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 degrees C. The accumulation of choline was inhibited by microwave irradiation of brain, or by heating of brain to 50 degrees C, and was maximal at 37 degrees 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 degrees 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.     

The enterohepatic recycling of bile choline in sheep

1. Measurement of unesterified choline in blood samples taken from five conscious multi-cannulated sheep indicated a significant production of unesterified choline by the alimentary tract, as judged by the portal venous minus arterial difference and significant uptake by the liver, as judged from the portal venous minus hepatic venous and arterial minus hepatic venous differences. 2. A mean liver blood flow rate of 1.68 +/- 0.22 1/min for the five sheep was determined by bromosulphophthalein clearance and, combined with the differences in unesterified choline across organs, gave a production rate of free choline of 9.1 mmol/day by the alimentary tract and an uptake by the liver of 13.2 mmol/day. 3. Infusion of [methyl-3H]choline chloride into the portal vein of a sheep over 1 hr and subsequent isolation of the bile for several days showed over 70% cumulative recovery of the radioactivity in the choline moiety of bile phosphatidylcholine over a 120 hr period. 4. Subsequent infusion 17 days later of bile lipid [3H]choline via a duodenal fistula also gave approx. 70% cumulative recovery of radioactivity in the choline moiety of newly secreted bile phosphatidylcholine in 120 hr. 5. These results show a very extensive enterohepatic recirculation of bile choline in the sheep, which is in contrast to the situation in monogastric animals.     

The uptake and metabolism of plasma lysophosphatidylcholine in vivo by the brain of squirrel monkeys

1. Adult squirrel monkeys were injected intravenously with doubly labelled lysophosphatidylcholine (a mixture of 1-[1-(14)C]palmitoyl-sn-glycero-3-phosphorylcholine and 1-acyl-sn-glycero-3-phosphoryl[Me-(3)H]choline; (3)H:(14)Cratio 3.75) complexed to albumin, and the incorporation into the brain was studied at times up to 3h. 2. After 20min, 1% of the radioactivity injected as lysophosphatidylcholine had been taken up by the brain. 3. Approx. 70% of the doubly labelled lysophosphatidylcholine taken up by both grey and white matter was converted into phosphatidylcholine, whereas about 30% was hydrolysed. 4. The absence of significant radioactivity in the phosphatidylcholine, free fatty acid and water-soluble fractions of plasma up to 30min after injection of doubly labelled lysophosphatidylcholine rules out the possibility that the rapid labelling of these compounds in brain could be due to uptake from or exchange with their counterparts in plasma. 5. The similarity between the (3)H:(14)C ratios of brain phosphatidylcholine and injected lysophosphatidylcholine demonstrates that formation of the former occurred predominantly via direct acylation. 6. Analysis of the water-soluble products from lysophosphatidylcholine catabolism revealed that appreciable glycerophosphoryl-[Me-(3)H]choline did not accumulate in the brain and that radioactivity was incorporated into choline, acetylcholine, phosphorylcholine and betaine. 7. The role of plasma lysophosphatidylcholine as both a precursor of brain phosphatidylcholine and a source of free choline for the brain is discussed.     

Measurement of choline and choline metabolite concentrations using high-pressure liquid chromatography and gas chromatography-mass spectrometry

