Paired-ion high-performance liquid chromatographic assay for plasma choline

Choline was isolated from deproteinized plasma by cation-exchange chromatography. Isolated choline was directly converted to the 3,5-dinitrobenzoate derivative and was analyzed by paired-ion high-performance liquid chromatography with UV detection at 254 nm. An internal standard, 3-hydroxy-N,N,N-trimethylpropanaminium iodide was used for quantitation of plasma choline.Linearity was achieved from 1–500 nmole/ml with a reproducibility of ± 6%. Plasma choline concentrations below 1 nmole/ml could not be accurately measured while plasma choline concentrations in the μmole/ml range deviated from linearity.     

Monoclonal antibodies to choline acetyltransferase of rat brain

Monoclonal antibodies to rat brain choline acetyltransferase were produced by the hybridoma technique. Two stable cell lines, Ab-57 and Ab-60, secreted immunoglobulin of subclass IgG1. The monoclonal antibodies bound to choline acetyltransferase without blocking catalytic activity. Affinity of Ab-57 was 100-times higher than that of Ab-60. Both antibodies bound to the rat enzyme in a mutually exclusive fashion. The antibodies showed cross-species reactivity with choline acetyltransferase from several mammalian brains.     

Arterio-venous differences of choline and choline lipids across the brain of rat and rabbit.

The concentration of unesterified choline in the plasma in the jugular vein of the rat (0.85 nmol/ml) was found to be three times that of the arterial supply to the brain (0.25 nmol/ml), indicating a higher efflux than uptake of unesterified choline by the brain. No such difference was found for the rabbit and no arterio-venous difference for phosphatidylcholine or lysophosphatidylcholine was observed in either species. No arterio-venous difference was found for choline in blood cells. The infusion of [Me-3H]choline into the circulation of the rat or rabbit indicated an uptake of radioactive choline by the brain and an efflux of non-radioactive choline. In the rabbit such an infusion produced a steady rise in the labelling of phosphatidylcholine and lysophosphatidylcholine in the plasma. When [14C2]ethanolamine was injected intraperitoneally into the rat there was a labelling of phosphatidylcholine, lysophosphatidylcholine and sphingomyelin in the plasma and cells of blood from the jugular vein and the arterial supply, as well as in the brain tissue. However, no labelling of unesterified choline in these tissues could be detected. Unesterified choline was shown to be liberated into the plasma when whole blood from the rat or man, but not the rabbit, was incubated for short periods at 30 degrees C.     

Evidence for the extreme overestimation of choline acetyltransferase in human sperm, human seminal plasma and rat heart: a case of mistaking carnitine acetyltransferase for choline acetyltransferase

Detection of choline acetyltransferase (ChAc) in a number of non-neuronal tissues has been extremely overestimated. There are two major types of errors encountered. Type 1 error occurs when endogenous substrates (e.g. L-carnitine) are acetylated by acetyltransferase enzymes (e.g. carnitine acetyltransferase ( CarAc ) ) yielding an acetylated product mistaken for acetylcholine (AcCh). In the past, human sperm and human seminal plasma putative ChAc activity has been extremely overestimated due to Type 1 error. This study demonstrates (1) an endogenous acetyltransferase and substrate activity in human sperm and human seminal plasma forming an acetylated product that is not AcCh but probably acetylcarnitine ( AcCar ); (2) that the addition of 5 mM choline substrate does not significantly increase acetyltransferase activity; (3) that boiled seminal plasma contains an endogenous acetyltransferase substrate which is not choline, but probably L-carnitine. Type 2 error occurs when endogenous carnitine acetyltransferase synthesizes true AcCh, resulting in mistaken evidence for ChAc. This is demonstrated by the fact that the choline substrate Km-value for the neuronal or true ChAc from mouse brain is 0.73 +/- 0.06 mM while the Km-value of choline substrate for purified CarAc from pigeon breast muscle is 108 +/- 4 mM. Type 2 error has occurred for the estimation of putative ChAc in rat heart. The rat heart ChAc was measured in previous studies utilizing a concentration of 30 mM choline substrate. While saturation of neuronal ChAc is observed at 2-5 mM choline, saturation of the rat heart CarAc enzyme is not reached until over 800 mM. Purified CarAc significantly synthesizes AcCh at 30 mM choline. Thus, putative ChAc has been greatly overestimated in the scientific literature for mammalian sperm, human seminal plasma and rat heart.     

