Dietary Phosphatidylcholine As a Precursor of Brain Acetylcholine

Dietary Phosphatidylcholine as a Precursor of Brain Acetylcholine

Jean Mauron and Peter Leathwood

Nestle Research Centre, Nestec Ltd., CH-1000 Lausanne 26, Switzerland

Degeneration of cholinergic neurons and decreases in cholinergic function occur in several clinical syndromes. In tardive dyskinesia, senile dementia of the Alz- heimer type (SDAT), Huntington's disease, myasthenia gravis, and even in the normal process of growing old, more or less specific cholinergic deficits have been described (1-4). In some cases, treatment with cholinomimetic drugs such as physostigmine have brought improvement (5-7), but the unpleasant side effects, short duration of action, and extreme variability of individual sensitivity to cholinomimetics means that their use is clinically impractical. Recently, an alternative strat- egy for increasing cholinergic function has been suggested. Several research groups (8-12) have shown in animals that, under appropriate circumstances, treatment with choline or phosphatidylcholine can accelerate acetylcholine synthesis. This has led to the hope that ingestion of these neurotransmitter precursors might influence cholinergic function, without producing the side effects seen with drugs. Studies on elderly animals have produced quite promising results (13—15), but so far, clinical trials have not been very successful (16). Some improvements have been seen in patients with tardive dyskinesia (17) and Alzheimer disease (18), but most have been negative. It is, however, worth remembering that initial trials with L-dopa in the treatment of Parkinson's disease were not very successful either (see ref. 19 for a review). In this chapter, we will examine the rationale for using phosphatidylcholine in the treatment of cholinergic deficiency (with particular reference to treating senile mental decline and senile dementia) and critically review some of the clinical trials.

SYNTHESIS OF ACETYLCHOLINE

Synthesis of the neurotransmitter acetylcholine is catalyzed by the enzyme choline acetyltransferase (Fig. 1). Reported values of the KM for choline and for acetyl coenzyme A are 400 to 600 |XM and 6.6 to 18 JJLM, respectively (20). Whole brain levels of choline are 133 134 PHOSPHATIDYLCHOUNE AND ACETYLCHOLINE

RCETYLCOENZYME R CHOLINE

Choiine Rcetyltransferase

COENZYME H * ^ RCETYLCHOLINE

FIG. 1. Synthesis of acetylcholine. The reaction between acetyl coenzyme A and choline is catalyzed by the enzyme choline acetyltransferase. For precursor control to operate, the Mi- chaelis constant (KM) of the enzyme for choline must be similar to or greater than the normal intracellular concentration of choline. usually about 30 |XM, while acetyl coenzyme A varies between 2 and 20 (XM. This combination of observations has suggested to some that choline acetyltransferase must therefore be unsaturated in vivo so that the rate of acetylcholine synthesis will be precursor-dependent (21,22); others disagree (23). As Leathwood and Schlosser (24) have pointed out, the uncertainty about the real values of substrate concentration and kinetic constants within the microenvironment of the cell pre- cludes any strong conclusion from enzyme kinetic analyses about the possibility (or not) of precursor control of acetylcholine synthesis.

SOURCES OF CHOLINE FOR THE CHOLINERGIC NEURON

The cholinergic neuron can obtain choline from (a) de novo synthesis of choline in the brain; (b) reuptake of choline produced in the synaptic cleft by hydrolysis of released acetylcholine; and (c) uptake from the systemic circulation (via the interstitial fluid).

Synthesis

It has recently been confirmed that the brain can methylate phosphatidylethanolamine to phosphatidylcholine, which can then be broken down to yield choline. The estimated V^ for the synthesis is between 15 and 100 pmol/min/g of brain (25). This is far less than the average rate of synthesis of acetylcholine (about 6 nmol/min/g) (26) or the estimated rate of efflux of choline from the brain (about 4 nmol/min/g) (27). The phosphatidylcholine formed by this synthesis can be broken down to release choline by the action of phospholipase D, by base exchange, or by stepwise deacylation and subsequent breakdown of lysophosphatidylcholine or phosphocholine. The relative importance of these pathways in vivo is still not PHOSPHATIDYLCHOUNE AND ACEJYLCHOLINE 135 clear (Fig. 2) (25). Blusztajn et al. (21) have speculated that in Alzheimer's disease the (presumed) depletion of intracellular choline due to the high rate of acetylcholine synthesis in the remaining neurons may lead to autocannibalism of structural phosphatidylcholine. This might explain the specific vulnerability of cholinergic neurons.

