High-performance liquid chromatography of water-soluble choline metabolites

doi:10.1016/0003-2697(85)90069-7

Analytical Biochemistry

Volume 151, Issue 1, 15 November 1985, Pages 182-187

https://doi.org/10.1016/0003-2697(85)90069-7 Get rights and content

Abstract

We have developed a new method for the separation of [³H]choline metabolites by high-performance liquid chromatography. Using this method it is possible to separate, in one step, all of the known major water-soluble choline metabolites present in crude acid extracts of cells that have been incubated with [³H]choline, with baseline or near-baseline resolution. We use a gradient HPLC system with a normal-phase silica column as the stationary phase, and a linear gradient of increasing polarity and ionic strength as the mobile phase. The mobile phase is composed of two buffers: Buffer A, containing acetonitrile/water/ethyl alcohol/acetic acid/0.83 m sodium acetate (800/127/68/2/3), and buffer B (400/400/68/53/79), pH 3.6. A linear gradient from 0 to 100% buffer B, with a slope of 5%/min, is started 15 min after injection. At a flow rate of 2.7 ml/min and column temperature of 45°C, typical retention times for the following compounds are (in min): betaine, 10; acetylcholine, 18; choline, 22; glycerophosphocholine, 26; CDP-choline, 31; and phosphorylcholine, 40. This procedure has been applied in tracer studies of choline metabolism utilizing the neuronal NG 108-15 cell line and rat hippocampal slices as model systems. While the compounds labeled in the NG108-15 cells were primarily phosphorylcholine and glycerophosphocholine, reflecting high rates of phospholipid turnover, in the hippocampal slices choline and acetylcholine were the major labeled species. Identification of individual peaks was confirmed by comparing the elution profiles of untreated cell extracts with extracts that had been treated with hydrolyzing enzymes of differing specificities. This HPLC method may be useful in studies of acetylcholine and phosphatidylcholine metabolism, and of the possible interrelationships of these compounds in cholinergic cells.

