Addition Of Choline Research Articles

Fluorescence Regeneration as a Signaling Principle for Choline and Carnitine Binding: A Refined Supramolecular Sensor System Based on a Fluorescent Azoalkane

AbstractThe fluorescent azoalkane, 2,3‐diazabicyclo[2.2.2]oct‐2‐ene (DBO), forms inclusion complexes with p‐sulfonatocalix[4]arene (CX4). The binding constants are on the order of 103 M–1 in water. The addition of CX4 to DBO solutions results in an efficient fluorescence quenching (up to 90 %). This supramolecular system can be used as a truly water‐soluble sensor system to signal the binding of organic ammonium ions over a large pH range. Addition of choline and carnitine derivatives and tetraalkylammonium ions results in regeneration of this fluorescence, from which the binding constants (KC = 103–105 M–1) are calculated by means of a competitive complexation model. Electrostatic effects are observed, namely, a more‐than‐one order of magnitude weaker binding of the carnitines in neutral solution.

Hofmeister Effects in Biology: Effect of Choline Addition on the Salt-Induced Super Activity of Horseradish Peroxidase and Its Implication for Salt Resistance of Plants

The effect of choline addition on the salt-induced super activity of horseradish peroxidase (HRP) is investigated. HRP is presented in the literature as an efficient H(2)O(2) scavenger, and choline is the precursor of glycine betaine, a strong osmoprotectant molecule. Both the regulations of H(2)O(2) and of osmoprotectant concentrations are implicated in plants in order to counteract salt-induced cell damage. For the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), sulfate anions were found to play a crucial role in the increase of HRP activity. This induced super activity can be strongly reduced by adding choline chloride. The phenomena provide an example of physicochemical Hofmeister effects playing a central regulatory role in an important biological system.

Regulation of the Slow Vacuolar Channel by Luminal Potassium: Role of Surface Charge

Voltage-dependent activation of slow vacuolar (SV) channels has been studied on isolated patches from red beet (Beta vulgaris L.) vacuoles. Isoosmotic variation of vacuolar K(+) from 10 to 400 mM in Ca(2+)-free solutions at the vacuolar side shifted the SV channel activation threshold to more positive voltages. The effect of K(+) could be mimicked by additions of choline or N-methyl D-glucamine and could be explained by unspecific screening of the negative surface charge. Fitting the dependence of voltage shift on K(+) concentration to the Gouy-Chapman model yields a surface charge density of 0.36 +/- 0.05 e(-)/nm(2). Negative surface potential also tended to increase the local concentration of permeable ions (K(+)), resulting in anomalously high single-channel conductance, approximately 200 pS in 10 mM KCl. An increase of ionic strength due to addition of impermeable cations greatly reduced the unitary conductance. Large positive shift of the SV channel voltage dependence, caused by physiological (0.5 mM) free vacuolar Ca(2+), was partly ameliorated by increasing luminal K(+). We interpreted these results as follows: K(+)induced a reduction of surface potential, hence i) causing a positive shift of the voltage dependence and ii) a dilution of Ca(2+) in the membrane vicinity, thus reducing the inhibitory effect of vacuolar Ca(2+) and causing a negative shift of the SV channel voltage dependence, with a sum of the two shifts being negative.

Salt-tolerance genes involved in cation efflux and osmoregulation of Sinorhizobium fredii RT19 detected by isolation and characterization of Tn5 mutants

Salt-tolerance genes of Sinorhizobium fredii RT19 were identified by the construction and screening of a transposon Tn5-1063 library containing over 30,000 clones. Twenty-one salt-sensitive mutants were obtained and five different genes were identified by sequencing. Eight mutants were found with disruptions in the phaA2 gene, which encodes a cation efflux system protein, while mutations in genes encoding other cation effux system proteins were found in seven ( phaD2), two ( phaF2) and two ( phaG2) mutants. A mutation in the metH gene, encoding 5′ methyltetrahydrofolate homocysteine methyltransferase, was found in two of the salt sensitive strains. Growth experiments showed that phaA2, phaD2, phaF2 and phaG2 mutants were hypersensitive to Na +/Li + and slightly sensitive to K + and not sensitive to sucrose and that metH mutants were highly sensitive to any of Na +, Li +, K + and sucrose. Na + intracellular content measurements established that phaA2, phaD2, phaF2 and phaG2 are mainly involved in the Na + efflux in S. fredii RT19. Recovery of growth of the metH mutants incubated with different concentrations of NaCl could be obtained by additions of methionine, choline and betaine, which showed that the metH gene is probably involved in osmoregulation in S. fredii RT19.

