Gas-chromatographic identification of fluoroorganic acids

Abstract

Gas chromatographic techniques are described for the separation and identification of fluoroacetate and fluorocitrate from fluoride-injured plant tissue and organs of animals suffering from severe fluorosis. Tissue was extracted with 9 5 z ethanol in a Soxhlet apparatus. The methyl esters of organic acids were separated on columns packed with 10% polyethylene glycol 6000 or 10% Reoplex 400 on Chromosorb W solid phase using a flame ionization detector. Evidence was presented for the occurrence of fluoroacetate and fluorocitrate in forage crops collected near a phosphate plant. The results obtained support the earlier findings suggesting that atmospheric HF may be incorporated into fluoroacetate and fluorocitrate by crested wheatgrass [Agropyron cristatum (L.) Gaerth]. luoroacetate and fluorocitrate have been shown to F occur in soybeans (Glycine max, Merr.) treated with atmospheric HF (Cheng, Yu, et al., 1968). More recently, forage crops collected near a phosphate plant were shown to contain these two compounds (Lovelace, Miller, et ai., 1968). Although these observations suggest that the biosynthesis of fluoroacetate and fluorocitrate may be a general property of plants, this has not yet been established. These analyses of the fluoroorganic compounds were conducted using paper chromatography, ashing, and fluoride determination. The concentrations reported for these compounds may be too high because of possible contamination with inorganic fluoride.Horses and cattle grazing near the above-mentioned industrial plant showed severe fluorosis. It was suspected that part of the injury might have been induced by the toxic fluoroorganic acids contained in the hay. A further investigation of the presence of fluoroorganic compounds in the organs of fluorotic animals was thus deemed of interest. Organs of these animals, control animals, crested wheatgrass growing near the phosphate plant, and control plants grown in a greenhouse were obtained and analyzed. ProceduresMethylfluoroacetate was prepared from monofluoroacetate (Na salt, Sigma Chemical Co.) by dissolving the latter in water, acidifying with 10% H2S04, extracting with sulfuric ether, drying the ethereal extract over anhydrous sodium sulfate, and methylating by the method of Colowick and Kaplan (1963).Trimethylfluorocitrate was prepared from monofluorocitrate (Ba salt, Calbiochem.) by dissolving the latter in 1 N HCl solution, and removing the barium by addition of NazSOl followed by centrifugation. The solution was then passed through a n ion-exchange column @owex 50-X, Hf form) and the eluate evaporated to dryness in a rotary evaporator at 40' C. The residue was taken up into methanol and esterified (Colowick and Kaplan, 1963). Treatment with ion-exchange column chromatography facilitated methylation of fluorocitrate and resulted in a higher yield than otherwise.Samples of crested wheatgrass [Agropyron cristatum (L.) Gaerth] were collected within 2 miles of a n industrial phosphate plant, air-dried, and ground in a mill. Plants grown in a greenhouse in the abse...

The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves

“…There are at least three possible reasons for this: (a) insensitivity of plant aconitase to fluorocitrate (31,32,45,49,50); (b) inability of either the thiokinase or citrate synthase to accept the fluoroderivative (16); and (c) defluorination of fluoroacetate (39,40,49,50). Plants, however, are capable of forming fluorocitrate, (32,49,50,55) and citrate does accumulate though to a lesser extent than in animal tissues (16,49, and this paper). The concentrations of fluoroacetate necessary to demonstrate aconitase inhibition will vary considerably depending upon the detoxification mechanism in operation.…”

Synthesis of fluoroacetate from fluoride, glycerol, and beta-hydroxypyruvate by Streptomyces cattleya

“…In this study, we show by 19 F nuclear magnetic resonance (NMR) spectroscopy that fluoroacetate is first produced and metabolized to 4-fluorothreonine by S. cattleya. We have shown by experiments of incorporation of [2-13 C]glycerol and [3- C]glycerol (0.37 GBq/mmol) and sodium [3-14 C]pyruvate (0.60 GBq/mmol) were from DuPont-NEN.…”

“…A few plants, Dichapetalum cymosum (6), Palicourea marcgravii (12), Acacia georginae (11,14,19), Gastrolobium grandiflorum (7), and Oxylobium species (1), are not only resistant to fluoroacetate but also synthesize it from fluoride at high concentrations. However, only speculative biosynthetic pathways of fluoroacetate in plants have been proposed, without evidence (8-10, 13, 18).…”

“…The culture broth (5 ml) was sampled at intervals, and mycelia were removed by centrifugation. The supernatant solution was concentrated under reduced pressure, dissolved in 0.5 ml of D 2 O (99.8%; CEA, Gif-sur-Yvette, France), and analyzed by 19 C]glycolate was purified by preparative HPLC as described below: the yield was 23%, on the basis of the recovery of radioactivity (2.16 MBq). The specific radioactivity was adjusted to 330 MBq/mmol by the addition of unlabeled glycolate.…”

“…The ether extract was analyzed by proton-decoupled 13 C NMR. Fluoroacetate was isolated by preparative HPLC as described above and analyzed by gas chromatography-mass spectrometry (GC-MS) and 1 H and 19 F NMR. GC-MS analysis.…”