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.     

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).     

Isolation of choline and choline esters from Krebs-Ringer solution for gas chromatographic determination

A simple and efficient novel method for isolating picomole amounts of choline and choline esters in milliliter volumes of Krebs-Ringer solution has been developed. The procedure is based on the observation that the solubility of choline esters in acetonitrile is 10(4)-10(5) times higher than that of the inorganic salt constituents of Krebs-Ringer solution. The glucose content of the medium, which prevented the one-step isolation of choline esters based on acetonitrile extraction from its lyophilizate, was removed using Amberlite CG-50 column chromatography. Bound compounds to the column were eluted in 0.25 N HCl and lyophilized. The lyophilizate was extracted with acetonitrile, which was then decanted and eliminated by evaporation to dryness. The resultant glucose and salt-free residue can be assayed by gas chromatography. Total recoveries of added choline and choline esters over the entire isolation procedure, measured isotopically and/or gas chromatographically, were 93 and 97%, respectively. Due to the high and close-to-equal recoveries of choline esters, and the high purity of the final product, this procedure is suitable for estimating acetylcholine and choline in neural tissue perfusates by gas chromatography, as was demonstrated by this method using hippocampal slices.     

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique

Choline enters brain by saturable transport at the blood-brain barrier (BBB). In separate studies, both sodium-dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re-examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (V:(max) = 2.4-3.1 nmol/min/g; K(m) = 39-42 microM) with an apparent affinity (1/Km)) for choline five to ten-fold greater than previously reported in vivo, but less than neuronal 'high-affinity' brain choline transport (K(m) = 1-5 microM). BBB choline uptake from a sodium-free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium-3 inhibited brain choline uptake with a K(i) (57 +/- 11 microM) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The V:(max) of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.     

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.     

Characterization of three choline transport activities in Rhizobium meliloti: modulation by choline and osmotic stress.

Choline has both a nutritional and osmoregulatory role in Rhizobium meliloti (T. Bernard, J. A. Pocard, B. Perroud, and D. Le Rudulier, Arch. Microbiol. 143:359-364, 1986). In view of this fact, choline transport was studied in R. meliloti 102F34 to determine how the rate of choline uptake is modulated. The effects of the cultural conditions on the kinetics of transport are presented. A high-affinity activity and a low-affinity activity were found in cells grown in minimal medium. The addition of 0.3 M NaCl or other osmolytes to the medium resulted in a marked decrease in the high-affinity activity, whereas the low-affinity activity remained fairly constant. Furthermore, results from osmotic upshock and downshock experiments indicate that the response of the cell to high osmolarity is rapid; hence, the mechanism of regulation by salt likely does not involve gene induction. A second high-affinity transport activity was induced by choline itself. Like the constitutive low-affinity transport activity, this activity was not greatly altered when the cells were grown in media of elevated osmotic strength. We conclude that although all three kinetically distinct transport systems are efficient at low osmolarity, only the induced high- and low-affinity activities are important for osmoregulation. The characteristics of the three transport activities from R. meliloti are compared with those from other bacterial species that use choline for growth and/or osmoregulation.     

High-performance liquid chromatography of water-soluble choline metabolites

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 degrees 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 NG108-15 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.     

Amperometric determination of choline with use of immobilized choline oxidase

Choline oxidase (choline: oxygen oxidoreductaserpar; was immobilized on a partially aminated polyacrylonitrile membrane. The enzyme electrode, consisting of an immobilized-enzyme membrane and an oxygen probe, was employed for the determination choline. Dissolved oxygen consumption by the enzymatic reaction was measured amperometrically. The rate assay method was used for the choline determination. The response time of the sensor was 7 sec for choline. The choline assay was done within 1 min. The choline calibration curve was linear from 0 to 0.1mM. The response was reproducible within an average relative error of 2.3% when 0.2mM choline was employed for experiments. The choline in the fermentation media was determined by the sensor. Furthermore, phospholipids in the serum were also determined with native phospholiphase D and the enzyme electrode.     

