A specific, sensitive, simple and rapid method for analyzing acetylcholine is described. Acetylcholine is precipitated as the enneaiodide in the presence of tetramethylammonium iodide as a co-precipitant and, for quantification, with the addition of a known amount of butyrylcholine as an internal standard. After sublimation of excess iodine, the precipitate is dissolved, concentrated in a pyrolyzing chamber connected to a gas chromatograph and flash-heated. Pyrolysis causes N-demethylation to yield dimethylaminoethyl acetate from acetylcholine and dimethylaminoethyl butyrate from butyryicholine. The relative ratios of the peak areas are linearly related to the amounts of acetylcholine. The method permits measurements of acetylcholine in quantities as low as 2 ng ( i.e. , 13.7 pmol) in 5 ml of Tyrode9s solution and 5 ng in 300 ml of Tyrode9s solution. The phrenic nerve-diaphragm preparation of the rat released material which is chromatographically indistinguishable from acetylcholine and which is destroyed by electric eel acetyicholinesterase. Electrical stimulation caused increased release of of acetylcholine from the phrenic nerve-diaphragm. No other differences between the effluents from the stimulated and unstimulated preparation were seen.
Abstract: Levels of histamine and its major metabolites in brain, tele ‐methylhistamine (t‐MH) and tele ‐methylimidazoleacetic acid (t‐MIAA), were measured in rat brains up to 12 h after intraperitoneal administration of l ‐histidine (His), the precursor of histamine. Compared with saline‐treated controls, mean levels of histamine were elevated at 1 h (+ 102%) after a 500 mg/kg dose; levels of t‐MH did not increase. Following a 1,000 mg/kg dose; levels mean histamine levels were increased for up to 7 h, peaked at 3 h, and returned to control levels within 12 h. In contrast, levels of t‐MH showed a small increase only after 3 h; levels of t‐MIAA remained unchanged after either dose. Failure of most newly formed histamine to undergo methylation, its major route of metabolism in brain, suggested that histamine was metabolized by another mechanism possibly following nonspecific decarboxylation. To test this hypothesis, other rats were injected with α‐fluoromethylhistidine (α‐FMHis; 75 mg/kg, i.p.), an irreversible inhibitor of specific histidine decarboxylase. Six hours after rats received α‐FMHis, the mean brain histamine level was reduced 30% compared with saline‐treated controls. Rats given His (1,000 mg/kg) 3 h after α‐FMHis (75 mg/kg) and examined 3 h later had a higher (+112%) mean level of histamine than rats given α‐FMHis, followed by saline. Levels of t‐MH and t‐MIAA did not increase. These results imply that high doses of His distort the simple precursor‐product relationship between histamine and its methylated metabolites in brain. The possibility that some His may undergo nonspecific decarboxylation in brain after His loading is discussed. These findings, and other actions of His independent of histamine, raise questions about the validity of using His loading as a specific probe of brain histaminergic function.