Reuptake

Acetylcholine released into the synaptic cleft is rapidly hydrolyzed and the choline taken up again by a high affinity (KT = 1 to 3 JAM) transport system and reuti- lized (28). Speth and Yamamura (29) have estimated that, under resting conditions, this cycle provides most of the choline needed for synthesis of acetylcholine. When the neuron is firing rapidly, reuptake is no longer sufficient.

Uptake from the Interstitial Fluid

The cholinergic neuron also contains a low affinity transport system for choline. At first it was thought that this system was only used to transport choline into the cell body for phospholipid synthesis. Later it was shown that in synaptosomes maintained under depolarizing conditions (to ensure that they were releasing acetylcholine rapidly), choline taken up by the low-affinity system also contributes to acetylcholine synthesis (30). Evidence that the low-affinity system feeds acetylcholine synthesis when the neuron is firing rapidly comes from several sources. Bierkamper and Goldberg (8) using the stimulated rat phrenic nerve-hemidiaphragm preparation showed that adding 30 or 60 JAM choline to the medium led to a dose-dependent increase in acetylcholine release. Adding choline to the unstimu- lated preparation had no effect on acetylcholine release. In slices of rat striatum, addition of 5 or 20 JAM choline to the bathing fluid produces a dose-dependent increase in acetylcholine release (21). Evidence for a similar relationship between firing frequency and precursor responsiveness in the whole brain has been obtained by unilaterally destroying some striatal neurons with kainic acid. After this treatment, systemic choline administration (sufficient to raise brain choline) had no effect on brain acetylcholine levels in the intact striatum but doubled them in the kainate-lesioned side (11). Similarly, as Wecker and Schmidt (12) showed, atro- pine (5-7 mg/kg) depletes caudal and hippocampal acetylcholine levels by 10% to 15%, presumably by accelerating release to such an extent that resynthesis cannot keep pace. Treatment with choline completely restores acetylcholine levels.

TRANSPORT OF CHOLINE ACROSS THE BLOOD-BRAIN BARRIER

Choline is carried bidirectionally across the blood-brain barrier (BBB) by a specific transport system (26). The carrier has a low affinity for choline with a KT of 0.44 mM so that it is unsaturated in the physiologically possible range of plasma Phos p Phos p

•co 3" 3" 3- O a id y o (B a o \ CO CO T o M B > T > z •^ i -dyl e c z I— • ro UOUI - U|p- |

S y n a p t I c

FIG. 2. Sources of choline for brain neurons. The choline in cholinergic neurons is shown as deriving from three main sources: (i) Choline in extracellular fluid, which is in equilibrium with circulating choline and the choline present in other brain cells and which presumably enters the neuron by a low-affinity transport mechanism (1); (ii) intrasynaptic choline formed by acetylcholinesterase (2) from the acetylcholine released by the neuron (3), and then taken up into it by a high-affinity transport mechanism (4); (iii) neuronal phosphatidylcholine, from which the choline is liberated by base exchange (5)—the enzymatic substitution of a serine or an ethanolamine for the choline—or phospholipase-mediated hydrolyses (6-13). (Other potential PHOSPHATIDYLCHOUNE AND ACETYLCHOUNE 137 choline concentrations. There is also abundant evidence that increasing plasma choline levels can raise brain choline. In rats, it is generally found that a twofold rise in plasma choline (from 10 u-M to 20 U,M) will produce a 20% to 50% rise in brain choline (i.e., from 30 nmol/g to 40 or 50 nmol/g). The exact values vary according to the details of the experimental design and the brain region used (see ref. 24 for a review). It is important to note that the BBB choline transport system is generally thought to produce a net efflux of choline from the brain (25,27,31)—but all authors do not agree (26)—and that increasing plasma choline levels raises brain choline by slowing the output. It is not clear where the excess choline leaving the brain actually comes from. The brain can synthesize choline but the amounts are small (usually estimated at less than 10% of the efflux) (26,31). One suggestion is that phosphatidylcholine or lysophosphatidylcholine in plasma may contribute to brain choline (27,31), but Pardridge et al. (26) claim that these molecules cannot be transported across the BBB. They suggest that the "efflux" of choline might be an artefact produced by contamination of venous (jugular) blood from noncerebral sources. Thus, the net loss of choline from the brain (if it really occurs) still lacks a convincing explanation.