Plasma and erythrocyte choline concentrations in rats following chronic treatment with lithium or choline

Rats were given daily injections of choline, lithium or lithium plus choline for either 11 or 18 days and red cell choline, glycine and glutathione levels were measured using proton nuclear magnetic resonance spectroscopy. In addition, plasma choline, plasma lithium and red cell lithium levels were measured 4 hr after the last dosage. Choline (1 mmol/kg) alone increased plasma but not red cell choline concentrations. Lithium (0.94 mmol/kg) elevated red cell choline levels but did not affect plasma choline concentrations. In contrast, red cell choline levels were not elevated in rats treated with a higher dose of lithium (1.88 mmol/kg). When choline was given in addition to the lower dose of lithium, a similar accumulation of red cell choline was observed suggesting that the lithium-induced choline accumulation was not enhanced by a greater availability of free choline. No differences were detected in red cell glycine or glutathione levels between any of the treatment groups. Therefore, lithium produced a specific (dose-dependent) accumulation of choline in rat erythrocytes. However, the 100% increase observed in rats was not as marked as the increased red cell choline levels reported in patients maintained on lithium (8 to 10-fold). This discrepancy supports the concept that species differences exist in red cell choline transport or metabolism.     

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K_m) of 16.5 μM and a maximal velocity (V_max) of 133.9 pmol/mg protein/min. The V_max value of choline uptake was strongly enhanced in the absence of Na⁺ without any change in K_m values. The increase in choline uptake under Na⁺-free conditions was inhibited by Na⁺/H⁺ exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.     

A simple, sensitive, and specific assay for free choline in plasma.

This paper describes a sensitive and specific enzymatic-radioisotopic method for determining plasma choline. Assays may be performed without prior extraction of the tissue. Plasma is first heated to destroy enzymes that would otherwise produce free choline from that which is normally bound. The free choline in plasma is then converted to phosphorylcholine [³²P], in the presence of ATP-γ-³²P, in a reaction catalyzed by choline kinase. Phosphorylcholine [³²P], isolated by ion-exchange chromatography, is measured as an index of the concentration of free choline. The concentration of plasma choline in man and in several species of laboratory animals was determined, and found to range from 5.5 nmoles/ml in dogs to 15.4 nmoles/ml in guinea pigs. The concentration of free choline in plasma of adult rats raised on a choline-deficient diet was half that of littermate controls raised on a control diet supplemented with free choline.     

Elevation of cerebrospinal fluid choline levels by nicotinamide involves the enzymatic formation of N1-methylnicotinamide in brain tissue

Nicotinamide administration can elevate plasma and brain choline levels and produce a marginal increase in striatal acetylcholine levels in the rat. We now report that subcutaneous nicotinamide produces a substantial and long-lasting rise in asternal cerebrospinal fluid (CSF) levels of choline in free-moving rats, possibly through the enzymatic formation of N¹-methylnicotinamide (NMN) in brain. CSF choline levels peaked 2 hours after nicotinamide administration and were accompanied by increases in striatal, cortical, hippocampal and plasma choline levels. The enzymatic formation of [³H]NMN in rat brain was evaluated by incubating aliquots of rat brain cytosol with unlabelled nicotinamide and the methyl donor [³H]S-adenosylmethionine. High performance liquid chromatography and radiochemical detection demonstrated that [³H]NMN was specifically formed by a brain cytosolic enzyme. The production of [³H]NMN was dependent on exogenous nicotinamide and could be prevented by denaturing the cytosol. The metabolism of nicotinamide to NMN in rat brain may explain the rise in CSF choline levels since NMN, a quaternary amine, can inhibit choline transport at the choroid villus and reduce choline clearance.     

