Determination of picomole quantities of acetylcholine and choline in physiologic salt solutions

An assay capable of detecting tens-of-picomole quantities of choline and acetylcholine in milliliter volumes of a physiological salt solution has been developed. Silica column chromatography was used to bind and separate 10–3000 pmol [¹⁴C]choline and [¹⁴C]acetylcholine standards made up in 3 ml of a bicarbonate-buffered Krebs-Ringer solution. The silica columns bound 95–98% of both choline and acetylcholine. Of the bound choline 84–87% was eluted in 1.5 ml of 0.075 n HCl, whereas 95–98% of the bound acetylcholine was eluted in a subsequent wash with 1.5 ml of 0.030 n HCl in 10% 2-butanone. Vacuum centrifugation of the eluants yielded small white pellets with losses of choline and acetylcholine of only 1%. Dried pellets of unlabeled choline and acetylcholine standards were assayed radioenzymatically using [γ-³²P]ATP, choline kinase, and acetylcholinesterase. The net disintegrations per minute of choline[³²P]phosphate product was proportional to both the acetylcholine (10–3000 pmol) and choline (30–3000 pmol) standards. The “limit sensitivity” was 8.5 pmol for acetylcholine and 11.4 pmol for choline. Cross-contamination of the choline assay by acetylcholine averaged 1.3%, whereas contamination of the acetylcholine assay by choline averaged 3.1%.     

Chemiluminescent assays for choline and phospholipase-D using a flow injection system

A flow injection chemiluminescent method is described for the determination of choline. The method is based on the production of hydrogen peroxide from choline using on-line covalently bound immobilized choline oxidase column. The product is mixed downstream and detected via the cobalt catalyzed chemiluminescent oxidation of luminol. The detection limit is 1×10⁻⁷ mol/L, with rsd 1.8 to 2.8% in the range 2–10×10⁻⁵ mol/L. The sample throughput is 30 per hour. The method was applied to the determination of choline produced off-line from phosphatidylcholine using phospholipase-D isolated from cabbage. © 1997 John Wiley & Sons, Ltd.     

Determination of choline in erythrocytes using high-resolution proton nuclear magnetic resonance spectroscopy: Comparison with a choline oxidase method

Detailed operating conditions are reported for the determination of choline in human erythrocytes using proton nuclear magnetic resonance spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence. The results of the NMR method were in excellent agreement with those obtained using an enzymatic (choline oxidase) assay; however, they were approximately three times higher than those reported using gas chromatography/mass spectrometry techniques. The differences may be partly due to the method of preparing or sampling cells since there is a distribution of choline in cells of different ages. However, choline levels were not affected by the methods used in the present study for storing or preparing cells.     

Cortical choline transporter function measured in vivo using choline-sensitive microelectrodes: clearance of endogenous and exogenous choline and effects of removal of cholinergic terminals

The capacity of the high-affinity choline transporter (CHT) to import choline into presynaptic terminals is essential for acetylcholine synthesis. Ceramic-based microelectrodes, coated at recording sites with choline oxidase to detect extracellular choline concentration changes, were attached to multibarrel glass micropipettes and implanted into the rat frontoparietal cortex. Pressure ejections of hemicholinium-3 (HC-3), a selective CHT blocker, dose-dependently reduced the uptake rate of exogenous choline as well as that of choline generated in response to terminal depolarization. Following the removal of CHTs, choline signal recordings confirmed that the demonstration of potassium-induced choline signals and HC-3-induced decreases in choline clearance require the presence of cholinergic terminals. The results obtained from lesioned animals also confirmed the selectivity of the effects of HC-3 on choline clearance in intact animals. Residual cortical choline clearance correlated significantly with CHT-immunoreactivity in lesioned and intact animals. Finally, synaptosomal choline uptake assays were conducted under conditions reflecting in vivo basal extracellular choline concentrations. Results from these assays confirmed the capacity of CHTs measured in vivo and indicated that diffusion of substrate away from the electrode did not confound the in vivo findings. Collectively, these results indicate that increases in extracellular choline concentrations, irrespective of source, are rapidly cleared by CHTs.     