Differential transcriptional regulation of two distinct S-adenosylmethionine synthetase genes (SAM1 and SAM2) of Saccharomyces cerevisiae.

Expression of a number of genes encoding enzymes involved in phospholipid biosynthesis in yeast Saccharomyces cerevisiae is known to be repressed on the addition of myo-inositol and choline to the culture medium (inositol-choline regulation). All genes subject to this inositol-choline regulation have an octamer sequence 5'-CATRTGAA-3' in their upstream regions and those octamer sequences play an important role in this regulation. To confirm the role of the octamer sequence further, we studied the transcriptional regulation of two distinct S-adenosylmethionine synthetase genes (SAM1 and SAM2) of S. cerevisiae. Quantitative RT-PCR analysis showed that only the SAM2 gene was subject to the inositol-choline regulation, consistent with the fact that only the SAM2 gene has two octamer sequences in its upstream region. Furthermore, functional promoter analysis revealed that the proximal octamer sequence of the SAM2 gene has an essential role for this regulation.

Parenteral nutrition

Parenteral nutrition remains a topic of intense research interest. It has now been shown to offer no advantage over, but to be associated with an increased frequency of complications, compared to enteral nutrition in patients with gastrointestinal cancer. Nutritional support administered via an intraportal route, combined with multimodal analgesia, appears to offer certain metabolic and clinical advantages compared with that delivered via systemic veins. Parenteral nutrition, however, does not prevent the decrease in antioxidant capacity seen after major surgery, and feeding lines present an additional risk factor for systemic candidiasis in the intensive care setting. Nonetheless, use of the conventional percutaneous access route is reconfirmed, even in infants, to be a safe technique. Addition of choline to intravenous nutrition admixtures may improve some of the hepatic abnormalities associated with parenteral nutrition, but clodronate has only limited ability to prevent the progression of osteoporosis in patients on long-term treatment.

Effect of equi-molar dietary betaine and choline addition on performance, carcass quality and physiological parameters of pigs

AbstractBetaine and its precursor choline were compared in their efficiency in affecting the performance, carcass traits, and liver betaine concentration of growing-finishing pigs. Individually penned Finnish Landrace and Yorkshire pigs and their crosses (30 kg; no. = 70) were offered the basal diet with no added betaine or choline, or the basal diet supplemented with low to moderate doses (250, 500 or 1000 mg/kg) of betaine (Betafin® S1), or with a similar molar amount of choline (578, 1155 or 2310 mg/kg of choline chloride). The maize-soya-bean-meal basal diet was formulated to contain 12·3 MJ/kg digestible energy, 155 g/kg crude protein and 7·4, 4·4 and 4·3 g/kg digestible lysine, threonine and methionine + cystine, respectively. Oat hull meal (100 g/kg) was added to reduce the dietary energy concentration. The pigs were on a restricted feeding level, 1·5 to 3·0 kg food per day (proportionately 0·8 of ad libitum intake) for 75 days. Daily weight gain and food-to-gain ratio improved linearly (P < 0·01) with increasing dietary betaine. Carcass weight increased linearly (P < 0·01) but slaughter loss proportion, backfat and sidefat thicknesses and lean proportions in ham and carcass were unaffected by dietary betaine level. Liver betaine level increased linearly (by up to a proportion of 0·62 in comparison with the control) with dietary betaine addition (F < 0·05) and betaine tended to improve linearly the tensile strength of the proximal ileum (P = 0·07). The presence of choline had no effect on any of these parameters. These results indicate that low to moderate doses of dietary betaine improved the growth and the efficiency of food utilization of growing-finishing pigs. Pigs on betaine diets had heavier carcasses without a relative increase in carcass fat. Choline had no such effects in pigs offered the restricted amount of diet. Liver betaine concentration increased with level of betaine in the diet whereas the betaine precursor choline did not affect hepatic betaine.

Active transport of high-affinity choline and nicotine analogs into the central nervous system by the blood-brain barrier choline transporter.

