Intrinsic deuterium labeling of spinach: a hydroponic method to enrich isotopic labeling of vitamins E and K suitable for human bioavailability studies
- 1Human Nutrition Program, Department of Human Sciences, The Ohio State University
- 2Department of Horticulture and Crop Science, The Ohio State University
- 3Laboratory for the Analysis of Metabolites from Plants (LAMP), The Ohio State University
Abstract
Introduction: Intrinsic stable isotope labeling enables assessment of nutrient bioavailability from intact plant food matrices by distinguishing nutrients derived from the test food from existing body pools. Although deuterium oxide (D ₂ O) has been used to intrinsically label green leafy vegetables under hydroponic growth conditions, well-characterized protocols for static hydroponic systems remain limited.
Methods: This study established and evaluated a novel hydroponic D ₂ O-labeling protocol using spinach to produce intrinsically labeled α-tocopherol (α-T) and phylloquinone (VK1). Three independent experiments were conducted in a controlled-environment growth chamber. Spinach seedlings were transferred to a static hydroponic system containing 0% (control) or nominal 30% (v/v) D ₂ O nutrient solution. Plants grown under controlled environmental conditions were harvested at Weeks 4, 5, and 6 to evaluate leaf biomass, nutrient content, and isotopomer distributions.
Results: No labeling condition × harvest time interactions occurred for leaf biomass, α-T content, or VK1 content ( p > 0.05). Leaf biomass, α-T content, and VK1 content increased from Week 4 to Week 6 ( p < 0.05) but were unaffected by labeling condition ( p > 0.05). Compared with non-D ₂ O-grown spinach, D ₂ O-grown spinach demonstrated significantly higher relative abundance of the summed d ₃ –d ₂₀ mass isotopomers for α-T (98.7–99.0% vs 3.6–4.6%; p = 0.0002) and VK1 (97.0–97.5% vs 1.8–2.1%; p = 0.0008). The dominant labeled isotopomers were d ₉ for α-T and d ₈ –d ₉ for VK1. Inter-experiment coefficients of variation for the summed d ₃ –d ₂₀ relative abundance ranged from 0.1–0.3% for α-T and 0.1–0.4% for VK1 across harvest times.
Discussion: These findings provide a detailed framework for a static hydroponic D ₂ O-labeling method that produced similar α-T and VK1 isotopomer distributions across three experiments conducted in the same laboratory, with potential application in future bioavailability and metabolic tracing research.
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BibTeX
@article{rezaei2026intrinsic,
title = {{Intrinsic deuterium labeling of spinach: a hydroponic method to enrich isotopic labeling of vitamins E and K suitable for human bioavailability studies}},
author = {Shahabeddin Rezaei and Sisi Cao and Min Zeng and Maria L. Antonius and Allyson Q. McHenry and Richard S. Bruno and Joshua J. Blakeslee},
journal = {Frontiers in Plant Science},
year = {2026},
month = oct,
doi = {10.3389/fpls.2026.1915355},
url = {https://www.frontiersin.org/articles/10.3389/fpls.2026.1915355/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Intrinsic deuterium labeling of spinach: a hydroponic method to enrich isotopic labeling of vitamins E and K suitable for human bioavailability studies
AU - Shahabeddin Rezaei
AU - Sisi Cao
AU - Min Zeng
AU - Maria L. Antonius
AU - Allyson Q. McHenry
AU - Richard S. Bruno
AU - Joshua J. Blakeslee
PY - 2026
DA - 2026/10/14
JO - Frontiers in Plant Science
DO - 10.3389/fpls.2026.1915355
UR - https://www.frontiersin.org/articles/10.3389/fpls.2026.1915355/full
AB - Introduction: Intrinsic stable isotope labeling enables assessment of nutrient bioavailability from intact plant food matrices by distinguishing nutrients derived from the test food from existing body pools. Although deuterium oxide (D ₂ O) has been used to intrinsically label green leafy vegetables under hydroponic growth conditions, well-characterized protocols for static hydroponic systems remain limited. Methods: This study established and evaluated a novel hydroponic D ₂ O-labeling protocol using spinach to produce intrinsically labeled α-tocopherol (α-T) and phylloquinone (VK1). Three independent experiments were conducted in a controlled-environment growth chamber. Spinach seedlings were transferred to a static hydroponic system containing 0% (control) or nominal 30% (v/v) D ₂ O nutrient solution. Plants grown under controlled environmental conditions were harvested at Weeks 4, 5, and 6 to evaluate leaf biomass, nutrient content, and isotopomer distributions. Results: No labeling condition × harvest time interactions occurred for leaf biomass, α-T content, or VK1 content ( p > 0.05). Leaf biomass, α-T content, and VK1 content increased from Week 4 to Week 6 ( p < 0.05) but were unaffected by labeling condition ( p > 0.05). Compared with non-D ₂ O-grown spinach, D ₂ O-grown spinach demonstrated significantly higher relative abundance of the summed d ₃ –d ₂₀ mass isotopomers for α-T (98.7–99.0% vs 3.6–4.6%; p = 0.0002) and VK1 (97.0–97.5% vs 1.8–2.1%; p = 0.0008). The dominant labeled isotopomers were d ₉ for α-T and d ₈ –d ₉ for VK1. Inter-experiment coefficients of variation for the summed d ₃ –d ₂₀ relative abundance ranged from 0.1–0.3% for α-T and 0.1–0.4% for VK1 across harvest times. Discussion: These findings provide a detailed framework for a static hydroponic D ₂ O-labeling method that produced similar α-T and VK1 isotopomer distributions across three experiments conducted in the same laboratory, with potential application in future bioavailability and metabolic tracing research.
SN - 1664-462X
ER -