Andrea Köppen

Free choline and choline metabolites in rat brain and body fluids: sensitive determination and implications for choline supply to the brain

In the central nervous system, choline is an essential precursor of choline-containing phospholipids in neurons and glial cells and of acetylcholine in cholinergic neurons. In order to study choline transport and metabolism in the brain, we developed a comprehensive methodical procedure for the analysis of choline and its major metabolites which involves a separation step, selective hydrolysis and subsequent determination of free choline by HPLC and electrochemical detection. In the present paper, we report the levels of choline, acetylcholine, phosphocholine, glycerophosphocholine and choline-containing phospholipids in brain tissue, cerebrospinal fluid and blood plasma of the untreated rat. The levels of free choline in blood plasma (11.4 microM), CSF (6.7 microM) and brain intracellular space (64.0 microM) were sufficiently similar to be compatible with an exchange of choline between these compartments. In contrast, the intracellular levels of glycerophosphocholine (1.15 mM) and phosphocholine (0.59 mM) in the brain were considerably higher than their CSF concentrations of 2.83 and 1.70 microM, respectively. In blood plasma, glycerophosphocholine was present in a concentration of 4.58 microM while phosphocholine levels were very low or absent (< 0.1 microM). The levels of phosphatidylcholine and lyso-phosphatidylcholine were high in blood plasma (1267 and 268 microM) but very low in cerebrospinal fluid (< 10 microM). We concluded that the transport of free choline is the only likely mechanism which contributes to the supply of choline to the brain under physiological conditions.

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

Abstract: 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 μM 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 >23 μM. Net choline release and uptake were linearly dependent on the plasma choline level in the physiological range of 10‐50 μM, whereas the CSF choline level was significantly increased only at plasma choline levels of >50 μM. The bolus injection of 60 mg/kg of [3H]choline chloride caused the net uptake of > 500 μMol/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.

Small rises in plasma choline reverse the negative arteriovenous difference of brain choline.

Abstract: 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 (<10 μM) as observed under fasting conditions, the arteriovenous difference was negative (about ‐2 μM), indicating a net release of choline from the brain of about 1.6 nmol/g/min. In rats with spontaneously high arterial blood levels (> 15 μM), 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. Moreover, the often described release of choline from the brain as reflected by the negative arteriovenous difference of brain choline is not a steady‐state phenomenon. Instead, the uptake of choline into and the release of choline from the brain seem to be in dynamic equilibrium that is closely related to the plasma choline level and, consequently, to nutritional choline uptake.

Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high‐choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (>96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet‐induced changes of the net uptake and release of choline.

Chapter 23: Choline, a precursor of acetylcholine and phospholipids in the brain

Publisher Summary The plasma level of free choline is remarkably constant at about 10 pM in animals and human. Ingestion of food, especially when rich in choline or lecithin, transiently elevates the plasma choline level up to 20 pM or more. In contrast, choline-deficient diet leads to a reduction of the plasma level by about 50%. Choline is considered an essential nutrient, which is predominantly supplied as phosphatidylcholine (lecithin). For a long time, neuroscientists have been intrigued by the fact that choline is a precursor for the biosynthesis of both acetylcholine (ACh) and phospholipids. For 50 years, lecithin has been marketed in Europe as a drug that was claimed to prevent exhaustion of membrane phospholipids in the CNS and therefore could reinforce “neuronal strength”. More recently, Wurtman and his colleagues suggested that partial degeneration of a cholinergic pathway may lead to an over-activity of the remaining viable neurones and consequently to exhaustion of the ACh pools of these neurons. The enhanced ACh turnover may in turn attack those synaptic phospholipid stores that serve as a source of choline for transmitter synthesis. Administration of choline or lecithin might then ameliorate the symptoms of chronic diseases that are caused by cholinergic hypofunction. These speculations stimulated a number of research activities in the cholinergic field. Unequivocal experimental evidence for the validity of the hypotheses in animals and especially in man is still lacking. Among the theoretical arguments against the above hypotheses was the general belief that the “milieu interne” of the brain, which is maintained by the blood-brain-barrier (BBB) and other mechanisms, protects the brain from fluctuations in the plasma levels of essential nutrients. In the case of choline, however, the BBB does not buffer fluctuations in the plasma level, because the carrier-mediated transport through the BBB is highly unsaturated at physiological plasma levels of choline. Therefore, homeostatic mechanisms of brain choline, if they exist, should be localized beyond the BBB within the CNS. This chapter discusses the existence and the nature of these homeostatic mechanisms and the results are summarized.