Cigarette smoking is strongly implicated in the development of cardiovascular disorders. Recently identified nicotinium analogs may have therapeutic benefit as smoking cessation therapies but may have restricted entry into the central nervous system by the blood-brain barrier (BBB) due to their physicochemical properties. Using the in situ perfusion technique, lobeline, choline, and nicotinium analogs were evaluated for binding to the BBB choline transporter. Calculated apparent K(i) values for the choline transporter were 1.7 microM N-n-octyl choline, 2.2 microM N-n-hexyl choline, 27 microM N-n-decylnicotinium iodide, 31.9 microM N-n-octylpyridinium iodide, 49 microM N-n-octylnicotinium iodide (NONI), 393 microM lobeline, and >/=1000 microM N-methylnicotinium iodide. Nicotine and N-methylpyridinium iodide, however, do not apparently interact with the BBB choline transporter. Given NONI's apparent K(i) value determined in this study and its ability to inhibit nicotine-evoked dopamine release from superfused rat brain slices, potential brain entry of NONI via the BBB choline transporter was evaluated. [(3)H]NONI exhibited a BBB transfer coefficient value of approximately 1.6 x 10(-3) ml/s/g and a K(m) of approximately 250 microM. Unlabeled choline addition to the perfusion fluid reduced [(3)H]NONI brain uptake. We hypothesize the N-n-octyl group on the pyridinium nitrogen of NONI facilitates brain entry via the BBB choline transporter. Thus, NONI may have utility as a smoking cessation agent, given its ability to inhibit nAChRs mediating nicotine-evoked dopamine release centrally, and to be distributed to brain via the BBB choline transporter.

Choline Deficiency–Induced Liver Damage Is Reversible in Pemt−/− Mice

Hepatic tissue has two pathways for phosphatidylcholine (PC) synthesis, i.e., the cytidinediphosphocholine (CDP-choline) pathway and the methylation pathway, which utilizes phosphatidylethanolamine-N-methyltransferase (PEMT). Fatal liver damage occurs in Pemt(-/-)mice fed a choline-deficient (CD) diet. We investigated whether liver damage can be reversed by the addition of dietary choline. Mice (8 wk old) were fed the CD purified diet for 4 d, a choline-supplemented (CS) diet (CD diet + 0.4% choline chloride) for 4 d, or the CD diet for 3 d and a CS diet for 1 d (CD/CS). Pemt(-/-)mice fed the CD diet for 3 d exhibited liver damage as assayed by plasma aminotransferase levels. The livers appeared normal after subsequent feeding of the CS diet for 1 d (CD/CS). The activities of plasma aminotransferases of CD/CS fed mice were comparable to Pemt(-/-)mice fed the CS diet. Hepatic PC and triacylglycerol levels as well as plasma PC levels in the CD/CS-fed Pemt(-/-)mice were lower than those of mice fed the CD diet and began to approach normal levels. Although the CD diet induces liver damage in Pemt(-/-)mice, this damage can be rapidly reversed by the addition of dietary choline.

Influence of dietary methionine level on the liver metallothionein mRNA level in rats.

The effects of some methyl-containing compounds added to a choline-deficient diet on the metallothionein mRNA level in the rat liver were studied. The addition of choline or carnitine to the choline-deficient diet did not induce a gain in body weight, while the addition of either betaine or methionine to the choline-deficient diet, or of methionine to the choline-deficient diet with choline significantly increased the body weight. The metallothionein mRNA level in the liver of rats fed on the choline-deficient diet was similar to that of rats fed on the choline-deficient diet with choline, betaine or carnitine. However, the addition of methionine to the choline-deficient diet with or without choline caused a marked suppression in the metallothionein mRNA level in the liver. It is thus surmised that the metallothionein mRNA level in the liver might be regulated by the dietary content of methionine.

Choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes

A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a K m of 2.2×10 −5 M and a V max of 0.15 nmol 10 min −1 mg −1 cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated mycoplasmas and could be reversed by the addition of free choline to the growth medium.

Choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes.

A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a K(m) of 2.2x10(-5) M and a V(max) of 0.15 nmol 10 min(-1) mg(-1) cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated mycoplasmas and could be reversed by the addition of free choline to the growth medium.

The effect of equi-molar dietary betaine and choline addition on performance and carcass quality of pigs

Betaine has three chemically reactive methyl groups attached to the nitrogen atom of the glycine molecule. Therefore, it can be used as a methyl group donor partially to replace methionine in poultry and pig diets. Recent work also suggests that betaine has an energy sparing role by reducing maintenance requirement of the pig (Schrama and Gerrits, 2000). Betaine has improved performance and carcass leanness in some studies but the results are variable and seem to depend on age and sex of the animal, feeding level and diet composition.Choline can also be used as methyl donor in animal feeds. In poultry, methyl groups are available after the conversion to betaine in the liver. However, dietary betaine is twice as efficient as the equi-molar dietary choline for increasing liver betaine levels in broiler chick (Saarinen et al., 2000). The aim of this study was to compare the response of pigs fed equi-molar betaine and choline in terms of pig performance and carcass characteristics.