The influence of lecithin on plasma choline concentrations in triathletes and adolescent runners during exercise

An investigation was carried out on the effect of lecithin (phosphatidylcholine, 90%) on the plasma choline concentrations during continuous strain in 10 top level triathletes (4 women and 6 men), trial I, and 13 excellent adolescent runners (3 girls and 10 boys), trial II. Venous blood, collected before and immediately after the race, was separated and plasma was assayed by an improved high performance liquid chromatography method for choline. Each study comprised three experiments. In trial I the triathletes performed two periods of bicycle exercise each lasting 2 h at an average speed of 35 km · h^–1, and in the second study (trial II) the subjects were subjected to severe physical stress on two occasions during cross-country races of durations between 30–60 min according to their ages. The participants received either a placebo or 0.2 g lecithin · kg body mass^–1, 1 h before each exercise. As a control the same dose of lecithin was administered without any exercise (both trials I and II). Bicycle exercise without lecithin supply decreased plasma choline concentrations in all the triathletes, on average by 16.9% (P0.01). When lecithin was given before exercise, average plasma choline concentrations remained at the same level as the initial values. The supply of lecithin without exercise led to a significant increase of the plasma choline concentrations, on average by 26.9% (P0.01). In trial II, when running without a supply of lecithin, the mean plasma choline concentrations in the adolescent runners remained stable which may have been due to the duration of the physical stress. When lecithin was given before exercise, plasma choline concentrations increased, on average by 18.9% (P0.01). The administration of lecithin without exercise led in these participants to an increase in plasma choline concentrations, on average by 54% (P0.001).It was found from the present study that a combination of both lecithin intake and hard physical stress prevented in most subjects a decrease in plasma choline and this could affect performance.     

Plasma concentrations of choline in man following choline chloride

Plasma choline levels were measured in patients being treated with choline chloride for movement disorders. Following single doses of 5 g given orally in aqueous solution, plasma concentrations rose to a peak within four hours and then rapidly declined. The degree of increase was variable both between and within patients. During chronic treatment, plasma choline concentrations tended to rise as the dose increased, although the relationship was not strong. The highest concentrations attained by patients were always at a dose of 16 or 20 g daily. Following chronic treatment, the disappearance of choline from plasma was rapid, with most patients reaching baseline by four days. Choline chloride is generally given in four divided doses, which seems reasonable in the early stages of treatment. Most therapeutic effect is seen when patients are treated with daily doses in the 12 to 20 g range, doses likely to produce substantial increases in plasma choline concentration. However, the relationship of plasma choline concentration to clinical efficacy may be tenuous. Following discontinuation of treatment, clinical improvement tends to persist long after plasma choline has returned to baseline concentrations.     

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.     

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in sub-microliter samples of rat airway surface liquid (ASL), a thin (10–30 μm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (1–3 μl per cm² of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 μM for nitrate and 30 μM for nitrite (S/N=3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102±12 μM in rat ASL and 70±1.0 μM in rat plasma, whereas nitrite was 83±28 μM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387±16 μM, and to 377±88 μM in plasma. The concentration of nitrite increased to 103±7.0 μM for ASL and 138±17 μM for plasma.     

Determination of free choline in plasma and erythrocyte samples and choline derived from membrane phosphatidylcholine by a chemiluminescence method

A sensitive chemiluminescence method for assay of choline which has been developed for analysis of erythrocyte and plasma levels of choline is reported here. This method includes a charcoal purification step which yields consistent results with plasma and erythrocyte extracts. Further, choline derived from membrane phosphatidylcholine may also be measured by an extension of this method following digestion with phospholipase D. This method has been used to study abnormal levels of erythrocyte choline that occur in cluster headache patients compared to control subjects and migraine patients. In addition, the time course of changes in plasma and erythrocyte choline following a fatty meal have been monitored. Plasma choline levels rise to a maximum between 1 and 3 h after the meal and this is followed by a rise in erythrocyte choline levels which are maximal 3 h after the meal.     

Control of choline oxidation in rat kidney mitochondria

Choline is a quaternary amino cationic organic alcohol that is oxidized to betaine in liver and kidney mitochondria. Betaine acts as an intracellular organic osmolyte in the medulla of the kidney. Evidence is provided that kidney mitochondria have a choline transporter in their inner membrane. The transporter has a Km of 173 ± 64 μM and a Vmax of 0.4 ± 0.1 nmol/min/mg mitochondrial protein (at 10 °C). Uptake of choline is not coupled to betaine efflux. Transporter activity demonstrates a dependence on membrane potential and choline transport is inhibited by hemicholinium-3. Steady-state oxygen consumption due to choline oxidation in kidney mitochondria was measurable at 37 °C (125 ± 6 pmolO₂/min/mg mitochondrial protein), in the absence of other mitochondrial electron transport chain substrates and the choline transporter was shown to be the major site of control (96 ± 4%) over choline oxidation flux in isolated kidney mitochondria. We conclude that the choline transporter in rat kidney mitochondria is the major site of control over the production of the organic osmolyte, betaine.     

