Formation Of Choline Research Articles

Formation of unesterified choline by rat brain

Two preparations of rat brain (ischemic intact brain and homogenized whole brain) formed large amounts of unesterified (free) choline when incubated at 37°C. The accumulation of choline was inhibited by microwave irradiation of brain, or by heating of brain to 50° C, and was maximal at 37° C at pH 7.4–8.5. Choline formation was only observed in subcellular fractions of brain that contained membranes. In homogenates of brain, choline accumulated at a rate exceeding 10 nmol/mg protein per h. There was a significant decrease in brain phosphatidylcholine concentration (of 50 nmol/mg protein) during incubation for 1 h at 37° C. Concentrations of phosphocholine rose (by 2.3 nmol / mg protein), and concentrations of glycerophosphocholine and sphingomyelin did not change during this period. We used radiolabeled phospholipids to trace the fate of phosphatidylcholine and sphingomyelin during incubations of homogenates of brain. Phosphatidylcholine was degraded to form phosphocholine, glycerophosphocholine and free choline. No lysophosphatidylcholine accumulated. Sphingomyelin was degraded to form phosphocholine and a small amount of free choline. Magnesium ions stimulated choline production, while zinc ions were a potent inhibitor. Other divalent cations (calcium, manganese) had little effect on choline accumulation. ATP concentrations in brain homogenates were less than 5 nmol/mg protein (rapidly microwaved brain contained 27 nmol/mg protein). Addition of ATP or ADP to brain homogenates increased ATP concentrations and significantly inhibited choline accumulation. ATP diminished the formation of choline from added phosphatidylcholine, lysophosphatidylcholine, phosphocholine and glycerophosphocholine. The effects of ATP, zinc ion, or magnesium ion upon choline accumulation were not mediated by changes in the rates of utilization of choline for formation of phosphocholine or phosphatidylcholine. In summary, we showed that there was enhanced formation of choline when ATP concentrations within brain were low. This choline was derived, in part, from the degradation of phosphatidylcholine, and we suggest that phospholipase A activity was the primary initiator of choline release from this phospholipid.

Release of acetylcholine in the isolated heart.

This article summarizes methods for studying release of acetylcholine (ACh) in the heart and reviews the literature on release, synthesis, and inactivation of ACh. Cholinergic mechanisms involved in cardiac neuroeffector transmission are, in principle, the same as in ganglia and motor endplates, but their relative functional significance exhibits unique features. First, release of ACh evoked by nerve stimulation is maintained at a high level due to rapid formation of free extracellular choline from phospholipids. Choline derived from ACh hydrolysis plays a minor role in ACh synthesis. The constant efflux of choline allows continuous monitoring of changes in formation and removal of extracellular choline, e.g., by activation of neuronal uptake during nerve stimulation. Second, ACh released from terminal nerve fibers is rapidly washed into the circulation and thereby escapes hydrolysis by cholinesterase activity to a functionally significant extent. Hydrolysis and diffusion appear as equally important mechanisms of transmitter inactivation in the heart. The cardiac neuroeffector junction is not a morphological or functional entity restricted to adjacent pre- and postsynaptic elements.

In vivo formation and post-mortem changes of choline and acetylcholine in the brain of mice

In vivo formation and post-mortem changes of choline and acetylcholine in the brain of mice

Free choline formation by cerebral cortical slices from rat brain

Changes in free choline levels of cerebral cortical slices and their incubation fluid were examined under conditions of active ACh synthesis. Free choline was found to increase tenfold during two hours of incubation. Most of the free choline, which was presumably formed from esterified choline precursors, was found in the extracellular medium.

Sphingomyelinase in normal human spleens and in spleens from subjects with Niemann-Pick disease

This paper describes the purification and some of the properties of an enzyme from human spleen that catalyzes the hydrolysis of sphingomyelin with the formation of ceramide and phosphoryl choline. The enzyme, which is located in the subcellular particulate fraction that sediments between 700 and 8500 g, is readily made soluble and has been partially purified. Its pH optimum is between 4.5 and 5.0. It is unaffected by divalent cations, chelating agents, and sulfhydryl reagents, but is inhibited by phosphate. The enzyme attacks sphingomyelin and dihydrosphingomyelin, but is inactive toward sphingosine phosphoryl choline, O-acetylsphingomyelin, and lecithin. In some of its properties, the enzyme from human spleen is different from the previously studied sphingomyelinase from rat tissues. The enzyme is absent or markedly reduced in spleens from patients with classical and visceral varieties of Niemann-Pick disease, but is present in normal amounts in the late infantile type of the disease. In the present study another enzyme, this one magnesium-dependent, capable of catalyzing the cleavage of sphingomyelin has been detected in the spleens of patients with the classical form of Niemann-Pick disease. Some implications of these findings for theories of the metabolic defect in Niemann-Pick disease are discussed.