Effect of supplemental methionine, choline and their combinations on the performance and immune response of broilers

1. A study was conducted with broilers to find out the effect of addition of methionine (0.0, 1.5, 3.0 and 4.5 g/kg) and choline (0.0, 1000 and 2000 mg/kg) to a basal diet containing crude protein, 221 g/kg, ME, 12.25 MJ/kg, methionine, 3.6 g/kg and choline, 1300 mg/kg. 2. Supplemental levels of methionine and choline were found to be ineffective in improving the growth, food consumption and food conversion efficiency of broilers. 3. However, a higher value for leucocyte migration inhibition was observed in chicks fed on the diet containing 6.5 g/kg methionine and 1300 mg/kg choline, indicating a significantly improved cellular immune response. 4. HI test and ELISA indicated enhanced antibody titres in chicks receiving 3.0 g/kg methionine and 3300 mg/kg choline, showing a significantly better humoral immune response. 5. Methionine and choline should be supplemented at levels higher than the recommended level of BIS for better health and production in chicks.

Enhancement of free fatty acid incorporation into phospholipids by choline plus cytidine

Cytidine and choline, present in cytidine 5′-diphosphate choline (CDP-choline), are major precursors of the phosphatidylcholine found in cell membranes and important regulatory elements in phosphatide biosynthesis. Administration of CDP-choline to rats increases blood and brain cytidine and choline levels; this enhances the production of endogenous CDP-choline which then combines with fatty acids (as diacylglycerol), to yield phosphatidylcholine. We examined the effect of providing cytidine and choline on incorporation of free fatty acids into phosphatidylcholine and other major phospholipids in PC12 cells. Addition of equimolar cytidine and choline (100–500 μM) to [ 3 H ]-arachidonic acid (50 μM, 0.2 μCi, bound to bovine serum albumin) dose-dependently increased the accumulations of [ 3 H ]-phosphatidylcholine (PtdCho), [ 3 H ]-phosphatidylinositol (PtdIno) and [ 3 H ]-phosphatidylethanolamine (PtdEtn) (by up to 27±3%, 16±3% and 11±3%, respectively, means±S.E.M.). This effect was seen with 8–18 h of incubation. The incorporation of [ 3 H ]-oleic acid into [ 3 H ]-PtdCho was even more enhanced (by up to 42±3%) as were the incorporations of [ 14 C ]-choline and [ 3 H ]-glycerol. The effects of choline and cytidine were enhanced by 12- O-tetradecanoylphorbol-13-acetate (TPA, 1 μM), which activates CTP:phosphocholine cytidylyltransferase (CT) and facilitates choline uptake. Replacing choline by ethanolamine also enhanced the incorporation of [ 3 H ]-arachidonic acid into [ 3 H ]-PtdEtn, [ 3 H ]-PtdIno and [ 3 H ]-PtdCho. Arachidonic acid (10–200 μM) alone failed to affect the incorporation of [ 14 C ]-choline into phosphatidylcholine. We suggest that the increases in phospholipid synthesis caused by concurrent cytidine and choline supplementation enhance the incorporation of arachidonic acid and certain other fatty acids into the major glycerophospholipids. Removing these fatty acids as source of potentially toxic oxidation products could contribute to the beneficial effects of CDP-choline in treating stroke or other brain damage.

Synthesis of glycine betaine from exogenous choline in the moderately halophilic bacterium halomonas elongata

The role of choline in osmoprotection in the moderate halophile Halomonas elongata has been examined. Transport and conversion of choline to betaine began immediately after addition of choline to the growth medium. Intracellular accumulation of betaine synthesized from choline was salt dependent up to 2.5 M NaCl. Oxidation of choline was enhanced at 2.0 M NaCl in the presence or absence of externally provided betaine. This indicates that the NaCl concentration in the growth medium has major effects on the choline-betaine pathway of H. elongata.

