PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl-CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis-aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were 1-¹⁴C]acetate, 2-¹⁴C]acetate, 4-¹⁴C]acetoacetate, 1-¹⁴C]butyrate, 1, 5-¹⁴C]citrate, 2-¹⁴C]glucose, 5-¹⁴C]glutamate, 3-hydroxy3-¹⁴C]butyrate, 2-¹⁴C]lactate, U-¹⁴C]leucine, 2-¹⁴C]pyruvate and ³H]acetylaspartate. The highest specific radioactivity of the acetyl group of ACh was observed 4 min after the injection of 2-¹⁴C]pyruvate. The contribution of pyruvate, lactate and glucose to the biosynthesis of ACh is considerably higher than the contribution of acetoacetate, 3-hydroxybutyrate and acetate; that of citrate and leucine is very low. No incorporation of label from 5-¹⁴C]glutamate into ACh was observed. Pyruvate appears to be the most important precursor of the acetyl group of ACh. The incorporation of label from 1, 5-¹⁴C]citrate into ACh was very low although citrate did enter the cells, was metabolized rapidly, did not interfere with the metabolism of ACh and the distribution of radioactivity from it in subcellular fractions of the brain was exactly the same as from 2-¹⁴C]pyruvate. It appears unlikely that citrate, glutamate or acetate act as transporters of intramitochondrially generated acetyl groups for the biosynthesis of ACh. Carnitine increased the incorporation of label from 1-¹⁴C]acetate into brain lipids and lowered its incorporation into ACh. Differences in the degree of labelling which various radioactive precursors produce in brain glutamine as compared to glutamate, previously described after intravenous, intra-arterial, or intraperitoneal administration, were confirmed using direct administration into the cerebrospinal fluid. Specific radioactivities of brain glutamine were higher than those of glutamate after injections of 1-¹⁴C]acetate, 2-¹⁴C]acetate, 1-¹⁴C]butyrate, 1,5-¹⁴C]citrate, ³H]acetylaspartate, U-¹⁴C]leucine, and also after 2-¹⁴C]pyruvate and 4-¹⁴C]acetoacetate. The intracisternal route possibly favours the entry of substrates into the glutamine-synthesizing (‘small’) compartment. Increasing the amount of injected 2-¹⁴C]pyruvate lowered the glutamine/glutamate specific radioactivity ratio. The incorporation of ¹⁴C from 1-¹⁴C]acetate into brain lipids was several times higher than that from other compounds. By the extent of incorporation into brain lipids the substrates formed four groups: acetate > butyrate, acetoacetate, 3-hydroxybutyrate, citrate > pyruvate, lactate, acetylaspartate > glucose, glutamate. The ratios of specific radioactivity of ‘citrate’ over that of ACh and of glutamine over that of ACh were significantly higher after the administration of 1-¹⁴C]acetate than after 2-¹⁴C]pyruvate. The results indicate that the 1-¹⁴C]acetyl-CoA arising from 1-¹⁴C]acetate does not enter the same pool as the 1-¹⁴C]acetyl-CoA arising from 2-¹⁴C]pyruvate, and that the cholinergic nerve endings do not form a part of the acetate-utilizing and glutamine-synthesizing (‘small’) metabolic compartment in the brain. The distribution of radioactivity in subcellular fractions of the brain after the injection of 1-¹⁴C]acetate was different from that after 1, 5-¹⁴C]citrate. This suggests that 1-¹⁴C]acetate and 1, 5-¹⁴C]citrate are utilized in different subdivisions of the ‘;small’ compartment.