Effects of chronic (dietary) choline availability on the transport of choline across the blood-brain barrier

The effects of dietary choline availability on the transport of choline across the blood-brain barrier (BBB) were investigated using the intracarotid injection technique. Maintenance of rats on choline-deficient, basal choline, or choline-supplemented diets for 28-32 days led to respective increases in blood levels of choline and correlative increases in the velocity of transport of choline measured using a buffer injectate. When serum from these rats was included in the injectate and transport determined in control animals, there was a marked inhibition of choline transport that was related to the concentration of choline in the diets. Results suggest that the activity of the choline carrier at the BBB is antagonized by an inhibitory substance in serum whose concentration or activity may be modified by chronic alterations in circulating levels of choline and whose presence may normally regulate the velocity of choline transport.     

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.     

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.     

Substrate specificity of choline kinase

The substrate specificity of choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from brewer's yeast has been examined using multiple analogs of choline, most of which have been reported to be a substrate of one or another choline-using system from other sources. In contrast to many such systems, choline kinase from brewer's yeast has been found to have relatively stringent and straight-forward structural requirements for its substrates. It is hypothesized that there are at least four points of interaction of the substrate with the enzyme--one for the hydroxyalkyl side chain and one for each of the three substituents on the quaternary nitrogen. Of the latter, one site seems relatively more sterically hindered than the other two. Short, single or double alkyl substitutions on the quaternary nitrogen are possible without a large loss of substrate capacity of the analog. Thus N,N-dimethyl-N-propylethanolamine had a relative Vmax of 116% and a relative Vmax 96% that of choline and a Km of 68 +/- 15 microM [nearly four times that of choline itself (18 microM)]. However, N-butyl-N,N-dimethylethanolamine and N,N,N-triethylethanolamine were very poor substrates. Analogs with substituents on the quaternary nitrogen of longer chain length were without activity as were aromatic derivatives. None of the bisquaternary compounds of the general structure HOCH2CH2N+(CH3)2-(CH2)n-N+(CH3)2CH2CH2OH (n = 2-10) showed any substrate capacity, as well. Restrictions on the hydroxyethyl side chain were also severe. One additional methylene group in this chain greatly reduced substrate capacity of the analog and two additional ones eliminated it entirely, as did almost any substituent on the beta carbon. A single (but not a double) substituent on the alpha carbon was moderately tolerated, however. Thus alpha-methylcholine and N-methyl-2-hydroxymethylpiperidine were substrates (although the latter one was a poor one) but beta-methylcholine and N-methyl-3-hydroxypiperidine were not. Such information may be of use toward designing cholinergic probes targeting specific enzyme or metabolic functions.     

Enzymatic determination of choline acetyltransferase by coenzyme A cycling and its application to analysis of single mammalian neurons

Abstract: An enzymatic assay for choline acetyltrans-ferase was developed by measuring acetyl-coenzyme A (acetyl-CoA) formed from CoASH and acetylcholine (ACh). This method is extremely sensitive and may be applied to the analysis of microgram to nanogram crude samples. The method is, however, not useful when choline acetyltransferase is present in very low concentrations. The basis of this method is to amplify a small amount of synthesized acetyl-CoA in the assay mixture by using an enzymatic amplification reaction, CoA cycling. This amplification mechanism made it possible to perform microassays (13 nl-2.2 μl of assay volume) of freeze-dried sections prepared from cerebral cortex, striatum, and hippocampus of mice and single cell bodies isolated from freeze-dried sections of rabbit spinal cords. These samples were weighed and added directly to the reaction mixture. The activities of the above cerebral regions, assayed with 1,500–2,000-fold amplification, corresponded well to the results previously reported by other workers. The average activity of single anterior horn cells, determined with 64,000–420,000-fold amplification, was 40-fold higher than that of rabbit cerebral cortex, and the specific activities on a dry weight basis were widely distributed among individual neurons. No activity was detected in the noncholinergic dorsal root ganglion cells or in cerebellar cortex.     

