Work

Intrinsic deuterium labeling of spinach: a hydroponic method to enrich isotopic labeling of vitamins E and K suitable for human bioavailability studies

Year: 2026

Type: article

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.

Source: Frontiers in Plant Science

DOI: 10.3389/fpls.2026.1915355