We have developed a reproducible and sensitive procedure for the isolation and measurement of choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, lysophosphatidylcholine and acetylcholine in a single 100-mg sample of biological tissue. Tissues were spiked with 14C-methyl- and 2H-methyl- or 15N-choline labeled internal standards for each compound. They were extracted with chloroform/methanol/water and the aqueous and organic phases were dried. The organic phase was resuspended in chloroform/methanol (1/1, v/v) and an aliquot was applied to a silica-gel thin-layer chromatography plate. The plate was developed in chloroform/methanol/water (65/30/4, v/v). Segments which cochromatographed with external standards of phosphatidylcholine and lysophosphatidylcholine were stained, scraped, and hydrolyzed in 6 M methanolic-HCl at 80 degrees C for 60 min, liberating free choline. The aqueous phase was resuspended in methanol/water and injected onto a silica HPLC column. Choline and its metabolites were eluted using a binary nonlinear gradient of acetonitrile/ethanol/acetic acid/1 M ammonium acetate/water/0.1 M sodium phosphate (800/68/2/3/127/10, v/v changing to 400/68/44/88/400/10, v/v). Peaks were detected with an on-line radiometric detector, collected, and dried under vacuum. Each choline ester was digested in 6 M HCl at 80 degrees C to form choline. Choline was then converted to the propionyl ester and demethylated with sodium benzenethiolate. This volatile derivative was then isolated using gas chromatography and measured with a mass selective detector. Deuterated internal standards were used to correct for variations in recovery. Choline, glycerophosphocholine, phosphocholine, phosphatidylcholine, lysophosphatidylcholine, and acetylcholine were measured in rat liver, heart, muscle, kidney, plasma, red blood cells, and brain and in human plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the origin of choline in the brain of the rat

1. Labelled precursors of choline, namely ethanolamine, dimethylaminoethanol and methionine and also labelled choline itself were injected intraperitoneally into the adult female rat and the incorporation into lipids and water-soluble fractions was traced in liver, blood and brain. 2. No significant free choline was detected and no labelling of the phosphorylcholine of blood. There was, however, considerable labelling of the phosphorylcholine of brain and liver. 3. After intracerebral injection, [1,2-(14)C]dimethylaminoethanol was rapidly phosphorylated and converted into phosphatidyldimethylaminoethanol, presumably by the cytidine pathway. 4. In view of the pattern of labelling and the amount of phosphatidylcholine in the tissues examined, it seems highly likely that choline is transported to the brain by the blood in a lipid-bound form.     

The importance of the lysophosphatidylcholine and choline moiety of bile phosphatidylcholine in lymphatic transport of fat

A luminal supply of biliary phosphatidylcholine is important in the translocation of absorbed fat into lymph and in the amount and composition of phosphatidylcholine concurrently synthesized. This study was undertaken to determine whether the effect was due to absorbed lysophosphatidylcholine, to a specific (1-palmitoyl) biliary lysophosphatidylcholine or to extra choline supplied by lysophosphatidylcholine. Rats with bile fistulae and thoracic duct lymph fistulae were given test meals of oleic acid and monoolein (molar ratio 2 : 1) infused duodenally for 8 h. Addition of choline chloride to the test meal increased lymphatic output of triglyceride and phospholipid but not to values found previously in rats with supplements of bile phosphatidylcholine or with bile ducts intact. Addition of dioleoyl phosphatidylcholine increased triglyceride and phospholipid output to values found in rats with intact bile ducts. Since dioleoyl phosphatidylcholine was as efficient as biliary phosphatidylcholine it was concluded that a luminal supply of 1-palmitoyl lysophosphatidylcholine was not essential. It seemed likely from the smaller effect of supplemented choline and from the fatty acid composition of lymph phosphatidylcholine that the essential requirement was a supply of absorbed lysophosphatidylcholine for rapid reacylation to phosphatidylcholine.     

Uptake and Metabolism of Choline by Rat Brain After Acute Choline Administration

The present study is concerned with the uptake and metabolism of choline by the rat brain. Intraperitoneal administration of choline chloride (4-60 mg/kg) caused a dose-dependent elevation of the plasma choline concentration from 11.8 to up to 165.2 microM within 10 min and the reversal of the negative arteriovenous difference (AVD) of choline across the brain to positive values at plasma choline levels of greater than 23 microM. Net choline release and uptake were linearly dependent on the plasma choline level in the physiological range of 10-50 microM, whereas the CSF choline level was significantly increased only at plasma choline levels of greater than 50 microM. The bolus injection of 60 mg/kg of [3H]choline chloride caused the net uptake of greater than 500 nmol/g of choline by the brain as calculated from the AVD, which was reflected in a minor increase of free choline level and a long-lasting increase of brain phosphorylcholine content, which paralleled the uptake curve. Loss of label from phosphorylcholine 30 min to 24 h after choline administration was accompanied by an increase of label in phosphatidylcholine, an indication of a delayed transfer of newly taken-up choline into membrane choline pools. In conclusion, homeostasis of brain choline is maintained by a complex system that interrelates choline net movements into and out of the brain and choline incorporation into and release from phospholipids.     