EFFECT OF INGESTED PHOSPHATIDYLCHOLINE ON PLASMA CHOLINE LEVELS

Plasma choline is influenced both by phosphatidylcholine (and choline) in food and by synthesis and breakdown of choline in the liver. The diet usually furnishes 0.1 to 1 g of choline per day (22,32), with most in the form of phosphatidylcholine or sphingomyelin and less than 5% as free choline (Fig. 3). sources not shown include membrane plasmalogens and sphingomyelin.) The phosphatidylcholine in neurons is shown as being formed either by the incorporation of preexisting choline [by way of base-exchange (5) or cytidine diphosphatidyl-choline (14-16) pathways] or by its de novo synthesis. The latter process can be initiated by the base-exchange substitution of a serine molecule for the choline in phosphatidylcholine (5) yielding phosphatidylserine, which is then decarboxylated to phosphatidylethanolamine (17). The latter, which can also be formed from ethanolamine through the base-exchange or CDP-ethanolamine pathway (18-20) is con- verted to phosphatidylcholine by phosphatidylethanolamine A/-methyltransferase, which cata- lyzes its stepwise methylation (21, a and b), with S-adenosylmethionine as methyl donor. Cho- line is acetylated to acetylcholine by choline acetyltransferase (22). The numbers on the figure and in parenthesis in this legend refer to the following enzymes and transport processes: (1) Low-affinity uptake of choline; (2) acetylcholinesterase (E.C. 3.1.1.7); (3) mechanism of acetylcholine release into the synapse; (4) high-affinity uptake of choline; (5) base-exchange enzyme; (6) phospholipase D (E.C. 3.1.4.4); (7) phospholipase C (E.C. 3.1.4.3); (8) phospholipases A, (E.C. 3.1.1.32) or A2 (E.C. 3.1.1.4); (9) lysophospholipase D (E.C. 3.1.4.39); (10) lysophospholipase (E.C. 3.1.1.5); (11) glycerophosphocholine cholinephosphodiesterase (E.C. 3.1.4.38); (12) glycerophosphocholine phosphodiesterase (E.C. 3.1.4.2); (13) alkaline phos- phatase (E.C. 3.1.3.1); (14) choline kinase (E.C. 2.7.1.32); (15) phosphocholine cytidylyltransferase (E.C. 2.7.7.15); (16) cholinephosphotransferase (E.C. 2.7.8.2); (17) phosphatidylserine decarboxylase (E.C. 4.1.1.65); (18) ethanolamine kinase (E.C. 2.7.1.82); (19) phosphoetha- nolamine cytidylyltransferase (E.C. 2.7.7.14); (20) ethanolaminephosphotransferase (E.C. 2.7.8.1); (21) phosphatidylethanolamine W-methyltransferase (forms 1 and 2) (E.C. 2.1.1.17); (22) choline acetyltransferase (E.C. 2.3.1.6). Reactions that are believed to be reversible are indicated with two-way arrows. (From ref. 25.) 138 PHOSPHATIDYLCHOUNE AND ACETYLCHOLINE

