Stable‐isotope method for determining the gastrointestinal handling of [1‐13C]Palmitic acid

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Abstract
The 13C enrichment in individual fatty acids extracted from human feces following the oral administration of [1-13C]palmitic acid has been determined using a novel approach based upon gas chromatography-isotope ratio mass spectrometry. The method was established and tested for precision and repeatability. Analytical precision was determined from 10 repeated injections of a sample containing 16∶0 and 18∶0 with levels of δ13C abundance measured at −34.01±0.60 and −23.62±0.95 delta per mil (parts per thousand) (‰), respectively (mean±SD). For the repeatability study, measurement of enrichment of the same mixture of unlabeled fatty acid methyl ester (FAME) standards (13∶0, 14∶0, 16∶0, and 18∶0) was found to have standard deviations (0.45, 0.56, 1.46 and 1.54‰ respectively). When labeled [1-13C]palmitic acid was serially diluted with naturally enriched palmitic acid, a linear relationship was obtained to a dilution of 10% enriched compound (530‰). FAME were prepared from two fecal samples from a normal healthy adult; the first, a baseline specimen, containing no added label and the second, followed a single oral dose of [1-13C]palmitic acid and was enriched. Enrichment in 13C was confined to the solvent-soluble fraction following lipid extraction, and was only identified with prior acidification. The enrichments were measured in triplicate, baseline sample −32.66±0.5‰ enriched sample +268.61±8.0‰ Enrichment was restricted to the labeled species consumed, 16∶0. The methodology described here allows for the separation of compounds prior to the determination of enrichment and can be utilized to contribute to a more complete description of the gastrointestinal handling of labeled substrates than previously obtained.