Characterisation of bound residues of [3H]triforine in barley grain grown in the field

Barley was grown in an experimental field and at growth stage J (during the stem extension stage when the second node of the stem was formed and the next-to-last leaf was just visible), the aerial part of the plants was sprayed with an aqueous emulsion of a mixture of triforine and [³H]triforine (uniformly labelled in the piperazine ring) at the normal rate of 250 g triforine ha^?1. The barley was harvested when ripe, and the grain was analysed separately. Extraction of the grain with methanol left methanol-insoluble solids containing an amount of radioactivity (the bound residue) which represented 75% of the total radioactivity incorporated into the grain. Methanol acidified with hydrochloric acid extracted a further 7% of the triforine-derived bound residues in the form of radioactive iminodiacetic acid (1.1%), glycine (3.3 %), serine (0.9%), ethanolamine (0.2%) and unidentified compounds (1.5 %); in the grain, these compounds or their precursors had thus been complexed to grain constituents. Aqueous 0.03M sodium hydroxide extracted a further 27% of the total tritium which had been incorporated by means of α chemical bonds into the protein fraction; acid hydrolysis of the proteins yielded radioactive glycine (9.2%), serine (3.9%) and unidentified compounds (13.9%) which could have been a mixture of a large number of other amino-acids. The plant solids (which contained 41% of the total tritium) left after the alkaline aqueous extractions, were processed and separated into tritiated cellulose (4%) and starch (37%) fractions. The starch was hydrolysed and the resulting glucose was converted into the osazone (34%). After being recrystallised several times, the osazone contained a constant specific radioactivity, indicating that [³H]glucose was present. This glucose may be considered as having a carbon skeleton mainly originating biochemically from some metabolites of [³H]triforine; this tritiated glucose may also be considered as indirect evidence of the biochemical oxidation, by transamination, of the metabolites of [³H]triforine; in barley grain, the tritiated glucose (or at least a part of it) was anabolised into proteins. No piperazine was observed in the bound residues in the grain.