Effects of ruminally protected choline and dietary fat on performance of finishing heifers (1998)

A 120-day finishing study utilizing 318 heifers (753 lb initial body weight) examined the effects of ruminally protected choline in diets with no tallow or 2 or 4% of supplemental tallow. Heifers were fed a finishing diet based on a mix of steam-flaked and dry- rolled corn; encapsulated choline (20, 40, or 60 g/head/day, supplying 5, 10 or 15 g choline/head/day) was top-dressed to the diet or not added. Dry matter intake decreased linearly with inclusion of fat (P<.05) but was not affected significantly by addition of choline. Daily gains also decreased linearly (P<.05) with fat addition. Choline supplementation increased gain (linear, P<.1; qua dratic, P<.05), with the greatest increase occurring for the first 20 g increment encapsulated choline/day. Likewise, feed efficiency improved (P<.1) with supplemental choline. Again, the greatest response occurred for the first 20 g/day. Kidney, pelvic, and heart fat and yield grade both increased linearly (P<.1) with fat supplementation. The percentage of carcasses grading USDA Choice decreased (linear, P<.05; quadratic, P<.1) when choline was added at 60 g/day. Hot carcass weight, marbling, dressing percent, and 12th rib fat thickness were not affected significantly by either fat or choline. Ruminally protected choline can improve average daily gain and feed efficiency of finishing cattle.

Phosphatidylcholine biosynthesis is required for secretion of truncated apolipoprotein Bs from McArdle RH7777 cells only when a neutral lipid core is formed

Decreased phosphatidylcholine biosynthesis inhibits the secretion of very low density lipoproteins from hepatocytes (Yao, Z. and D. E. Vance. 1988. J. Biol. Chem. 263: 2998-3004). We have now investigated whether or not phosphatidylcholine biosynthesis is required for secretion of carboxyl-truncated apoBs 15, 18, 23, 28, and 37 from transfected McArdle RH7777 cells. Transfected cells were choline-deprived for 1 day and then choline-supplemented or maintained choline-deficient. Pulse-chase experiments showed that in the presence of 100 microM choline, the secretion of apoBs 28 and 37 was increased by up to 50 and 45%, respectively, whereas the secretion of apoBs 15, 18, and 23 was not affected by the addition of choline. Immunoblot analyses also showed that choline in the medium increased the secretion of apoBs 28 and 37 by 30-50% whereas the secretion of apoBs 15, 18, and 23 was unaffected over 24 h. As only carboxyl-terminal truncations with a size greater than 23% of apoB-100 are able to assemble a neutral lipid core (McLeod, R. S., Y. Zhao, S. L. Selby, J. Westerlund, and Z. Yao. 1994. J. Biol. Chem. 269: 2852-2862), we conclude that only apoB-truncations that assemble a neutral lipid core require phosphatidylcholine synthesis. Other experiments established that the requirement of phosphatidylcholine for apoB secretion is related to the presence of neutral lipid associated with a truncation, rather than the length of apoB.

Autolysis and cell wall degradation in a choline-independent strain of Streptococcus pneumoniae.

Streptococcus pneumoniae has an auxotrophic requirement for choline, and choline residues that incorporate into the wall and membrane teichoic acids are intimately involved with the control of autolytic phenomena of this bacterium. We report here the re-examination of the role of choline in autolytic cell wall degradation using the choline-independent S. pneumoniae strain R6Cho- recovered from a heterologous cross with DNA from Streptococcus oralis. S pneumoniae Cho- cultured in choline-free medium grew with normal generation time but formed long chains, failed to undergo stationary-phase autolysis, and was also resistant to lysis induced by deoxycholate or penicillin. Cell walls produced under these conditions had reduced phosphorus content, contained no choline residues detectable by nuclear magnetic resonance, and had reduced binding capacity for the pneumococcal autolytic amidase, and complete hydrolysis of such walls by the amidase required prolonged incubation with high concentrations of the enzyme. Addition of choline to the growth medium reversed at these phenomena. High-performance liquid chromatography analysis of amidase digests of cell walls prepared from strain R6Cho- grown with or without choline produced identical stem peptide profiles, which were also similar to that of the parental S. pneumoniae strain R6. Peptidoglycans prepared by hydrofluoric extraction of cell walls from Cho- growth with or without choline or from the parental strain R6 were uniformly susceptible to the autolytic amidase and were fully degraded to the normal family of stem peptides, indicating that, in sharp contrast to the case of cell walls, the amidase degradation of teichoic acid-free peptidoglycan did not require the presence of choline residues in the substrate.

Structural Characterization of the Unligated and Choline-bound Forms of the Major Pneumococcal Autolysin LytA Amidase: CONFORMATIONAL TRANSITIONS INDUCED BY TEMPERATURE

The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I' band, results in 19% of alpha-helix and tight loops, 47% of beta-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module.