Ethanolamine inhibits choline uptake in the isolated hamster heart

The effect of exogenous ethanolamine on phosphatidylcholine biosynthesis in the isolated hamster heart was investigated. Hamster hearts were perfused with [Me-3H]choline in the presence of 0.05-0.5 mM ethanolamine. Incorporation of label into phosphatidylcholine was decreased 26-63% at 0.1-0.5 mM ethanolamine. Similar decreases in the labelling of the metabolites of the CDP-choline pathway were observed at these ethanolamine concentrations. The observed decrease in phosphatidylcholine labelling at 0.1-0.5 mM ethanolamine was attributed to an inhibition of labelled choline uptake by ethanolamine. The inhibitory role of ethanolamine to choline uptake was examined by comparison to hemicholinium-3. Both compounds inhibited choline uptake in a competitive manner. Intracellular choline, phosphocholine and CDP-choline concentrations were not altered under all experimental conditions. It can be concluded that exogenous ethanolamine has no immediate effect on the rate of phosphatidylcholine biosynthesis in the isolated hamster heart. The reduced labelling of phosphatidylcholine in the presence of ethanolamine is a direct result of the reduction of labelled choline taken up by the heart.     

Uptake and output of various forms of choline by organs of the conscious chronically catheterized sheep.

The net uptake and output of plasma unesterified choline, glycerophosphocholine, phosphocholine and lipid choline by organs of the conscious chronically catheterized sheep were measured. There was significant production of plasma unesterified choline by the upper- and lower-body regions and the alimentary tract and uptake by the liver, lungs and kidneys. The upper- and lower-body regions drained by the venae cavae provided the bulk (about 82%) of the total body venous return of plasma unesterified choline. Production of plasma unesterified choline by the alimentary tract was approximately balanced by the plasma unesterified choline taken up by the liver, and was almost equal to the amount of choline secreted in the bile. There was a considerable amount of glycerophosphocholine in the liver and there was production of plasma glycerophosphocholine by the liver and uptake by the lungs and kidneys. Glycerophosphocholine was higher in the plasma of sheep than in that of rats. Plasma phosphocholine was produced by the alimentary tract and kidneys. There was production of plasma lipid choline by the upper- and lower-body regions drained by the venae cavae. The results suggest that the sheep synthesizes substantial amounts of choline in ectrahepatic tissues and has the capacity for extensive retention and recycling of bile choline. These observations, coupled with a slow turnover of the endogenous choline body pool, explain the low requirement of sheep for dietary choline in contrast with non-ruminant species.     

Utilization of High-Density Lipoprotein Sphingomyelin by the Developing and Mature Brain in the Rat

Utilization of very long chain saturated fatty acids by brain was studied by injecting 20-day-old and adult rats with high-density lipoprotein containing [stearic or lignoceric acid-14C, (methyl-3H)choline]sphingomyelin. Labeling was followed for 24 h. Very small amounts of 14C were recovered in the brain of all rats, and there was no preferential uptake of lignoceric acid. Approximately 20% of the entrapped 14C was located in the form of unchanged sphingomyelin 24 h after injection. This result shows that the rat brain utilizes very little very long chain fatty acids (greater than or equal to 20 C atoms) from high-density lipoprotein sphingomyelin, even during the myelinating period. The [3H]choline moiety from sphingomyelin was recovered in brain phosphatidylcholine in a higher proportion in comparison with the 14C uptake. The brain 3H increased throughout the studied period in all experiments, but was much higher in the myelinating brain than in the mature brain. From the radioactivity distribution in liver and plasma lipids, it is clear that the choline 3H in the brain originates from either double-labeled phosphatidylcholine of lipoproteins or tritiated lysophosphatidylcholine bound to albumin, both synthesized by the liver.     

