Bioaccumulation and isotopomer distribution of ±-tocopherol in deuterium-labeled spinach: A tool for bioavailability studies in humans

Shahabeddin Rezaei
Graduate (PhD)
Richard S. Bruno (OSU Nutrition Program)

Introduction: Most Americans have inadequate intakes and circulating concentrations of vitamin E (±-tocopherol; ±-T). While strategies to improve its bioavailability could help achieve ±-T nutriture, accurate measures of ±-T bioavailability from ±-T-rich dietary sources requires the development of isotopically-labeled plants. Our objective was to establish deuterium-labeled spinach as a tool to study of ±-T bioavailability in humans. We hypothesized that: (1) ±-T in spinach would preferentially bioaccumulate in the leaf compared with the petiole and (2) maturation of spinach under deuterium oxide conditions would generate an isotopomer distribution of ±-T suitable for human bioavailability studies.
Materials and Methods: Spinach was grown hydroponically using growth media. In the first experiment, spinach was harvested at 4-weeks post-germination. HPLC-ECD was performed to quantify -T bioaccumulation in raw and cooked leaves and petioles. In the second experiment, spinach was grown in the growth media containing 30% deuterium oxide prior to harvesting at 6-weeks post-germination. Targeted LC-MS studies were conducted to assess the isotopomer profile of ±-T in spinach at 6-weeks post-germination. 
Results: ±-T bioaccumulated ~5-times greater in spinach leaf tissue compared with petiole tissue regardless of cooking. Isotopomer studies indicated that 100% of ±-T in spinach was deuterium-labeled, and that 66% of all deuterated ±-T was hexa- (d6-) to deca- (d10)-deuterium labeled ±-T (mass-to-charge ratios, 435 to 439).  
Conclusions and Discussion: These findings indicate that deuterium-labeled spinach is isotopically enriched in a manner suitable for clinical bioavailability studies, and that ±-T leaves are the most appropriate plant tissue for use in these future human studies.
Acknowledgments: Funded by Egg Nutrition Center