2-Fluorocitrate

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase) the reaction occurs in two steps dehydration to r-aconitate, some of which remains bound to the enzyme and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. The poison fluo-roacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate. 

The mechanism of fluoroacetate toxicity in mammals has been extensively examined and was originally thought to involve simply initial synthesis of fluorocitrate that inhibits aconitase and thereby the functioning of the TCA cycle (Peters 1952). Walsh (1982) has... 

There are several examples in which metabolites that toxify the organism responsible for their synthesis are produced. The classic example is fluoroacetate (Peters 1952), which enters the TCA cycle and is thereby converted into fluorocitrate. This effectively inhibits aconitase—the enzyme involved in the next metabolic step—so that cell metabolism itself is inhibited with the resulting death of the cell. Walsh (1982) has extensively reinvestigated the problan and revealed both the complexity of the mechanism of inhibition and the stereospecihcity of the formation of fluorocitrate from fluoroacetate (p. 239). It should be noted, however, that bacteria able to degrade fluoroacetate to fluoride exist so that some organisms have developed the capability for overcoming this toxicity (Meyer et al. 1990). 

Intraperitoneal injection of 4-methylpyrazole to rats at 90 mg/kg BW, given 2 h prior to 1080 administration, offered partial protection against accumulations of citrate or fluorocitrate in the kidney (Feldwick et al. 1994). The antidotal effects of 4-methylpyrazole are attributed to its inhibition of NAD+-dependent alcohol dehydrogenase that converts l,3-difluoro-2-propanol to difluoro-acetone, an intermediate in the pathway of erythrofluorocitrate metabolism (Feldwick et al. 1994). A disadvantage of 4-methylpyrazole is that it needs to be administered before significant exposure to fluoroacetate. 

Lovelace, J., G.W. Miller, and G.W. Welkie. 1968. The accumulation of fluoroacetate and fluorocitrate in forage crops collected near a phosphate plant. Atmospher. Environ. 2 187-190. 

Savarie, P.J. 1984. Toxic characteristics of fluorocitrate, the toxic metabolite of compound 1080. Pages 132-137 in D.O. Clark (ed.). Proceedings Eleventh Vertebrate Pest Conference. Univ. California, Davis, CA. 

See, however, work on fluorocitrate to which fluoroacetate gives rise in the animal body (p. 142). 

Working with kidney homogenates from guinea-pigs, Peters has isolated an inhibitor containing a C—F link as the result of the enzymic action of kidney upon fumarate and fluoroacetate. This inhibitor is almost certainly fluorocitrate because... 

Aconitase, an unstable enzyme,4 is concerned with the reversible conversion of cis-aconitate to either citric acid or isocitric acid. It may be noted that the entire system of tricarboxylic cycle enzymes are present in the mitochondria separated from cells, and, furthermore, it has been found that the mitochondrial enzymes differ from the isolated enzymes in that the former require no addition of D.P.N. (co-enzyme I) or T.P.N. (co-enzyme II) for activity. Peters suggests that the citrate accumulation is caused by the competitive reaction of the fluorocitrate with aconitase required for the conversion of citrate to isocitrate. This interference with the tricarboxylic acid... 

The proof that fluorocitrate is indeed the toxic substance is shown by injecting 30 mg. of enzymically produced fluorocitrate within the skull of an anaesthetized pigeon. Some 10 min. after the bird came round, convulsions and death followed. On the other hand, an intracranial injection of fluoroacetate in larger amounts produces no convulsions. This fact indicates that brain tissue does not synthesize fluorocitrate from fluoroacetate, and suggests that convulsions occurring after intraperitoneal injections by fluoroacetate are due to penetration to the brain by fluorocitrate synthesized elsewhere. 

The above observations, taken together with the facts set out on pp. 133-6, support the view that fluoroacetate poisoning is brought about by the building up of FCH2COOH into fluorocitrate by enzymes in vivo. 

Finally, we may add that fluorocitrate interferes with fat metabolism in vivo, because it leads to rapid and marked urinary appearance of ketone bodies.1 Unlike fluoroacetate, intraperi-toneal fluorocitrate (20 mg./kg.) (synthetic), though increasing the citrate in the brain, produces no convulsions in 2 hr. 

Sherwood-Jones et al.2 have demonstrated the presence of the tricarboxylic acid cycle in mammalian reticulocytes by observing citrate accumulation in the presence of sodium fluoroacetate. They also demonstrated a substantial inhibition of respiration by fluorocitrate. 

The synthetic fluorocitric acid, to which reference has been made in the foregoing pages, has been prepared3 in low yield (12 per cent) by the following method from ethyl fluoroacetate 4... 

Such high selectivity is interesting, for it does not appear to be explainable solely on the basis of the incorporation of a fluorinated 2-carbon radical into fluorocitrate (see p. 141). 

Chenoweth believes that an explanation of the above results may lie in the reactions occurring before the entrance of fatty acid metabolites into the citric acid cycle. Activated acetate, i.e. acetyl coenzyme A (AcCoA) is the end-product of fatty acid metabolism prior to its condensation with oxalacetate to form citrate. Possibly fluoro-fatty acids behave like non-fluorinated fatty acids. The end-product before the oxalacetate condensation could be the same for all three fluorinated inhibitors, viz. fluoroacetyl coenzyme A (FAcCoA). Fluorocitrate could then be formed by the condensation of oxalacetate with FAcCoA, thereby blocking the citric acid cycle. The specificity of antagonisms must therefore occur before entrance of the metabolites into the citric acid cycle. 

The catalytic efficiency of this enzyme to hydrolyze 5-fluoro-5,6-dihydro-uracil was found to be approximately twice that toward 5,6-dihydrouracil [152], 2-Fluoro-/3-alanine can either be eliminated via the bile after conjugation with bile acids, or be converted to fluoroacetate (4.238) [153], The latter metabolite is transformed to fluorocitrate, a potent inhibitor of the aconi-tase-catalyzed conversion of citrate to isocitrate. This inhibition probably explains the clinical neurotoxicity of 5-fluorouracil [154] [155],... 

---