Phosphatidylcholine and choline homeostasis

Phosphatidylcholine (PC) is made in mammalian cells from choline via the CDP-choline pathway. Animals obtain choline primarily from the diet or from the conversion of phosphatidylethanolamine (PE) to PC followed by catabolism to choline. The main fate of choline is the synthesis of PC. In addition, choline is oxidized to betaine in kidney and liver and converted to acetylcholine in the nervous system. Mice that lack choline kinase (CK) alpha die during embryogenesis, whereas mice that lack CKbeta unexpectedly develop muscular dystrophy. Mice that lack CTP:phosphocholine cytidylyltransferase (CT) alpha also die during early embryogenesis, whereas mice that lack CTbeta exhibit gonadal dysfunction. The cytidylyltransferase beta isoform also plays a role in the branching of axons of neurons. An alternative PC biosynthetic pathway in the liver uses phosphatidylethanolamine N-methyltransferase to catalyze the formation of PC from PE. Mice that lack the methyltransferase survive but die from steatohepatitis and liver failure when placed on a choline-deficient diet. Hence, choline is an essential nutrient. PC biosynthesis is required for normal very low density lipoprotein secretion from hepatocytes. Recent studies indicate that choline is recycled in the liver and redistributed from kidney, lung, and intestine to liver and brain when choline supply is attenuated.     

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post‐mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used ¹⁵N‐choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before ¹H‐NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT‐mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real‐time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non‐invasive manner using NMR spectroscopic techniques.     

Substitution of choline by related compounds in the diet of Argyrotaenia velutinana and Heliothis virescens

Argyrotaenia velutinana, the red-banded leaf roller, and Heliothis virescens, the tobacco budworm, both require choline for growth and development when reared on semisynthetic diets. The optimum level for A. velutinana is $\text{50 mg}\text{100 g}$ of diet whereas that for H. virescens exceeds $\text{100 mg}\text{100 g}$ of diet.No choline analog tested can adequately replace choline in the diet. One compound, dimethylethylcholine, will permit some adut emergence but development is slower and mortality is greater than on the corresponding diet containing choline. This is in sharp contrast to a number of Diptera in which mary choline analogs can not only replace choline in the diet but are also incorporated into phospholipids analogous to phosphatidylcholine.In A. velutinana, dimethylisopropylcholine and β-methylcholine, although inadequate as choline replacements, can spare the dietary choline requirement, Isopropylethanolamine is a growth inhibitor for A. velutinana but not for H. virescens.     

Molecular and functional characterization of an Na+-independent choline transporter in rat astrocytes

In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (Km) of 35.7 +/- 4.1 microm and a maximal velocity (Vmax) of 49.1 +/- 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17beta-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.     

Choline oxidation and choline dehydrogenase

The oxidation of choline by both freshly prepared and aged rat liver mitochondria is inhibited by amytal. Whereas rotenone inhibits choline-cytochromec reductase only in the case of freshly prepared mitochondria, the extent of inhibition is influenced by preincubation, but the inhibition is not secondary to the inhibited oxidation of betaine aldehyde, the product of choline oxidation. Evidence shows that rotenone is able to inhibit the swelling of rat liver mitochondria and the inhibition of choline-cytochromec reductase by rotenone is related to the inhibition of mitochondrial swelling. Nine inhibitors of choline dehydrogenase have been reported. Among those, some belong to the category of acetylcholine esterase inhibitor. In view of the structure of those inhibitors, it seems quite likely that there is an anionic site at the active center of choline dehydrogenase. Purification of choline dehydrogenase in its native form has been accomplished by solubilization with Lubrol WX, hydroxyapatite, and DEAE-Sepharose chromatography and sucrose gradient ultracentrifugation. The preparation is pure as judged by SDS-PAGE and Ultrogel AcA 34 column chromatography. The molecular weight determined by SDS-PAGE is approximately 61,000. There is 0.23 mg phospholipid/mg protein and the Stokes' radius of protein-Lubrol-phospholipid mixed micelles is about 59 A.     

Chemiluminescent determination of choline in cerebrospinal fluid and red blood cells

The concentration of choline in the cerebrospinal fluid (CSF) of patients affected by primary dementia and in red blood cells (RBC) of depressed patients before and after treatment with lithium salts was determined using a chemiluminescent assay. The mean CSF concentration of choline was found to be 60 pmoles/ml (SD = 20 pmoles/ml) and this was lower than values obtained previously by spectrophotometric-colorimetric methods. Mean RBC choline concentrations before and after therapy with lithium salts were 20 nmoles/ml (SD = 16 nmoles/ml and 328 nmoles/ml (SD = 206 nmoles/l) respectively and these are similar to those reported previously (obtained by chemiluminescent and non-chemiluminescent methods).     

