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Nutritional selenium compounds are transformed to the common intermediate selenide and then utilized for selenoprotein synthesis or excreted in urine mostly as 1β-methylseleno-N-acetyl-Dd-galactosamine (selenosugar). Since the biological significance of selenosugar formation is unknown, we investigated their role in the formation of selenoenzymes in selenium deficiency. Rats were depleted of endogenous natural abundance selenium with a single stable isotope (82Se) and then made Se-deficient. 76Se-Selenosugar was administered intravenously to the rats and their urine, serum, liver, kidneys and testes were subjected to speciation analysis with HPLC inductively coupled argon plasma mass spectrometry. Most 76Se was recovered in its intact form (approximately 80% of dose) in urine within 1 h. Speciation analysis revealed that residual endogenous natural abundance selenium estimated by 77Se and 78Se was negligible and distinct distributions of the labeled 76Se were detected in the body fluids and organs without interference from the endogenous natural abundance stable isotope. Namely, intact 76Se-selenosugar was distributed to organs after the injection, and 76Se was used for selenoprotein synthesis. Oxidation to methylseleninic acid and/or hydrolysis of the selenoacetal group to methylselenol were proposed to the transformation of selenosugar for the reuse. Effective use of an enriched stable isotope as an absolute label in hosts depleted of natural abundance isotopes was discussed for application in tracer experiments.