Incorporation of choline and ethanolamine into phospholipids in germinating soya bean.

1. Incorporation of [Me-14C]choline and [2-14C]ethanolamine into lipids was studied in germinating soya bean (Glycine max L.) seeds. The precursors are only incorporated into phosphatidylcholine and into phosphatidylethanolamine respectively. 2. Base-labelling via a phospholipase-D type of reaction was eliminated as a significant factor. 3. Cyclo heximide inhibited labelling of phosphatidylcholine from [Me-14C]choline but did not affect labelling of the aqueous choline pool. It had no effect on [2-14C]ethanolamine uptake or incorporation into phosphatidylethanolamine. 4. Hemicholinium-15 at 10mM concentrations decreased uptake and lipid labelling from the both bases. 5. There was no evidence for base competition. 6. The endogenous pool of choline was much larger than that of ethanolamine, which resulted in higher specific radioactivities for phosphatidyl-ethanolamine than for phosphatidylcholine. 7. The results can be interpreted as indicating that the kinase and phosphoryltransferase enzymes of the CDP-base pathways are separate for each phospholipid.     

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.     

Brain and serum levels of choline and lecithin resulting from longterm administration

Choline and phosphatidylcholine tissue concentrations were examined in mice treated with long-term (18–22 month) dietary choline enrichment, choline deficiency, or phosphatidylcholine enrichment. There were no significant differences found in choline levels among the dietary groups in any of the tissues examined: plasma, erythrocytes, cortex, hippocampus, and striatum. In contrast, the concentration of phosphatidylcholine in both the choline enriched and phosphatidylcholine enriched groups were significantly increased in the cortex, compared to the choline deficient group, and in the striatum, compared to control. No differences in phosphatidylcholine concentration were found in the hippocampus or plasma between any of the dietary groups. These results are in contrast to the reported effects of acute or short-term choline and phosphatidylcholine treatment and indicate that there may be differences between the effects of short-term and long-term administration on the blood and brain levels of choline and phosphatidylcholine.     

Small Rises in Plasma Choline Reverse the Negative Arteriovenous Difference of Brain Choline

The concentrations of free choline in blood plasma from a peripheral artery and from the transverse sinus, in the CSF, and in total brain homogenate, have been measured in untreated rats and in rats after acute intraperitoneal administration of choline chloride. In untreated rats, the arteriovenous difference of brain choline was related to the arterial choline level. At low arterial blood levels (less than 10 microM) as observed under fasting conditions, the arteriovenous difference was negative (about -2 microM), indicating a net release of choline from the brain of about 1.6 nmol/g/min. In rats with spontaneously high arterial blood levels (greater than 15 microM), the arteriovenous difference was positive, implying a marked net uptake of choline by the brain (3.1 nmol/g/min). The CSF choline concentration, which reflects changes in the extracellular choline concentration, also increased with increasing plasma levels and closely paralleled the gradually rising net uptake. Acute administration of 6, 20, or 60 mg of choline chloride/kg caused, in a dose-dependent manner, a sharp rise of the arterial blood levels and the CSF choline, and reversed the arteriovenous difference of choline to markedly positive values. The total free choline in the brain rose only initially and to a quantitatively negligible extent. Thus, the amount of choline taken up by the brain within 30 min was stored almost completely in a metabolized form and was sufficient to sustain the release of choline from the brain as long as the plasma level remained low. We conclude that the extracellular choline concentration of the brain closely parallels fluctuations in the plasma level of choline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake and metabolism of choline in the organotypic culture of newborn mouse cerebellum