Fluorometric enzyme assay for choline and acetylcholine

A sensitive and specific assay for choline and ACh which may be applied directly to brain extracts is described. The method is based upon enzymic coupling to the oxidation of fluorescent NADH. The following enzymic sequence is utilized: acetylcholinesterase, choline phosphokinase, pyruvate kinase, and lactate dehydrogenase. The method detects as little as 0.1 mμmole of choline or ACh, which is the amount of metabolite present in 1 mg or 8 mg of whole rat brain, respectively. The specificity of the method is such that only choline and ACh of tissue samples react. Extraction of whole brain samples by either heating at pH 4 or by chloroform/methanol or perchloric acid were compared in order to find a single procedure which was useful for extraction of both ACh and free choline from brain samples. Perchloric acid extraction proved to be the most efficient of the three methods for extraction of the two constituents. By this procedure the ACh content of whole rat brain was found to be 11.5 mμmoles/gm and the choline content of the same samples was 107 mμmoles/gm. Both values are in agreement with other published results.     

Enzymatic determination of phospholipase D activity with choline oxidase

A new enzymatic method was developed for the assay of phospholipase D [phosphatidylcholine phosphatidohydrolase EC 3.1.4.4] from cabbage leaves using choline oxidase from Arthrobacter globiformis cells. The method was based on the estimation of choline by the following series of enzymatic reactions after ending the phospholipase D reaction: Choline + 202 + h2o Choline oxidase Betaine + 2H2O2 2H2O2 + Phenol + 4-Aminoantipyrine Peroxidase Quinoneimine dye + 4H2O The amount of choline was proportional to the amount of resulting quinoneimine dye with an absorbance maximum at 500 nm. The phospholipase D reaction (choline liberation) was carried out at pH 5.5 in the presence of Ca2+ ions and ended by adding EDTA in conc. Tris-HCl buffer, pH 8, to give a final pH of around 8. The initial rate of the phospholipase D reaction was proportional to the enzyme concentration over the absorbance change range of 0 to 0.25 (equivalent to 0-21 micron of choline) under the optimal reaction conditions.     

Disruption of choline methyl group donation for phosphatidylethanolamine methylation in hepatocarcinoma cells

Despite being widely hypothesized, the actual contribution of choline as a methyl source for phosphatidylethanolamine (PE) methylation has never been demonstrated, mainly due to the inability of conventional methods to distinguish the products from that of the CDP-choline pathway. Using a novel combination of stable-isotope labeling and tandem mass spectrometry, we demonstrated for the first time that choline contributed to phosphatidylcholine (PC) synthesis both as an intact choline moiety via the CDP-choline pathway and as a methyl donor via PE methylation pathway. When hepatocytes were labeled with d(9)-choline containing three deuterium atoms on each of the three methyl groups, d(3)-PC and d(6)-PC were detected, indicating that newly synthesized PC contained one or more individually mobilized methyl groups from d(9)-choline. The synthesis of d(3)-PC and d(6)-PC was sensitive to the general methylation inhibitor 3-deazaadenosine and were specific products of PE methylation using choline as a one-carbon donor. While the contribution to the CDP-choline pathway remained intact in hepatocarcinoma cells, contribution of choline to PE methylation was completely disrupted. In addition to a previously identified lack of PE methyltransferase, hepatocarcinoma cells were found to lack the abilities to oxidize choline to betaine and to donate the methyl group from betaine to homocysteine, whereas the usage of exogenous methionine as a methyl group donor was normal. The failure to use choline as a methyl source in hepatocarcinoma cells may contribute to methionine dependence, a widely observed aberration of one-carbon metabolism in malignancy.     

