A series of choline analogues and nitrogen mustard derivatives were evaluated as inhibitors of high‐affinity transport of choline in rat forebrain synaptosomes. When synaptosomes were preincubated for 10 min with choline mustard aziridinium ion, monoethylcholine and monoethylcholine mustard aziridinium ion, the agents appeared to be equipotent as inhibitors of high‐affinity uptake (Ki=2.63, 3.15 and 2.72 μm, respectively). Acetylcholine mustard aziridinium ion was less potent than these compounds (Ki= 27.8 μm), but it was more potent than ethoxycholine and ethoxycholine mustard aziridinium ion (Ki= 500 and 403 μm) as a blocker of choline transport. From study with these compounds it was concluded that the high‐affinity choline transport mechanism shows specificity for hydroxylated compounds over those in which the same hydroxyl has been acetylated (10‐fold) and that the carbonyl oxygen of the acetylated analogues is important, as its removal (to form the ethylether derivative) decreased affinity another 20‐fold. The presence of an aziridinium ring on the quaternary nitrogen in place of two methyl groups did not affect the blocking of transport at 10 min of inhibitor preincubation and replacement of a methyl group on the nitrogen by an ethyl group did not alter affinity for the high‐affinity carrier. The aziridinium ring on the nitrogen of the mustard analogues was important, however, in determining the extent of reversibility of the binding of these agents to the carrier protein. Choline transport was not restored by washing synaptosomes that were incubated with choline mustard aziridinium ion or monoethylcholine mustard aziridinium ion, but was readily obtained in washed synaptosomes preincubated with monoethylcholine, hemicholinium‐3, or pyrrolcholine. The results indicate that the mustard analogues may be potent alkylators of the high‐affinity choline carrier and thus, useful agents in monitoring acetylcholine turnover in systems where the carrier is blocked.

Abstract— The nitrogen mustard analogue of choline, choline mustard aziridinium ion, was studied as a substrate and inhibitor of choline acetyltransferase in crude homogenates of rat brain. The compound was acetylated at a maximum velocity (Vmax) of 1.51±0.33μmol/g tissue/h with an apparent Km of 4.63± 0.17 mM. The Vmax for choline was 5.25±0.15μmol/g tissue/h with a Km of 1.70±0.17mM indicating that the affinity and rate of acetylation of choline mustard aziridinium ion was about 3 times less. Choline mustard aziridinium ion was a competitive inhibitor of choline acetyltransferase with a Ki of 1.8 mM. The generation of aziridiniurn ion from acetylcholine mustard was determined at 25°C and the concentration of ion in aqueous solution was inversely related to the concentration or acetylcholine mustard; the optimum ion concentration of 70% was obtained at 10 mg acetylcholine mustard/ml solution. The ineffective generation of aziridiniurn ion at high concentrations of acetylcholine mustard placed limitations on the design and evaluation of the kinetic experiments with choline acetyltransferase. Nevertheless, the present experiments demonstrate that choline mustard aziridrnium ion interacts with choline acetyltransferase and they may provide evidence for a clearer understanding in the in vivo actions of certain nigrogen mustard analogues.

A compound thought to be choline mustard aziridimium ion was found previously in our laboratory to inhibit choline transport into human erythrocytes. Experiments with synaptosomes prepared from brain of rat now show that the aziridinium ion of choline mustard hydrochloride inhibited high- and low-affinity choline transport, with the inhibitory effect being about 10 times less than that of hemicholinium-3. Acetylcholine mustard aziridinium ion also inhibited the transport of choline, but it was less potent than its choline analogue. Studies with choline mustard aziridinium ion may yield important information about the transport of substances into the presynaptic nerve terminal.

Twenty-one patients with a diagnosis of Alzheimer's disease completed a double blind trial on lecithin 25 g daily. Equal improvement was noted in both the lecithin and placebo groups. Serum levels of lecithin measured 12-14 hours after its administration were also not increased compared with the levels in control patients. These findings of the ineffectiveness of lecithin inpatients with Alzheimer's disease are discussed in relation to the literature on this subject.

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