Biosynthesis of acetyl- l-carnityl choline

Earlier work had shown that acetyl-1-carnityl CoA was the material with acetylcholine-like activity which accumulated in the brain of narcotized rats. Accordingly, it was of importance to determine what normal function this material could have in the body. It appeared likely to be involved in acetylcholine and/or acetyl-1-carnityl choline formation. Since both these products are choline esters and it has been shown that the enzyme choline acetyltransferase is nonspecific for the esterification of choline, we have used the acetyl-1-carnityl CoA from narcotized brain extracts to determine which of these two products is produced when this substance is reacted with an extract of rat brain acetone powder as source of enzyme and choline. It was found that acetyl-1-carnityl choline could have been the only product formed under the particular reaction conditions studied. Product formation was indicated by increased acetylcholine-like activity as determined by bioassay. Acetylcholine formation could not be demonstrated with the particular reaction studied. These results also provide further evidence that the material with acetylcholine-like activity present in brain extracts from narcotized rats was not acetylcholine.

L-METHIONINE AS A MODULATOR OF ATRIAL RHYTHMICITY IN RABBIT AND GUINEA-PIG

By using the intracellular microelectrode technique, Toda (1) reported the effects of adrenaline, noradrenaline and reserpine on transmembrane potentials in pacemaker and non-pacemaker fibers of isolated rabbit atria, and suggested that endogenously liberated adrenaline and noradrenaline play an important physiological role in initiating and maintaining atrial rhythmicity. Further studies on cardiac mechanism were done by Matsuo and Tachi (2) who suggested that the initiating substance in atrial contraction may be adrenaline or noradrenaline in isolated atrial preparation. On the other hand, Bulbring and Burn (3, 4) concluded that the local formation of acetylcholine initiates auricular contraction. Kottegoda (5) also reported that acetylcholine in a high concentrations had a stimulant action on rabbit atria in the presence of atropine. In rabbit auricles, noradrenaline and acetylcholine were found present in about the same amounts of 1.3 to 1.48 μg/g [Burn (4), Matsuo (6)]. From these observations it may safely be assumed that endogenous acetylcholine is the trigger substance in the release of endogenous adrenaline or noradrenaline which, in turn, initiates atrial movement. Conversely, the depletion of endogenous acetylcholine or noradrenaline in atria may cause the arrest of atrial movement. Paasonen and Krayer (7) have shown that the administration of reserpine to a dog heart-lung preparation leads to a marked decrease in the noradrenaline content of the heart, but the adrenaline content normally 5 per cent that of noradrenaline shows no clear-cut decrease. This result would indicate that, rather than noradrenaline, endogenous adrenaline may be the more important hormone relative to atrial movement. Kirshner and Goodall (8) have shown that the soluble fraction of adrenal medullary hon ogenates form adrenaline from noradrenaline and L-methionine in the presence of ATP. Pilgeram et al. (9) suggested that the methyl groups of choline may be derived by transmethylation from L-methionine, and that the active formation of phospholipid choline from aminoethanol was obtained only through utilization of the methyl group of L-methionine. It may therefore be assumed that L-methionine is the substance essential both to endogenous adrenaline and acetylchholine, which are the initiators and modulators of atrial rhythmicity. The present experiment was carried out to study the effects of L-methionine on isolated normal, or reserpine or nicotine-treated atria, in order to discover the physiological role of L-methionine in the cardiac mechanism.

Phosphorus Compounds in Animal Tissues: VII. ENZYMATIC FORMATION OF DEOXYCYTIDINE DIPHOSPHATE CHOLINE AND LECITHIN BY TISSUE HOMOGENATES

Phosphorus Compounds in Animal Tissues: VII. ENZYMATIC FORMATION OF DEOXYCYTIDINE DIPHOSPHATE CHOLINE AND LECITHIN BY TISSUE HOMOGENATES

Determination of cholinesterase inhibition with the use of a cholinedependent strain of Neurospora crassa.

As small quantities of choline can be determined by the use of a cholineless mutant of Neurospora crassa1 and choline is a more powerful growth promoter of this organism than acetylcholine2, a method has been developed for the quantitative determination of organophosphorus cholinesterase inhibitors, by measuring the growth of this micro-organism in a medium, containing acetylcholine and cholinesterase, in the presence of the inhibitor. The latter diminishes or abolishes the formation of choline and thereby reduces the growth of this mutant. The mass of mycelium (dry weight) produced in a liquid medium and the rate of progression of the mycelium on an agar surface were used to measure the growth.

