves via the activation of the nuclear receptor PXR. Vitamin E is able to induce CYP3A-forms and to activate a PXR-driven reporter gene. It is shown here that K-type vitamins are also able to activate PXR. A ranking showed that compounds with an unsaturated side chain were most effective, as are tocotrienols and menaquinone-4 (vitamin K₂), which activated the reporter gene 8–10-fold. Vitamers with a saturated side chain, like tocopherols and phylloquinone were less active (2–5-fold activation). From the fact that CYPs commonly responsible for the elimination of xenobiotics are involved in the metabolism of fat-soluble vitamins and the ability of the vitamins to activate PXR it can be concluded that supranutritional amounts of these vitamins might be considered as foreign.     

Dietary supplementation of vitamin A, C and E prevents p-dimethylaminoazobenzene induced hepatic DNA damage in rats

The preventive effect of antioxidant vitamins A, C, E and their analogues against DNA damage induced by a hepatocarcinogen p-dimethylaminoazobenzene (DAB) was assessed by comet assay. For genotoxicity (DNA damage) study, male albino rats were divided into 11 groups, consisting of four rats each. Group I served as control. Group II to VII received 1, 10, 100, 200, 300 and 400 mg per kg body wt of DAB respectively; group VIII to XI received 500 mg/kg body wt of DAB. They were sacrificed by cervical decapitation 3, 6, 12 and 24 h after treatment; livers were excised immediately and subjected to comet assay to measure DNA damage. To study the effect of vitamins, experiments were conducted on a group of 275 rats divided into 3 sets of 25 rats each. First set served as control; second set received 0.06% DAB and third set received 0.06% DAB, along with analogues of vitamins A, C and E. Rats fed with 0.06% DAB were provided water ad libitum for a period of 4 months, followed by a normal (basal) diet for further 2 months. Vitamins A (10,000-50,000 IU), C (75-1000 mg) and E (50-500 mg) and their analogues were given (per kg body wt) to the third set of rats by gavage route once in a week for a period of 6 months. The DAB induced DNA damage only at the highest tested dose of 500 mg/kg body wt. Administration of high doses of vitamin A acid, L-ascorbic acid and vit. E succinate individually prevented the DNA damage. However, administration of a mixture of these vitamins at low doses prevented the DAB-induced DNA damage, which may be due to their synergistic effect. The results indicate that there is a significant advantage in mixed vitamins therapy at low dose over the treatment with individual vitamins.     

Choline fluxes in primary nerve cell cultures. Correlation with the endocellular metabolism of choline

The fluxes of choline across the plasma membrane were measured in primary nerve cell cultures from chick embryo cerebral hemispheres containing neurons and supporting cells.The incubation of cells with exogenous concentrations of choline far below the concentrations present in the growth medium (～30–50 μM) and in the range of the high affinity uptake mechanism (about 0.5 μM) profoundly affected the steady state of the endocellular free choline levels. The kinetics of the uptake were dependent upon the endocellular status of the choline pool since after preincubation in the absence of choline two K_ms are observed (K_m1: 0.8 μM; V_max1: 44.8 pmol/mg protein/2 min; K_m2: 14.3 μM, V_max2: 333.3 pmol/mg protein/2 min) while only one mechanism can be found when the endocellular pool of choline was kept in steady state conditions (K_m: 14.3 μM, V_max: 545.5 pmol/mg protein/2 min). The presence of an homoexchange phenomenon was suspected since choline efflux could be increased by increasing the concentrations of choline in the incubation medium.The results suggest that the movement of choline into nerve cells in culture appears to be mediated by a single mechanism which is regulated by the endocellular status of the choline pool.     

Adenosine transport systems on dissociated brain cells from mouse,guinea-pig,and rat

The kinetics and sodium dependence of adenosine transport were determined using an inhibitorstop method on dissociated cell body preparations obtained from mouse guinea-pig and rat brain. Transport affinity (K_T) values for the high affinity adenosine transport systems (K_T(H)) were significantly different between these three species; mean ±SEM values were 0.34 ±0.1 in mouse, 0.9 ±0.2 in rat, and 1.5±0.5 M in guinea-pig. The K_T values for the low affinity transport system (K_T(L)) were not different between the three species. Brain cells from rat displayed a significantly greater maximal capacity to accumulate [³H]adenosine (V_max) than did mouse or guinea-pig for the high affinity system, or than did mouse for the low affinity system. When sodium chloride was replaced in the transport medium with choline chloride, the K_T(H) values for guinea-pig and rat were both increased by approximately 100%; only in rat did the change reach statistical significance. The sodium-dependence of adenosine transport in mouse brain was clearly absent. The differences between K_T(H) values in mouse and those in guinea-pig or rat were accentuated in the absence of sodium. The differences in kinetic values, ionic requirements, and pharmacological characteristics between adenosine transporters in CNS tissues of mouse guinea-pig and rat may help account for some of the variability noted among species in terms of their physiological responses to adenosine.