Choline import into chloroplasts limits glycine betaine synthesis in tobacco: analysis of plants engineered with a chloroplastic or a cytosolic pathway

The biosynthesis of the osmoprotectant glycine betaine (GlyBet) is a target for metabolic engineering to enhance stress resistance in crops. Certain plants synthesize GlyBet in chloroplasts via a two-step oxidation of choline (Cho). In previous work, a chloroplastic GlyBet synthesis pathway was inserted into tobacco (which lacks GlyBet) by expressing spinach choline monooxygenase (CMO). The transformants had low CMO enzyme activity, and produced little GlyBet (less than or = 70 nmol g(-1) fresh wt). In this study, transformants with up to 100-fold higher CMO activity showed no further increase in GlyBet. In contrast, tobacco expressing a cytosolic GlyBet synthesis pathway accumulated significantly more GlyBet (430 nmol g(-1) fresh wt), suggesting that subcellular localization influences pathway flux. Modeling of the labeling kinetics of Cho metabolites observed when [14C]Cho was supplied to engineered plants demonstrated that Cho import into chloroplasts indeed limits the flux to GlyBet in the chloroplastic pathway. A high-activity Cho transporter in the chloroplast envelope may therefore be an integral part of the GlyBet synthesis pathway in species that accumulate GlyBet naturally, and hence a target for future engineering.     

Essentiality of ubiquinone for choline oxidation in rat liver mitochondria.

Rat liver mitochondria treated extensively with n-pentane are incapable of oxidizing choline. Choline oxidation is more sensitive than is succinate oxidation to serial n-pentane extraction of mitochondria. The ability to oxidize choline is restored by the addition of ubiquinone-2 or ubiquinone-10 to the oxidase assay medium.     

Measurement of Free Choline in Plant Leaves by Capillary Electrophoresis

A low pH capillary electrophoresis (CE) was used for the measurement of free choline in plant leaves. Choline in the leaf extract was first converted to the benzoyl ester and put into CE. A well-resolved peak in the electropherogram was easily obtained. Involvement of enzymes in a two-step oxidation of choline to glycine betaine was evaluated in different plant species with the same mehod developed for glycine betaine and betaine aldehyde.     

IGF-1、IGFBP-3在大鼠非酒精性脂肪性肝病进展中的变化

目的：观察非酒精性脂肪性肝病（NAFLD）大鼠血清胰岛素样生长因子1（IGF-1）、胰岛素样生长因子结合蛋白3（ IGFBP-3）的表达及多烯磷脂酰胆碱对其的影响。方法采用高脂饲料喂养建立大鼠NAFLD模型,以多烯磷脂酰胆碱进行干预。 HE染色动态观察大鼠4、8、12周时肝组织病理学变化,IRMA法动态监测血清IGF-1、IGFBP-3含量。结果正常组大鼠各时间点肝组织病理均无明显异常;模型组随高脂饮食时间延长,在4、8、12周3个时相点肝组织脂肪变、炎症程度、气球样变及NAFLD活动度积分逐渐增强,血清IGF-1、IGFBP-3逐渐明显下降,且模型组各时相点IGF-1、IGFBP-3的水平较正常组同一时相点明显降低;应用多烯磷脂酰胆碱干预后,大鼠肝组织炎症程度、NAFLD活动度积分较模型组显著降低,IGF-1、IGFBP-3的水平则较模型组明显增高。结论血清IGF-1、IGFBP-3水平随大鼠NAFLD进展而降低。     

Effects of hemicholinium-3 and sodium ions on choline uptake system in excised superior cervical sympathetic ganglia of rats

Active choline uptake by rat superior cervical sympathetic ganglia (SCG), which contain abundant cholinergic nerve terminals, was studied with respect to sensitivity to inhibition by hemicholinium-3 (HC-3) and dependence on extracellular Na⁺ under standard conditions of assay. Choline was taken up by a single saturable process with apparentK _m=3.07×10^–5 M and Vmax=286 pmoles/min/mg protein. Neither denervation followed by degeneration of cholinergic nerve terminals nor axotomy with successive neuronal degeneration significantly decreased in choline uptake by the ganglia in vitro. HC-3 dose-dependently inhibited ganglionic choline uptake more effectively at lower than at higher choline concentrations. HC-3 sensitive inhibition of ganglionic choline uptake was not seen in young rats one week after birth but appeared with maturity, attaining approximately 50% maximal inhibition in adult SCG. Extent of inhibition by HC-3 and Na⁺ dependence of ganglionic choline uptake was not altered by denervation or axotomy.Abbreviations used (HC-3) hemicholinium-3 - (HAChU) high affinity choline uptake - (LAChU) low affinity choline uptake - (SCG) superior cervical ganglia - (Ch) choline - (ACh) acetylcholine     