DIET- PCh Choiine

synthTesti s T

LIVER ^^ PCh ^n Choiine

breakdown

BLOOD PCh Choiine

BBB

synthes i s

ECF'NCN ^ pch Choiine bre akdoujn

(6)

synthes i s PCh Choiine breakdown NEURON

HCh

SYNRPTIC CLEFT RCh—Ch

FIG. 3. The pathway from phosphatidylcholine in the diet to acetylcholine in the brain. [1] Most of the choline in the diet occurs as phosphatidylcholine. [2] After ingestion phosphatidylcholine is deacylated by phospholipase A2 and the lysophosphatidylcholine absorbed across the mucosa where it may be reacylated or degraded. [3] For some authors plasma (lyso)phosphatidylcholine is a major source of brain choline. Others disagree. [4] There appears to be a net efflux of choline from the brain. [5] The brain can synthesize phosphatidylcholine but capacity is low. [6] It is unlikely that phosphatidylcholine per se is transferred across the neuronal membrane, but phospholipases in the membrane may release choline from phosphatidylcholine into the extracellular space where it becomes available for transport back into the cell. [8] Choline can enter the cell via high- or low-capacity transport systems. [7] Acetylcholine released into the synapse is hydroiyzed, and the choline is taken up by the high-affinity transport system: PCH, phosphatidylcholine; ECF, extracellular fluid; NCN, noncholinergic neurons; ACh, acetylcholine; Ch, choline. PHOSPHATIDYLCHOUNE AND ACETYLCHOUNE 139

In both rats and humans, consuming laige amounts of choline or phosphatidylcholine will increase plasma choline levels (24). Thus, in humans, a 3-g load of choline chloride will briefly double blood choline while phosphatidylcholine (containing the same amount of choline) produces a sustained rise persisting for about 12 hr (22) (Fig. 4). There is, however, a major problem with the use of choline salts as a means of raising plasma choline levels. When large amounts (2 g or more) are consumed, much of the choline is metabolized by intestinal bacteria to trimethylamine, which has an unpleasant rotten fish odor and which is toxic. Phosphatidylcholine does not produce this effect (10). On the other hand, the molecule contains only 12% to 15% choline (which means that large amounts must be consumed), and its waxy texture is difficult to adapt into a pleasant edible form.

LINK BETWEEN SENILE MENTAL DECLINE AND CHANGED CHOLINERGIC FUNCTION

There is strong evidence that a decrease in cholinergic function often occurs in elderly animals (16). Deficits have been observed in choline acetyltransferase, in muscarinic receptor densities, and, most convincing, in the measured rate of acetylcholine synthesis (2). There is some evidence of cholinergic deficits in normal elderly people (16), and patients with Alzheimer's disease show profound deficits in the number of cholinergic neurons in the nucleus basalis of Meynert, a brain area providing a major cholinergic input to the cortex (33). On the other hand, neurotransmitter deficits in old age (or in Alzheimer's disease) are not limited to the cholinergic system, and several research groups have reported age-related changes in other neurotransmitter systems (4,16), particularly in catecholamine levels. The tendency for deficits in mental function to occur in advanced age is also well established. Elderly monkeys show deficits in short-term memory (34), old

o 2.3g choline as ChCI • 2.3g choline as Lecithin E \ — 30 0 FIG. 4. Effects of consuming cho- E l line or phosphatidylcholine on plasma C choline. Ten healthy subjects consumed either 3 g of choline chloride " A— —"(ChCI) or 100 g of lecithin granules (containing about 16 g phosphatidylcholine and thus about 2.3 g choline). Subjects fasted for the follow- Ch o 1 i n

1 C ing 12 hours. (Adapted from ref. 22.) E 3 A 1. UI

6 12 Hours Hfter Choline Inqestion 140 PHOSPHAT1DYLCHOUNE AND ACETYLCHOUNE rats and mice show a decreasing ability to learn active and passive avoidance re- sponses or to learn multiple T mazes (24), and difficulty in remembering recent events and in orienting in time and space are common problems in old people. The work of Drachman and colleagues has provided a link between the known deficits in cholinergic and mental function with age. They first showed that the patterns of deficits in memory storage and in cognitive function in old people are surprisingly similar to those produced by giving young people scopolamine, an acetylcholine antagonist (35—36). In addition, the cholinergic agonist physostigmine improves scores both in elderly people and in young adults treated with scopolamine (5,37).