On the insensitivity of sheep to the almost complete microbial destruction of dietary choline before alimentary-tract absorption.

1. Injection of [Me-14C]choline into sheep indicated that the small amount of phosphatidylcholine present in abomasal digesta was largely (69%) of non-dietary or ruminal origin. 2. Long-term feeding of [Me-3H]choline to sheep produced insignificant labelling of plasma phosphatidylcholine, indicating that more than 99% of the choline body pool was of non-dietary origin. 3. In contrast, when rats were fed with [Me-3H]choline for similar periods, 18-54% of the tissue phosphatidylcholine was derived from dietary choline. 4. The loss of [14C]choline and 32P from the plasma phosphatidylcholine after a single injection of these isotopes indicated a markedly slower turnover of choline in the sheep compared with the rat. This observation, coupled with a lack of liver glycerophosphocholine diesterase, provides an explanation for the insensitivity of the sheep to an almost complete microbial destruction of dietary choline before alimentary-tract absorption.     

Developmental changes in rat blood choline concentration.

1. Serum choline concentration in the newborn rat is extremely high and declines as the rat matures until adult values are attained at 20 days of age. 2. Rat milk is a rich source of choline, and rat pups denied access to milk had significantly lower serum choline concentrations than did fed littermates. We conclude that dietary intake of choline contributes to the maintenance of high serum choline concentrations in the neonatal rat. 3. In vivo, choline disappears with a half-life of 70 min. It is converted into betaine, phosphocholine and phosphatidylcholine. The rate of phosphocholine formation is identical in 3- and 10-day-old rats (3.3 mumol/h), whereas the rate of betaine formation is slower in younger animals (0.15 mumol/h at 3 days versus 0.69 mumol/h at 10 days). In vitro, choline oxidase activity [choline dehydrogenase (EC 1.1.99.1) and betaine aldehyde dehydrogenase (EC 1.2.1.8)] increased between birth and 40 days of age. The age-related acceleration in choline's conversion into betaine probably tends to diminish unesterified choline concentration in the rat.     

Enzymic radioassay for acetylcholine and choline in brain

This assay for acetylcholine (ACh) or choline in extracts of rat brain involves the isolation of the choline ester by high-voltage paper electrophoresis, alkaline hydrolysis of ACh to choline, and the quantitative enzymic conversion of choline to a radioactive derivative, P³²-phosphorylcholine. The method is specific, is applicable to large numbers of tissue samples, and has a blank value of about 3 nanograms of choline.     

The crystal structure of the substrate-binding protein OpuBC from Bacillus subtilis in complex with choline

Bacillus subtilis can synthesize the compatible solute glycine betaine as an osmoprotectant from an exogenous supply of the precursor choline. Import of choline is mediated by two osmotically inducible ABC transport systems: OpuB and OpuC. OpuC catalyzes the import of various osmoprotectants, whereas OpuB is highly specific for choline. OpuBC is the substrate-binding protein of the OpuB transporter, and we have analyzed the affinity of the OpuBC/choline complex by intrinsic tryptophan fluorescence and determined a K_d value of about 30 μM. The X-ray crystal structure of the OpuBC/choline complex was solved at a resolution of 1.6 Å and revealed a fold typical of class II substrate-binding proteins. The positively charged trimethylammonium head group of choline is wedged into an aromatic cage formed by four tyrosine residues and is bound via cation-pi interactions. The hydroxyl group of choline protrudes out of this aromatic cage and makes a single interaction with residue Gln19. The substitution of this residue by Ala decreases choline binding affinity by approximately 15-fold. A water network stabilizes choline within its substrate-binding site and promotes indirect interactions between the two lobes of the OpuBC protein. Disruption of this intricate water network by site-directed mutagenesis of selected residues in OpuBC either strongly reduces choline binding affinity (between 18-fold and 25-fold) or abrogates ligand binding. The crystal structure of the OpuBC/choline complex provides a rational for the observed choline specificity of the OpuB ABC importer in vivo and explains its inability to catalyze the import of glycine betaine into osmotically stressed B. subtilis cells.