Sulphur tracer experiments in laboratory animals using 34S-labelled yeast
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We have evaluated the use of 34S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the 34S-labelled yeast to laboratory animals to follow the fate and distribution of 34S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of 34S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.
KeywordsSulphur metabolism Metabolic tracer Sulphur-labelled yeast HPLC-ICP-MS Urine
This work was supported by the Ministry of Science and Innovation, Madrid, Spain (project CTQ2009-12814) and the Education and Science Council of the Principado de Asturias (grant BP07-059). Teresa Fernández and Agustín Brea from the Biotery of the University of Oviedo are gratefully acknowledged for their help and kind suggestions. The authors thank Rafael Peláez for his assistance in the experimental work regarding gels.
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