USE OF PRECURSOR THERAPY

Animal Studies

Although cholinomimetics do tend to improve performance in several behavioral tests in both animals and humans, they also produce unpleasant side effects such as tachycardia and intestinal hypermotility. So even if they are behaviorally successful, they are therapeutically unusable. As pointed out in the earlier sections of this review, there does seem to be a chance that treatment with phosphatidylcholine may increase the rate of acetylcholine formation, especially when synthesis is marginally unable to keep pace with output. In turn, this might improve behavioral deficits due to reduced cholinergic function. We have used two-way avoidance learning in mice as a model for studying the effects of precursor treatment on behavior in old age. The rationale for this approach was as follows: the cholinergic agonist nicotine improves two-way avoidance acquisition in strains of mice that are poor avoidance performers, while high- performing strains are unaffected (38). Mice in the high-performing strains show a marked decline in avoidance acquisition with age and a decrease in the rate of brain acetylcholine synthesis (2,38). If this age-related deficit in avoidance performance is due to inadequate cholinergic function and if increasing availability of choline to the brain does improve inadequate acetylcholine synthesis, then treatment with large doses of phosphatidylcholine should improve performance in older mice. As can be seen from Fig. 5, phosphatidylcholine (8% in the diet) had no effect on avoidance performance in young mice but significantly improved it in older animals. The dose level required was extremely high (almost 9 g/kg/day—equivalent to 1.2 g choline/kg/day) (15). In subsequent studies, Golczewski et al. (14) measured learning in a multiple T maze in 23- to 31-month-old mice fed a 25% phosphatidylcholine diet for 4 days before testing began. They observed a significant decrease in both error scores and in the number of trials to learn the maze. More recently, R. Oettinger (in preparation, 1987) has shown that lysophosphatidylcholine (10% in the diet) and nicotine (0.2 mg/kg intraperitoneally) both increased locomotor activity of 27-month-old rats in a complex tunnel maze. PHOSPHATIDYLCHOUNE AND ACETYLCHOUNE 141

FIG. 5. Avoidance performance of male SEC/1 ReJ mice during five daily sessions of 100 trials each. Each point represents the mean (±SEM) of eight mice. Top: At 6 months; (o) controls; (A) 8% phosphatidylcholine. Bottom: At 17 months; (•) controls; (•) 8% phosphatidylcholine. (Adapted from ref. 15.)

Using a slightly different approach, Bartus et al. (13) have produced evidence that it might be possible to retard the onset of these changes by feeding choline from early middle age through into old age. They gave mice approximately 1 g/kg/day of choline beginnning at an age of 8.5 months. At 13 months, retention scores in the treated animals were significantly better than in controls (receiving 100 mg/kg/day of choline) and far superior to those of choline-deficient animals (less than 50 mg/kg/day) (Fig. 6). In summary, these animal studies confirm that it is possible to influence behavior in elderly animals by precursor therapy. The dose levels needed are, however, rather high, and, as choline is itself an effective cholinergic agonist (39), there is no absolute guarantee that the effect really is mediated by increases in acetylcholine synthesis.

Clinical Studies

As pointed out above, several clinical studies have shown improvement in memory or in learning in normal old people and in patients with Alzheimer's disease treated with cholinomimetic drugs (5,16,37,40-42), suggesting that if precursor treatment really does increase acetylcholine availability, it will be useful in treating both senile mental decline and the early stages of Alzheimer's disease. (In ad- 142 PHOSPHAT1DYLCHOUNE AND ACETYLCHOUNE

ChoI i ne enri ched u / diet u I VI FIG. 6. Latencies to enter the shock -200 Controls chamber 24 hr after training in C57B1/6 j mice. The 13-month-old choline deficient mice performed as poorly as 23- month-old controls, while with choline 100 enrichment, performance was no worse c than in 6-month-old controls. (Adapted 10 i et from ref. 13.) v r