Biosynthesis of Trimethylammonium Compounds in Aquatic Animals: III. Choline Metabolism in Marine Crustacea

The incorporation in vivo of radioactivity into phospholipid-bound choline has been studied in lobster (Homarus americanus) following intramuscular administration of Na-formate-C14, glycine-2-C14, DL-serine-3-C14, L-methionine-methyl-C14and choline-methyl-C14. Choline and methionine were utilized for formation of phospholipid choline at both metabolic periods of 24 and 72 hours, whereas the other compounds gave only a very limited labelling.When choline-methyl-C14was administered to crab (Cancer magister), after 24 hours 1.9% of the administered radioactivity was found in phospholipid choline and 0.1% in trimethylamine oxide.

Methyl transfering enzyme system of microsomes in the biosynthesis of lecithin (phosphatidylcholine)

The formation of choline from S-[ Me- 14C]adenosylmethionine in isolated microsomes has been studied. The enzyme system was found to be inhibited by mercury compounds, showing that free sulfhydryl groups are requied. Choline formation is inhibited by BAL and by mercaptoethanol. This inhibition is due to the presence of an enzyme system in microsomes catalyzing a transmethylation from S-adenosylmethionine to these “nonphysiological” sulfhydryl compounds. S-adenosylethionine inhibits choline formation as a competitive inhibitor of S-adenosylmethionine. Choline formation is stimulated by the addition of d, l(N,N-dimethyl)disteary l- α-cephalin in vitro, but not by dipalmitoyl-α-cephalin. The incorporation of the first methyl group is found to be the rate limiting step in choline biosynthesis. The choline-synthesizing enzyme system is found in several tissues of the rat and in the liver of 8 different animal species investigated. These results are discussed in relation to choline biosynthesis in vivo.

Hydrolysis of lecithins by venom phospholipase A I. Structure of the enzymically formed lysolecithins

Rat-liver [ 32P]lecithin was hydrolyzed by snake venom phospholipase A to give quantitative yield of [ 32P]lysolecithins. The lysolecithins were oxidized in buffered aqueous solutions in the pH range 2.8 to 7.0, with bromine or permanganate to yield a keto-lysolecithin derivative. The extent and rate of oxidation is pH dependent. Bromine oxidation is more rapid at pH 6.8 than at pH 5.5 or 2.8. Permanganate oxidation is rapid at pH 5.5 but very slow at pH 6.8. During the oxidation partial concomitant hydrolysis occurs. The keto-lysolecithin can be separated and measured by paper chromatography and thus permits the determination of the α-lysolecithin isomer. The amount of this isomer was found to vary between 54–94% depending on the pH at which the bromine oxidation is carried out. Definitive evidence for the formation of the lysolecithinic acid has not yet been obtained. Passage of lysolecithin through silicic acid causes ester migration from the beta to the alpha position of glycerol. This is reflected by an increase in the keto-lysolecithin upon bromine oxidation. A novel reaction occurs when lysolecithin is treated with bromine in chloroform or methanol. Brominolysis rather than oxidation takes place with the formation of lysophosphatic acids and choline. A discussion of these findings is presented.

魚介類のコリンに関する研究-I

Choline-content was estimated for white and red muscles and viscera of various fishes of migrating and demersal habits, including sharks and cuttle fish. Its seasonal variation was also measured, values fairly characteristic of each fish being obtained. The amounts of choline and trimethylaminoxide(TMAO) increase generally in winter for demersal fishes and in summer for migrating ones. Decrease of freshness makes the chief cause for the increases of choline and trimethylamine (TMA) in common with different kinds of fishes. In cuttle fish and sharks, choline and TMAO contents were especially of large values, their rates of change being somewhat remarkable. Similarly to the case of salt-water fish, the choline content in fresh-water fish increased at first but decreased afterwards with the advance of putrifaction. It is noticed that TMA was formed even in the absence of TMAO, the known mother substance of TMA. The choline formation in this study was confirmed to be a bacterial doing.

THE ROLE OF DIETARY BETAINE AND VITAMIN B12 IN CHOLINE FORMATION BY THE RAT

THE ROLE OF DIETARY BETAINE AND VITAMIN B12 IN CHOLINE FORMATION BY THE RAT

New synthesis of lecithin in an isolated enzyme system.

WORK in this Laboratory1 has shown that the biosynthesis of lecithin and of phosphatidyl ethanolamine involves the intermediary formation of cytidine diphosphate choline and cytidine diphosphate ethanolamine. The enzymatic synthesis of lecithin is described in the following simplified scheme:

THE FORMATION OF CHOLINE AND BETAINE IN LEAF DISKS OF BETA VULGARIS

THE FORMATION OF CHOLINE AND BETAINE IN LEAF DISKS OF BETA VULGARIS

Cholesterol stimulation of phosphatidyl choline formation.