Release of choline from rat brain under hypoxia: contribution from phospholipase A2 but not from phospholipase D

Moderate hypoxia induced in rats by inhalation of 10% oxygen led to an increase of the concentration of free choline in the brain and caused a large net-release of choline from the brain into the venous blood as determined by the measurement of the arterio-venous difference. In hippocampal slices from rat brain, hypoxia increased the release of choline into the superfusion medium. The activity of phospholipase D, as measured by the formation of phosphatidylpropanol in the presence of propanol, was not stimulated under these conditions. However, the mobilization of choline was completely depressed by lowering extracellular calcium and by 0.1 mM mepacrine. We conclude that hypoxia leads to a selective activation of phospholipase A2 in the brain and, consequently, to a net loss of choline-containing phospholipids and membrane structures.

Modulation of hippocampal acetylcholine release after fimbria-fornix lesions and septal transplantation in rats

Female Long-Evans rats sustained electrolytic lesions of the fimbria and the dorsal fornix causing a partial lesion of the septohippocampal pathway. Two weeks later, the rats received intra-hippocampal grafts of fetal septal cell suspensions. Nine to twelve months later, the release of acetylcholine (ACh) in the hippocampus of sham-operated, lesion-only and grafted rats was measured by microdialysis. The extent of cholinergic (re)innervation was determined by acetylcholinesterase (AChE) staining and densitometry. In both lesion-only and grafted rats, the ratio of ACh release to AChE staining intensity was increased as compared to sham-operated rats, indicating a loss of endogenous inhibitory mechanisms. Scopolamine (0.5 mg/kg i.p.), a muscarinic antagonist, increased ACh release in all treatment groups. 8-OH-DPAT (0.5 mg/kg s.c.), an agonist at serotonergic 5HT1A-receptors, induced an increase of hippocampal ACh release in sham-operated rats. This effect was lost in lesion-only rats, but was fully restored by neuronal grafting. As 8-OH-DPAT influences hippocampal ACh release by a postsynaptic action, this finding indicates that the host brain exerts a serotonergic influence on the grafted cholinergic neurons.

Effects of nicotinamide on central cholinergic transmission and on spatial learning in rats

High-dose nicotinamide (1000 mg/kg) leads to a minor increase of plasma choline but to a major increase of the choline concentrations in the intra- and extracellular spaces of the brain. In the hippocampus, the nicotinamide-induced increase in choline was associated with an increase in the release of acetylcholine under stimulated conditions. In young rats, nicotinamide in doses between 10 and 1000 mg/kg did not influence spatial learning, as tested in the Morris water maze. In old rats, low doses of nicotinamide were ineffective whereas the high dose of 1000 mg/kg even impaired spatial learning. The combined administration of choline and nicotinamide had a synergistic effect on brain choline levels but had similar effects as nicotinamide given alone in the behavioral experiments. Additional tests for spontaneous behaviour and locomotion revealed procholinergic and sedative effects of the compound. We conclude that the ineffectiveness of the putative cognition enhancer nicotinamide in the learning task may be due to the observed sedative effect. Therefore, the development of nonsedative nicotinamide derivatives is recommended.

The Homeostasis of Brain Choline

The interest in the homeostasis of brain choline is reinforced by the role of choline as immediate precursor of acetylcholine, phosphatidylcholine and other phospholipids in the brain. In order to obtain a comprehensive view of the mochanisms of homeostasis it appeared necessary to elucidate the negative arteriovenous difference of choline across the brain (net release), a phenomenon that has been known for 20 years and is present in mammals and in man. This finding prompted an intense search for a de novo synthesis of choline in the brain. We detected in anaesthetized rats a reversal of the net release into a net uptake (positive arterio-venous difference), when the plasma level of choline was elevated spontaneously (presumably due to dietary intake) or after i.p. injection of choline. In these experiments, the newly taken up choline was rapidly removed from the extracellular space of the brain by cellular uptake and subsequent phosphorylation. The newly generated phosphocholine was trapped in brain cells and was only slowly incorporated into phosphatidylcholine, which in turn appears to serve as a reservoir for free choline. Surplus choline in a free or bound form may be cleared from the brain by net release.