Metabolism in Rats in Viuo of RS-2-Methylbutyrate and n-Butyrate Labeled with Stable Isotopes at Various Positions

[l-‘3C]Butyric acid and four different racemic 2-methylbutyric acids labeled with 13C or ‘H at various positions were synthesized to study the mechanism of 2ethylmalonic acid formation as well as several other steps of the R pathway of isoleucine metabolism in rats in uiuo. These are [2-‘HI-, [methyZ-‘3C]-, [methyl-‘HJ-, and 2-methyl[l-‘3C]butyric acid. These labeled precursors were administered intraperitoneally to normal rats. Some of the rats were also treated with a small amount of hypoglycin (L-2-amino-(methylenecyclopropyl)propionic acid) to trap the butyrate intermediate as urinary N-butyrylglycine by partially inhibiting butyryl coenzyme A dehydrogenase. Urinary organic acids were analyzed by gas chromatography and gas chromatography-mass spectrometry to determine the amount of urinary excretion and isotope enrichment of metabolites from the labeled precursors. Four normal rats which were loaded with [l-l?]butyric acid excreted 54 to 460 pg of N-butyrylglycine which is not detectable in urine from normal rats without such loading or hypoglycin treatment. Both urinary ethylmalonic acid and butyrylglycine were significantly enriched either at one of the carboxyl carbons in the former or at the amide carbonyl in the latter, indicating that urinary ethylmalonic acid was formed by carboxylation of butyrate although the vast majority of butyrate given was oxidized by p oxidation. Since the 13C enrichment in ethylmalonic acid was about onethird lower than that of butyrylglycine, it was apparent that ethylmalonic acid was also produced from other sources. Urinary N-butyrylglycine excreted by two hypoglytin-treated rats after loading with RS-2-([‘%Imethyl)butyric acid was enriched with ‘C at the amide carbonyl while that excreted by two other hypoglycintreated rats after loading with RS-2-methyl[l-13C]butyric acid was not enriched. Urinary 2-ethylhydracrylic acid (2-ethyl-3-hydroxypropionic acid) was highly enriched structurally in both cases. These results indicate that the butyrate intermediate was produced by decarboxylation of 2-ethylhydracrylic acid, probably via 2-