The synthesis of fluorocitric acid and its inhibition in acetate.

The theory that the citric acid formation induced by the poison fluoroacetate in vitro (Liebecq & Peters, 1949; Martius, 1949) and in vivo (Buffa & Peters, 1949) is due to the synthesis of a fluorotricarboxylic acid has been proved by the isolation ofa crystalline fluorocitric acid (Peters, Wakelin, Buffa & Thomas, 1953) and by the observation that this substance inhibits the enzyme aconitase in vitro (Lotspeich, Peters & Wilson, 1952; Morrison & Peters, 1954). Two forms of aconitase are known, one present in mitochondria, the other obtainable in solution. Working with the soluble aconitase, Peters & Wilson (1952) and Morrison & Peters (1954) found that there is a competition between the inhibitor and the tricarboxylic acid substrates when the inhibition is induced by enzymically made fluorocitric acid. With synthetic fluorocitric acid (Rivett, 1953) the relations are more complex. More information on the mechanism of synthesis of the fluorocitric acid from the fluorinated C2 fragment was needed to place this upon a firrn biochemical basis, partly for theoretical reasons and partly for the practical objective of understanding protective therapy. Bartlett & Barron (1947) advanced the hypothesis that fluoroacetate interfered with the metabolism of acetate, a hypothesis which was shown by Liebecq & Peters (1949) not to apply to the accumulation of citric acid, because they found conditions in which this accumulated in the presence of fluoroacetate without a change in acetic acid content. In connexion with the protective action of C2 substances, it was early found (Hutchens, Wagner, Podolsky & McMahon, 1949) that acetate with or without ethanol protected against fluoroacetate poisoning in some animals. Farah, West & Angel (1950) also found that acetate prevented the pharmacological toxicity of fluoroacetate to preparations ofrabbit small intestine, and there have been other instances since of such protective effects (see Peters, 1957). The failure of acetate in some animals might well be due to failure of the initial activation stage; it was later established (Chenoweth, Kandel, Johnson & Bennett, 1951) that monoacetin was highly protective, presumably owing to its liberation of active acetyl groups in situ. On similar lines Gitter, Blank & Bergman (1953) (see also Gitter, 1956) found that acetamide was protective; this might be expected to penetrate mitochondria. None of these researches established whether acetate itself interfered at some active stage with the synthesis of fluorocitric acid. Peters (1952) reported the results of some preliminary experiments by R. W. Wakelin and himselfon the effect of acetate on the synthesis of fluorocitric acid in vitro, and on the conditions leading to the best synthesis of fluorocitric acid using kidney preparations from the guinea pig. The experiments suggested that in fact it is acetate which interferes with the synthesis of fluorocitric acid from fluoroacetate. Subsequently these experiments were extended and more carefully controlled, partly because in this earlier work the apparent effects of acetate stopped short of the maximum. Difficulties were found on account of the small amounts of fluorocitric acid synthesized in experiments of this type; this makes it necessary to estimate the fluorocitric acid by the inhibition produced upon citrate metabolism. Estimations were done with kidney particles in the first instance, but later the results were confirmed with brain tissue.