Choline transporter‐like protein 4 (CTL4) links to non‐neuronal acetylcholine synthesis

Synthesis of acetylcholine (ACh) by non‐neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na⁺‐dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non‐neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non‐neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter‐like proteins, a five gene family choline‐transporter like protein (CTL)1–5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na⁺‐independent and CTL1–5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non‐neuronal cell lines and presents a mechanism to target non‐neuronal ACh synthesis without affecting neuronal ACh synthesis.     

Sexually Dimorphic Activation of Liver and Brain Phosphatidylethanolamine N-Methyltransferase by Dietary Choline Deficiency

Phosphatidylethanolamine N-methyltransferase (PEMT) activity was measured by a radioenzymatic assay in homogenates of brain and liver obtained from Sprague Dawley rats fed a choline-free or control (0.3 g/kg of choline chloride) diet for seven days. Choline deficiency increased PEMT activity in the liver of male rats by 34% but had no effect on hepatic PEMT in females. In contrast, brain PEMT activity was increased in brain of choline deficient females (by 49%) but was unaltered in males. Activation of the PE methylation pathway in female brain may constitute a compensatory mechanism to sustain PC synthesis during choline deficiency.     

A possible mechanism for the increased oxidation of choline after chronic ethanol ingestion.

An attempt has been made to determine the location of the site at which the metabolism of ethanol interacts with that of choline to produce an increase in the oxidation of choline. The first enzyme in the oxidation pathway for choline, choline dehydrogenase, was assayed using a newly developed spectrophotometric assay and freshly isolated intact rat liver mitochondria. No changes were observed in either 'apparent' V or the 'apparent' Km values of choline dehydrogenase for choline after ethanol ingestion. However, when the choline oxidase system was assayed, a 28% decrease in 'apparent' Km for choline and a 53% increase in 'apparent' V was observed. The effects of ATP on choline oxidase were studied further, and a 29.4% decrease was observed in mitochondrial ATP levels from freshly isolated mitochondria from the ethanol-treated rats. In vitro aging of mitochondria further decreased the level of ATP, and the rate of decrease was considerably faster during the first hour in the mitochondria from the ethanol-treated animals. The decreases in ATP from both control and experimental mitochondria were accompanied by increases in choline oxidase activity. The initial decrease in ATP was correlated with an increase in mitochondrial ATPase activity which may be related to an increase in mitochondria Mg2+. Because chronic ethanol ingestion has resulted in decreased oxidation rates of succinate and beta-hydroxybutyrate while at the same time increasing the oxidation rates of choline, the studies reported here suggest that the effect of chronic ethanol ingestion is primarily on a step that is unique to choline and which probably exists prior to the electron transport chain.     

Choline is transported by vesicular acetylcholine transporter

Previously published results appeared to show that vesicular acetylcholine transporter (VAChT) does not transport choline (Ch). Because it is uniquely suited to detect transport of weakly bound substrates, a recently developed assay that detects transmembrane reorientation of the substrate binding site was used to re-examine transport selectivity. Rat VAChT was expressed in PC12(A1237) cells, postnuclear supernatant-containing microvesicles was prepared, and the reorientation assay was conducted with unlabeled Ch and tetramethylammonium (TMA). Also, [(14)C]Ch and [(3)H]acetylcholine (ACh) were used in an optimized accumulation assay. The results demonstrate that Ch is transported at least as well as ACh is, but with sevenfold lower affinity. Even TMA is transported, but with 26-fold lower affinity. Ch transport by VAChT is of interest in view of the possibilities that Ch (i) occurs at higher concentration than ACh does in terminal cytoplasm under some conditions, and (ii) is an agonist for alpha 7 nicotinic receptors.     

A spectrophotometric method for the assay of phospholipase D activity

Phosphatidylcholine phosphatidohydrolase (EC 3.1.4.4, phospholipase D) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. We have developed a spectrophotometric assay for phospholipase D using choline kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of choline with the oxidation of NADH. The assay was linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzyme activity, determination of initial rates of reaction, and detailed kinetic studies of phospholipase D. The method is limited to analysis of purified preparations of phospholipase D lacking competing activities to the coupled system.