Effect of ethyl choline mustard on choline dehydrogenase and other enzymes of choline metabolism

The effect of ethyl choline mustard (ECMA), and effective irreversible inhibitor of choline transport, was investigated on the enzymes of choline metabolism. ECMA at concentrations of 50 microM hardly affected choline acetyltransferase and caused only a 20% inhibition of choline kinase at a concentration of 1 mM. However, the mustard was an extremely effective inhibitor of choline dehydrogenase, producing 50% inhibition at concentrations of 6 microM. The inhibition was prevented by incubation in the presence of choline or by prior reaction of the mustard with thiosulphate. Separation of the components of the ECMA solution on TLC suggested that only the compound with an aziridine ring was an effective inhibitor of choline dehydrogenase. The inhibition was resistant to the washing out of excess unreacted mustard. The rate constant of inhibition was 395 M-1 X S-1. By the use of [3H]ECMA a single polypeptide in the enzyme preparation having a MW of 67,000 was labelled. The labelling was thiosulphate-sensitive and prevented by incubation with choline. It is concluded that ECMA is an irreversible inhibitor of choline dehydrogenase. It is at least as effective an inhibitor of choline dehydrogenase as of the choline transport system, although it does not appreciably inhibit choline acetyltransferase or choline kinase in the micromolar range.     

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.     

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.     

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.     

A simplified method of production of choline oxidase suitable for choline assay

A method of production of choline oxidase from Cylindrocarpon-didymum which is of sufficient purity to be used in the enzymatic assaying of choline has been described. The extraction and purification techniques used were ultrasonic disintegration, tangential ultrafiltration, and chromatography on DEAE-Sepharose Fast-Flow and on Sephacryl S 200 gel. The specific activity of the preparation obtained was 7.4 UI/mg of protein. The activity recuperation yield from the cellular extract was 56%. In the course of this purification, a protein displaying catalase activity was also obtained.     

Repression of choline kinase by inositol and choline in Saccharomyces cerevisiae.

The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae. The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity. Additional supplementation of choline caused a further reduction in the activity. The coding frame of the choline kinase gene, CK1, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody. Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CK1 probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance. The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene. The coding region of CK1 was replaced with that of lacZ, and CK1 expression was measured by assaying beta-galactosidase. The expression of beta-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal. The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance.     

Inhibition of choline transport into human erythrocytes by choline mustard aziridinium ion.

Acetylcholine mustard aziridinium ion inhibited the transport of [3H]choline into human erythrocytes. Treatment of the erythrocytes with 1 X 10(-4) M tetraethylpyrophosphate prevented the inhibition of [3H]choline transport by acetylcholine mustard aziridinium ion. Hydrolyzed acetylcholine mustard aziridinium ion inhibited choline transport both in the presence and absence of 1 X 10(-4) M tetraethylpyrophosphate. The product of hydrolysis was equipotent with acetylcholine mustard in its ability to inhibit choline transport; incubation of this product with sodium thiosulfate prevented inhibition of choline transport thereby indicating the presence of an aziridinium ion. The hydrolysis product is likely to be choline mustard aziridinium ion. Results on the efflux of [3H]choline from erythrocytes in the presence of the proposed choline mustard aziridinium ion showed that the mustard moiety was transported into the red cells on the choline carrier. The rate of efflux of [3H]choline produced by choline mustard aziridinium ion was 55% of that produced by the same concentration of choline. It is concluded that acetylcholinesterase (EC 3.1.1.7) of red cells rapidly hydrolyzes acetylcholine mustard aziridinium ion to acetate and choline mustard aziridinium and the latter compound can act as a potent inhibitor of choline transport. This finding would indicate that the hemicholinium-like toxicity of acetylcholine mustard in the mouse is due to the formation of choline mustard aziridinium ion.     

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.     

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.