6 9 13 23 Rge (months) vanced Alzheimer's disease, marked structural changes in the brain have occurred and the patient is almost certainly beyond help.) Of the 15 clinical trials reported that have used choline (see ref. 16 for summary), in only one (43) was there an improvement reported. This should not really be surprising, since consuming choline is not a very efficient method of increasing plasma choline levels. Nevertheless, choline has produced some positive results in the treatment of tardive dyskinesia (3). Results of the trials using phosphatidylcholine to treat Alzheimer's disease are ambiguous (see refs. 16 and 44 for a detailed review). Most studies have found no detectable effect. Some reported no further deterioration (40), while in others, objective tests showed no detectable improvement although families and caretakers reported improvements (45,46). In still others, there were no clearcut treatment effects, although subgroups of the test population showed improvements (18,47) that developed over several months. This is not what one would expect if phosphatidylcholine was acting as an imme- diate choline source for acetylcholine synthesis. The slow response could be due to the progressive repair of the neuronal membrane structure and be interpreted in terms of the autocannibalism hypothesis of Maire and Wurtman (48) for the patho- genesis of Alzheimer's disease. The latter attributes the selective vulnerability of certain cholinergic brain neurons that characterizes the disorder to the breakdown of their choline-phospholipids in order to provide choline for acetylcholine synthesis. Be that as it may, it is of course impossible to draw conclusions about therapeutic efficiency using post hoc (and unpredicted) groupings. The number of conditions that must be satisfied so that phosphatidylcholine could work is large and complex. In addition, cholinergic deficits are certainly not the only causes of senile mental decline. In these circumstances, it is to be expected that only a small sub- population of patients should respond to phosphatidylcholine therapy. Nevertheless there is seductive evidence from drug studies that increasing cholinergic release may improve mental function. This means that it is still worth trying to identify PHOSPHAT1DYLCHOUNE AND ACETYLCHOUNE 143 subgroups of older people who may benefit from precursor therapy and, at the same time, to manipulate the phosphatidylcholine molecule so that it is more easily administered and is more efficient as a source of choline. Lastly, it should be remembered that so far, none of the clinical trials have at- tempted to improve nutritional status in the patients. A variety of nutrition defi- ciencies common in old age could influence the outcome of treatments with phosphatidylcholine. For example, thiamine is involved in synthesis of acetyl coenzyme A, which in turn is a precursor of acetylcholine (49). There is little use in trying to accelerate the rate of acetylcholine synthesis by feeding choline precursors if the other precursor is rate limiting. In conclusion, although most clinical trials of phosphatidylcholine have not been very successful there still seems to be a reasonable possibility that by using a more nutritional approach and with improved forms of phosphatidylcholine, neurotransmitter therapy may one day be useful in treating senile mental decline.

REFERENCES

1. Gerlach J, Reisby N, Randrupp A. Dopaminergic hypersensitivity and cholinergic hypofunction in the pathology of tardive dyskinesia. Psychopharmacologia 1974;34:21—34. 2. Gibson GE, Peterson C, Jenden DJ. Brain acetylcholine synthesis declines with senescence. Sci- ence 1981;213:674-5. 3. Growdon JH. Neurotransmitter precursors in the diet: their use in the treatment of brain diseases. In: Barbeau A, Growdon JH, Wurtman RJ, eds. Nutrition and the brain, volume 3. New York: Raven Press, 1979:117-83. 4. Wurtman RJ. Alzheimer's disease. Sci Am 1984;252:62-74. 5. Drachman DA, Sahakian GH. Memory and cognitive function in the elderly. A preliminary trial of physostigmine. Arch Neurol 1980;37:674—5. 6. Klawans HL, Rubovits R. Central cholinergic-anticholinergic antagonism in Huntington's chorea. Neurology 1972;22:107-12. 7. Klawans HL, Rubovits R. Effect of cholinergic and anticholinergic agents in tardive dyskinesia. J Neurol Neurosurg Psychiatry 1974;27:941-7. 8. Bierkamper GG, Goldberg AM. Release of acetylcholine from the vascular perfused phrenic nerve-hemidiaphragm. Brain Res 1980;202:234—7. 9. Haubrich DR, Gerber NH, Pflueger AB. Choline availability and the synthesis of acetylcholine. In: Barbeau A, Growdon JH, Wurtman RJ, eds. Choline and lecithin in brain disorders. Nutrition and the brain, volume 5. New York: Raven Press, 1979:57-71. 10. Hirsch MJ, Wurtman RJ. Lecithin consumption elevates acetylcholine concentrations in rat brain and adrenal gland. Science 1978;207:223-5. 11. London ED, Coyle JT. Pharmacological augmentations of acetylcholine levels in kainate-lesioned rat striatum. Biochem Pharmacol 1978;27:2962-5. 12. Wecker L, Schmidt DE. Neuropharmacological consequences of choline administration. Brain Res 1973;184:234-8. 13. Bartus RT, Dean RL, Goas JA, Lippa AS. Age-related changes in passive avoidance retention and modulation with chronic dietary choline. Science 1969;209:301—3. 14. Golczewski JA, Hiramoto RN, Ghanta VK. Enhancement of maze learning in old C57BL/6 mice by dietary lecithin. Neurobiol Aging 1982,3:223-6. 15. Leathwood PD, Heck E, Mauron J. Phosphatidylcholine and avoidance performance in 17-month old SEC/lReJ mice. Life Sci 1982;30:1065-70. 16. Bartus RT, Dean RL, Beer B, Lippa AS. The cholinergic hypothesis of geriatric memory dys- function. Science 1982;217:408-17. 17. Growdon JH, Hirsch MJ, Wurtmann RJ, Weiner W. Oral choline administration to patients with tardive dyskinesia. N Engl J Med 1977;297:524-7. 18. Levy R, Little A, Chuaqui P, Reith M. Early results from a double-blind, placebo-controlled trial of high dose phosphatidylcholine in Alzheimer's disease. Lancet 1983;ii:987-8. 144 PHOSPHAT1DYLCHOUNE AND ACETYLCHOUNE