High affinity choline uptake by spinal cord neurons in dissociated cell culture

Spinal cord-myotube cultures prepared with dissociated embryonic chick spinal cord cells and myoblasts exhibit a high affinity mechanism for accumulating choline. The uptake mechanism has a K_m of 3.4 ± 0.5 μM (7) and a V_m of 40.0 ± 0.1 (7) pmoles/min/mg of protein (mean ± SEM; number of determinations in parentheses). It is inhibited 90–95% by 10 μM hemicholinium-3 or by replacement of Na⁺ in the incubation solution with Li⁺. Part of the choline (10–20%) accumulated by the high affinity system is converted to acetylcholine (ACh). Uptake studies on spinal cord cells and myotubes grown separately demonstrate that the spinal cord cells can account for virtually all of the choline uptake observed in the mixed cultures. Myotubes are unnecessary under these conditions for the expression of the high affinity uptake mechanism by spinal cord cells. Neurons are not the only cell type in culture to exhibit high affinity choline uptake. Chick fibroblasts in both rapidly growing and stationary phase can accumulate choline with kinetics similar to those observed for the high affinity uptake by spinal cord cells. Little if any of the choline accumulated by fibroblasts, however, is converted to ACh. In most uptake studies with spinal cord cells, contributions from fibroblasts were minimized by carrying out the analysis at a time when few non-neuronal cells were present in the spinal cord cultures. These observations suggest that a population of chick central nervous system (CNS) neurons develop a high affinity choline uptake mechanism in cell culture that has many of the properties described for uptake by cholinergic neurons in vivo and that at least part of the choline accumulated by the system can be used for neurotransmitter synthesis.     

Studies about the specificity of the histochemical localization of choline acetyltransferase.

1. Tissues examined for the histochemical localization of choline acetyltransferase are best fixed by acetone. 2. Topographical identification of choline acetyltransferase by its reaction products is not only substrate-dependent because a slight staining also occurred in the absence of the substrates choline and acetyl-coenzyme in the incubation medium. 3. Histochemical localization of choline acetyltransferase was inhibited by sarin but not by DFP or eserine. 4. According to Burt and Silver's method cells and cell organelles, of which the enzymatic content is doubtful, where stained. 5. Chloroacetylcholine-perchlorate did not inhibit the histochemical localization of choline acetyltransferase. 6. The staining of acetylcholinesterase showed a different topographical distribution although both methods were inhibited by sarin.     

Concentrative accumulation of choline by human erythrocytes

Influx and efflux of choline in human erythrocytes were studied using ¹⁴C-choline. When incubated at 37°C with physiological concentrations of choline erythrocytes concentrate choline; the steady-state ratio is 2.08 ± 0.23 when the external choline is 2.5 µM and falls to 0.94 ± 0.13 as the external concentration is raised to 50 µM. During the steady state the influx of choline is consistent with a carrier system with an apparent Michaelis constant of 30 x 10^-6 and a maximum flux of 1.1 µmoles per liter cells per min. For the influx into cells preequilibrated with a choline-free buffer the apparent Michaelis constant is about 6.5 x 10^-6 M and the maximum flux is 0.22 µmole per liter cells per min. At intracellular concentrations below 50 µmole per liter cells the efflux in the steady state approximates first order kinetics; however, it is not flux through a leak because it is inhibited by hemicholinium. Influx and efflux show a pronounced exchange flux phenomenon. The ability to concentrate choline is lost when external sodium is replaced by lithium or potassium. However, the uphill movement of choline is probably not coupled directly to the Na⁺ electrochemical gradient.     

Neurochemical effects of choline supplementation

Whether or not the brain can use supplemental choline to enhance the synthesis of acetylcholine (ACh) is an important consideration for assessing the merits of using choline or phosphatidylcholine (lecithin) for the treatment of neuropsychiatric disorders postulated to involve hypocholinergic activity. While it is well documented that administered choline is incorporated into ACh, the ability of supplemental choline to increase the synthesis and release of ACh has been questionable. Studies in my laboratory have demonstrated that acute or chronic choline supplementation does not, by itself, enhance the levels of ACh in brain under normal biochemical and physiological conditions. However, supplemental choline prevents the depletion of ACh in brain induced by numerous pharmacological agents that increase the firing of cholinergic neurons. Since the levels of free choline in brains from supplemented rats were not different from controls prior to drug challenge, evidence suggested that the observed effects of choline were mediated by alterations in the mobilization of choline from choline-containing compounds. Studies investigating the release of choline from brain indicated that more choline was released per unit time in tissues from choline-supplemented rats than from controls. In addition, brain tissue from choline-supplemented rats had increased concentrations of total lipid phosphorus as compared with controls. Hence, although choline supplementation does not alter the levels of ACh in brain under normal conditions, it does appear to support ACh synthesis during drug-induced increases in neuronal activity, an effect most likely mediated by alterations in the metabolism of choline-containing phospholipids.