The conversion of ethanolamine to phosphatidyl choline was studied in liver slices from rats exposed to a high dietary intake of cholesterol in the presence of cholic acid. Cholesterol stimulated phosphatidyl choline formation per unit weight of liver tissue and increased the total liver mass thereby increasing further the total yield of phosphatidyl choline. These results are contrasted with the opposite effect of cholesterol on the liver phosphatides of the rabbit. A relationship between phosphatides and cholesterol metabolism and between species susceptibility to cholesterol induced atherosclerosis and phosphatide metabolism is pointed out.

Vitamin B 12 and folic acid in the synthesis of choline in the rat

1. 1. Vitamin B 12 deficiency in rats reduced the utilization of the α-carbon of glycine, but not the utilization of the β-carbon of serine, for the synthesis of both moieties of choline. 2. 2. Folic acid deficiency reduced the utilization of the β-carbon of serine, and, to a smaller extent, the utilization of the α-carbon of glycine for choline formation. 3. 3. The results are interpreted as indicating the involvement of vitamin B 12 or of its physiological derivative in the synthesis of serine from glycine. Folic acid or its physiological derivative is involved in the synthesis of both moieties of choline from serine, and, through serine, from glycine.

The Antagonistic Action of Choline and its Triethyl Analogue.

In recent years, considerable interest has been developed in the interference with biological processes through the use of analogues of essential metabolites. Triethyl choline chloride, the ethyl analogue of choline, is an interesting compound from this point of view. Triethyl choline is not capable of replacing choline in biological processes in which choline acts as a methyl donor, but its lipotropic activity has been found to be substantially equal to that of choline itself. Since the formation of acetyl choline presumably would not involve transmethylation directly, the triethyl derivative might possibly be handled similarly to choline in the systems involving its acetylation. The acetyl derivative of triethyl choline, however, does not have any of the stimulating actions of acetyl choline. A competition with choline in these systems should therefore lead to diminished formation of active acetyl derivatives. Triethyl choline chloride, when injected into animals in sufficient amounts, is acutely toxic. The symptoms demonstrated include apparent muscular weakness, apparent exophthalmos, and convulsions, followed by death within a few minutes. On the assumption that this acute toxicity is due to competition with choline in an essential biological system, choline was administered to see whether it would protect animals against its triethyl analogue. Forty-four mice, ranging in weight from 16 to 23 g, were divided into 2 groups containing animals of corresponding weights. The mice of one group were injected in the hind leg with triethyl choline chloride, prepared according to the directions of Channon and Smith. Six out of 9 mice given 1.25 mg died. Twelve out of 13 mice given 1.75 mg died. The mice of the other group were treated with the same amounts of triethyl choline chloride and an equal weight of choline chloride. Out of 9 mice given 1.25 mg triethyl choline chloride together with 1.25 mg choline chloride, all survived. Out of 13 mice given 1.75 mg triethyl choline chloride together with 1.75 mg choline chloride, 12 survived.

Choline, Creatine and the "Labile Methyl" Supply

Hemorrhagic degeneration, characterized by renal hemorrhage and fatty livers, occurs in young rats on diets low in choline and in methylated compounds possessing choline-like activity. Supplements of choline, methionine or betaine readily prevent this dietary deficiency. The effect of creatine in decreasing the occurrence of renal lesions is shown in Table I. The liver fat is not significantly affected unless both creatine and choline are fed. Prevention of the fatty liver requires more choline than is necessary for the prevention of renal hemorrhage. The experiments in Table I demonstrate that supplements of creatine decrease the severity of but do not prevent hemorrhagic degeneration. This effect differs from that of methionine and betaine in that only partial protection occurs and the degree of protection is not increased by larger supplements of creatine. The following explanation, which deals with the transfer of essential methyl groups and not with the synthesis of the rest of a methylated molecule, is suggested for these results. An adequate supply of compounds containing labile methyl groups is indispensable in the diet of young rats. The occurrence of hemorrhagic degeneration is evidence of a deficiency of choline and of the labile methyl supply. Supplements of choline, methionine and betaine contribute to this reserve. On the other hand, the methyl group of creatine is not available for the synthesis of choline. However, dietary creatine does spare the labile methyl supply and makes available for choline formation those methyl groups which otherwise would be used in the synthesis of required creatine. Choline may or may not be directly involved in all of the various manifestations of hemorrhagic degeneration. It may be required as such for instance in the formation of choline phospholipids or of acetyl choline and it may function indirectly by increasing the labile methyl supply which is required for methylations in metabolism.,,,