19. Baibeau A. L-Dopa therapy in Parkinson's disease: a critical review of nine year's experience. Can Med Assoc J 1969;101:791-800. 20. White HL, Wu JC. Kinetics of choline acetyltransferases (ED 2.3.1.6) from human and other mammalian central and peripheral nervous tissues. J Neurochem 1973;20:297-307. 21. Blusztajn JK, Maire JC, Tacconi MT, Wurtman RJ. The possible role of neuronal choline metabolism in the pathophysiology of Alzheimer's disease: a hypothesis. In: Wurtman RJ, Corkin SH, Growdon JH, eds. Alhzeimer's disease: advances in basic research and therapy. Boston: Centre for Brain Sciences Charitable Trust, 1984:183-98. 22. Femstrom MH. Lecithin, choline and cholinergic transmission. In: Spiller GA, ed. Current topics in nutrition and disease. Nutritional pharmacology, volume 4. New York: Alan Liss, 1981:5—29. 23. Flentge F, Van den Berg CJ. Choline precursors and Alzheimer's disease. Lancet 1982;ii:1472. 24. Leathwood PD, Schlosser B. Phosphatidylcholine, choline and cholinergic function. Int J Vitam NutrRes 1986;29:[suppl]49-68. 25. Blusztajn JK, Wurtman RJ. Choline and cholinergic neurons. Science 1983;221:614-20. 26. Pardridge WM, Comford EM, Braun LD, Oldendorf WH. Transport of choline and choline ana- logues through the blood-brain barrier. In: Barbeau A, Growdon JH, Wurtman RJ, eds. Nutrition and the brain, volume 5. New York: Raven Press, 1979:25-34. 27. Ansell GB, Spanner S. Sources of choline for acetylcholine synthesis in the brain. In: Barbeau A, Growdon JH, Wurtman RJ, eds. Nutrition and the brain, volume 5. New York: Raven Press, 1979:35-64. 28. Yamamura HI, Snyder SH. High affinity transport of choline into synaptosomes of rat brain. J Neurochem 1973;21:1355-74. 29. Speth RC, Yamamura HI. Sodium dependent high affinity neuronal choline uptake. In: Barbeau A, Growdon JH, Wurtman RJ, eds. Nutrition and the brain, volume 5. New York: Raven Press, 1979:129-40. 30. Caroll PT, Goldberg AM. Relative importance of choline transport to spontaneous and potassium depolarised release of acetylcholine. J Neurochem 1975;25:523-7. 31. Jenden DS. An overview of choline and acetylcholine metabolism in relation to the therapeutic uses of choline. In: Barbeau A, Growdon JH, Wurtman RJ, eds. Nutrition and the brain, volume 5. New York: Raven Press, 1979:13-24. 32. Wurtman RJ. Sources of choline and lecithin in the diet. In: Barbeau A, Growden JH, Wurtman RJ, eds. Nutrition and the brain, volume 5. New York: Raven Press, 1979:73—81. 33. Whitehouse PJ, Price DL, Struble RG, Clark AW, Coyle JT, Delong MR. Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science 1982;215:1237-8. 34. Bartus RG, Fleming D, Johnson HR. Aging in the rhesus monkey: debilitating effects on short- term memory. J Gerontol 1978;33:858-71. 35. Drachman DA, Leavitt JL. Memory impairment in the aged: storage versus retrieval deficit. J Exp Psychol 1972;93:302-19. 36. Drachman DA, Leavitt JL. Human memory and the cholinergic system. A relationship to aging? Arch Neurol 1974;30:113-21. 37. Davis KL, Mohs RC, Tinklenberg JR, Pfefferbaum A, Hollister LE, Kopell BS. Physostigmine: improvement of long-term memory processes in normal humans. Science 1978;201:272—4. 38. Bovet D, Bovet-Nitti F, Oliverio A. Genetic aspects of learning and memory in mice. Science 1969;163:139-43. 39. Landinsky H, Consolo S, Pugnetti P. A possible central muscarine receptor agonist role for choline in increasing rat striatal acetylcholine content. In: Barbeau A, Growdon JH, Wurtman RJ, eds. Nutrition and the brain, volume 5. New York: Raven Press, 1979:227-32. 40. Christie JE, Blackburn IM, Glen AIM, Zeisel, S, Shering A, Yates GM. Effects of choline and lecithin on CSF choline levels and on cognitive function in patients with presenile dementia of the Alzheimer type. In: Barbeau A, Growdon JH, Wurtman RJ, eds. Nutrition and the brain, volume 5. New York: Raven Press, 1979:377-88. 41. Johns CA, Haroutunian V, Greenwald BS. Development of cholinergic drugs for the treatment of Alzheimer's disease. Drug Level Res 1985;5:77-96. 42. Sitaram N, Weingartner H, Gillin JC. Human serial learning: enhancement with arecholine and choline and impairment with scopolamine. Science 1978;201:274-6. 43. Fovall P, Dysken MW, Lazarus LW, et al. Choline bitartrate treatment of Alzheimer-type demen- tias. Commun Psychopharmacol 1980;4(2): 141-5. 44. Corkin S, Davis KL, Growdon JH, Usdin E, Wurtman RJ, eds. Alhzeimef s disease: a report of progress in research. New York: Raven Press, 1982. PHOSPHAT1DYLCHOUNE AND ACETYLCHOLINE 145