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The kinetics and routing of choline incorporation to its metabolic pathways have been previously investigated in synaptosomes, neuronal cell culture, and brain slices using radioactive tracer methods [32,37,66]. The incorporated radioactive label is distributed among all of the choline metabolites, and specificity for acetylcholine synthesis was gained by separation methods [37]. Indirect determination of acetylcholine has been obtained by measuring the levels of free choline [46,65] and the concentration of choline acetyltransferase [27] in post-mortem human samples.
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Hippocampal acetylcholine increased by almost a factor of three. Other high performance liquid chromatography methods reported for the determination of acetylcholine are summarized (together with their conditions) in Table 3 [4, 117, 147–193]. Niwa et al. determined acetylcholine and choline with platinum-black ultramicroarray electrodes using liquid chromatography with a post-column enzyme reactor [194].
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In cholinergic neurons choline is directed to three main pathways; (1) conversion to phosphorylcholine (PCh) and cytidine diphosphate choline (CDP-choline) for the synthesis of phosphatidylcholine, (2) acylation to the neurotransmitter acetylcholine and (3) oxidation to betaine for the formation of methionine. Thus, the distribution of choline among the different metabolites is important for a better understanding of the regulation of these pathways in neurons. A non-HPLC method for the simultaneous separation of five choline metabolites found in neurons is described. High voltage electrophoresis (HVE) was combined with thin layer chromatography (TLC) to separate choline, PCh, CDP-choline, acetylcholine and betaine. This method is useful in studying the distribution of choline among its different metabolites in radiotracer experiments. Aqueous metabolites from leukemia inhibitory factor treated LA-N-2 cells labeled with [methyl-³H]choline were separated by HVE followed by TLC in the same dimension. Although the separation appeared to be complete, some ‘tailing’ by PCh significantly elevated the radioactivity measured in CDP-choline. This tailing of PCh was confirmed by subjecting radiolabeled PCh alone to this multiple separation method. Contamination of CDP-choline by PCh was eliminated by subjecting the samples to HVE followed by TLC in the second dimension. This two-dimensional approach was consistently reproducible and achieved excellent resolution of all five metabolites. In addition, this technique also resolved a sixth choline-containing metabolite, glycerophosphorylcholine (GPC), a breakdown product of phosphatidylcholine.
Agonist-stimulated alveolar macrophages: apoptosis and phospholipid signaling
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Bovine alveolar macrophages (BAM) were labeled with [³H]-choline or [³H]-ethanolamine and exposed to quartz dust, metal oxide-coated silica particles, Escherichia coli-derived lipopolysaccharide (LPS) or tumor promotor 12-O-tetradecanoyl phorbol 13-acetate (PMA). The activation of phospholipases A₂, C and D (PLA₂, PLC and PLD) acting on phosphatidylcholine and phosphatidylethanolamine was determined by high performance liquid chromatography (HPLC) separation and liquid scintillation counting of water- and lipid-soluble phospholipid metabolites. Exposure of BAM to quartz dust, metal oxide-coated silica particles, and LPS led to a transient PLD activation while treatment with PMA caused a prolonged rise in PLD activity. LPS and quartz dust induced a short-term increase of PLC cleavage products. All agonists caused a transient activation of PLA₂. To induce apoptosis, BAM were stimulated with C₈-ceramide, calcium-ionophore 23187, or gliotoxin. Apoptosis was investigated by qualitative and quantitative methods like flow cytometry, propidium iodide/Hoechst 33258 double staining, Cell Death Detection ELISA, and electrophoretical detection of DNA fragmentation. All three agonists led to apoptosis of BAM in a time- and concentration-dependent manner. After stimulation with gliotoxin an increase in ceramide and a drastic decrease in sphingosine-1-phosphate levels were observed, suggesting an involvement of these sphingolipids in gliotoxin-mediated apoptosis.
Choline's phosphorylation in rat striatal slices is regulated by the activity of cholinergic neurons
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The mechanism by which populations of brain cells regulate the flux of choline (Ch) into membrane or neurotransmitter biosynthesis was investigated using electrically stimulated superfused slices of rat corpus striatum. [Me-¹⁴C]Ch placed in the superfusion medium for 30 min during a 1-h stimulation period was incorporated into tissue [¹⁴C]phosphorylcholine (PCh) and [¹⁴C]phosphatidylcholine (PtdCh). Stimulation also caused a profound inhibition of PCh synthesis and a 10-fold increase in [¹⁴C]ACh release into the medium; it failed to affect tissue [¹⁴C]ACh levels. This effect was not explained by changes in ATP levels nor in the kinetic properties of Ch kinase (E.C. 2.7.1.32) or Ch acetyltransferase (ChAT) (E.C. 2.3.1.7). To investigate the mechanism of these effects, Ch uptake studies were performed with and without hemicholinium-3 (HC3), a selective inhibitor of high affinity Ch uptake. A two-compartment model accurately fit the observed data and yielded aK_m for Ch uptake of 5 μM into cholinergic structures and 72 μM into all other cells. Using this model it was estimated that cholinergic neurons account for 60% of observed uptake of Ch at physiologic Ch concentrations, even though they represent fewer than 1 % of the total cells in the slice. The model also predicts that an increase in Ch uptake within cholinergic neurons, reported to be associated with depolarization [4,27,32], would significantly inhibit Ch uptake into all other cells, and would account for the observed decrease in PCh synthesis.
Betaine generation in cardiac myocytes after adrenergic activation of phosphatidylcholine hydrolysis
1996, Journal of Molecular and Cellular Cardiology
In this report, effects ofα₁-adrenergic stimulation on phosphatidylcholine (PC) hydrolysis and the subsequent generation of water-soluble choline metabolites were investigated after preincubation of isolated cardiac myocytes of adult rats with [methyl-³H]choline. Choline uptake into cardiac myocytes was apparently mediated by a choline carrier which could be inhibited by hemicholinium-3. Analysis of the intracellular choline metabolites was performed by HPLC. Adrenergic stimulation of cardiac myocytes by (−)-phenylephrine, which is also known to activate the phosphoinositide signaling system, induced the generation of betaine as a selective signal transduction response. Agonist-induced generation of betaine in cardiac myocytes was maximal at 10 min after stimulation, and was optimal at physiologically relevant (−)-phenylephrine concentrations (1–10μm). Betaine accumulation was transient, and no betaine remained detectable after 15 min. CDP-choline, however, was still elevated after 15 min which is indicative of continued PC resynthesis after adrenergic stimulation. The source of betaine in cellular signaling appeared to be hydrolysis of membrane PC to phosphatidic acid and choline by phospholipase D with subsequent oxidation of choline to betaine. This is based on the observation that radioactivity in unstimulated cells is present only in the lipid phase (presumably as PC) or as phosphocholine in the aqueous phase of the cells. The latter finding suggests that choline is rapidly phosphorylated after uptake into cardiac myocytes. Collectively, these results suggest a hypothetical role of betaine in the cellular signal transduction response toα₁-adrenergic stimulation in cardiac myocytes.

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High-performance liquid chromatography of water-soluble choline metabolites

Abstract

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