The uptake and metabolism of [methyl-14C]choline in the organotypic culture of newborn mouse cerebellum was examined. Explants of 8 day in vitro (8 DIV) were incubated for 48 h under standard conditions with 21.0 microM [14C]choline at 35 degrees C. During the first hour of incubation, most of the [14C]choline incorporated was transferred to phosphocholine. The amount of [14C]phosphocholine increased gradually at the initial rate of 0.95 +/- 0.17 nmol/mg protein/h and saturated after 7 h (4.31 +/- 1.30 nmol/mg protein). The synthesis of [14C]phospholipids was observed after a distinct time lag. About 96% of the radioactivity in the lipids was incorporated into phosphatidylcholine. The amount of phosphatidylcholine increased linearly up to 48 h of incubation: 11.9 +/- 2.10 nmol/mg protein at 24 h and 21.9 +/- 2.43 nmol/mg protein at 48 h. From double-label studies it was found that phosphocholine was a precursor of phosphatidylcholine. The content of [14C]choline within explants remained nearly constant through the incubation period. Acetylcholine synthesis in mouse cerebellum culture was relatively low, and the content remained constant through the incubation period (0.006 +/- 0.003 nmol/mg protein). Activities of acetylcholine synthesis of cerebral and cerebellar homogenates were compared. Phosphatidylcholine synthesized in mouse cerebellum culture separated into two spots on thin layer chromatograph using silica gel G plates. Gas chromatographs suggested that the separation depends on the difference in fatty acid composition.     

Liquid chromatography with electrochemical detection for quantitation of bound choline liberated by phospholipase D hydrolysis from phospholipids containing choline in rat plasma

This report describes the optimal conditions for the determination of bound choline in rat plasma. The method used was based on the liberation of choline by phospholipase D from phospholipids containing choline in plasma, followed by high-performance liquid chromatographic analysis. Normal concentrations of total, free and bound choline in rat plasma were found to be 1278.7 ± 132.5, 11.5 ± 2.2 and 1267.2 ± 125.5 nmol/ml, respectively. The described procedure has the advantages of rapidity, specificity, excellent precision and the need for only a small amount of the sample.     

The uptake of choline by rat liver mitochondria.

1. Rat liver mitochondria can accumulate choline against a concentration gradient. Maximally about 30 nmol choline per mg mitochondrial protein are found in the matrix space. 2. The process of choline uptake is biphasic. After a rapid uptake of 1.5-15 nmol per mg protein, a slower uptake occurs if an energy supply is present. In the absence of energy, only the rapid uptake is found. 3. The inhibition of uncoupler-stimulated choline oxidation by cations is the result of an inhibition of choline uptake.     

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.     

Muscarinic Mobilization of Choline in Rat Brain In Vivo as Shown by the Cerebral Arterio-Venous Difference of Choline

In anesthetized rats, the choline levels of cerebrospinal fluid and plasma obtained from blood collected from peripheral vessels (carotid artery, cardiac vessels) and from the transverse sinus were determined with a radioenzymatic assay. Cortical release of choline was studied using the "cup technique." The plasma choline level of the peripheral blood (11.5 mumol/L) was lower than that of the sinus blood. The resulting cerebral arterio-venous difference of choline was negative (3.2 mumol/L) and reflected the net release of choline from the whole brain. The plasma choline levels were not different irrespective of whether the rats were anesthetized with ether, urethane, or pentobarbital. However, the choline level of the cerebrospinal fluid, which normally was lower than the plasma choline levels, was increased by urethane anesthesia to a level between the arterial and venous plasma concentrations of the brain. In old rats (24 months), the choline level of the cerebrospinal fluid was significantly lowered, when compared with the results obtained with younger rats (2-4 months). In rats kept on a low-choline diet for 2 weeks, the plasma choline level of the peripheral blood was reduced to 51% of the control. The effect on the choline level of the sinus blood was smaller; the cerebral arterio-venous difference of choline was not reduced (it was even slightly enhanced). Likewise, the choline level of the cerebrospinal fluid and the cortical release of choline were not altered. Intraperitoneal administration of oxotremorine in pentobarbital-anesthetized rats kept on a low-choline diet increased the plasma levels of choline.(ABSTRACT TRUNCATED AT 250 WORDS)