45. Etienne P, Dastoor D, Gauthier S, Holloway D, Hurwitz B. Lecithin in the treatment of Alz- heimer's disease. In: Corkin S, Davis KL, Growdon JH, Usdin E, Wurtman RJ, eds. Alhzeimer's disease: a report of progress in research. New York: Raven Press, 1982:369-72. 46. Sullivan EV, Shedlack KJ, Corkin S, Growdon JH. Physostigmine and lecithin in Alzheimer's disease. In: Corkin S, Davis KL, Growdon JH, Usdin E, Wurtman RJ, eds. Alheizmer's disease: a report of progress in research. New York: Raven Press, 1982:361-8. 47. Domino EF, Minor L, Duff IF, Tait S, Gershon S. Effects of oral lecithin in both blood choline levels and memory tests in geriatric volunteers. In: Corkin S, Davis KL, Growdon, JH, Usdin E, Wurtman RJ, eds. Alzeimer's disease: a report of progress in research. New York: Raven Press, 1982:393-8. 48. Maire JC, Wurtman RJ. Choline production from choline containing phospholipids: a hypotheti- cal role in Alzheimer's disease and aging. Prog Neuropsychopharmacol Biol Psychiatry 1984:8:637^2. 49. Sacchi O, Ladinsky H, Prigioni I et al. Acetylcholine turnover in the thiamine-depleted superior cervical ganglion of the rat. Brain Res 1978;151:609-14.