Production of Uniformly Labeled 13C‐Lutein and 13C‐α‐Tocopherol in vitro Using Carrot Cell Suspension Culture

Abstract

The xanthophyll lutein (Lut) is present in high concentrations in primate retinae, protecting against photooxidation and enhancing visual performance. Interestingly, in the elderly, diets high in Lut slow cognitive decline and circulating levels of both Lut and the lipid‐soluble antioxidant α‐tocopherol (αT) are inversely associated with cognitive impairment. However, Lut deposition, metabolism, and function in the brain are poorly understood, and cost‐effective isotopically labeled Lut tracers are not commercially available. The goal of this work, therefore, is to establish a system for efficient bioproduction of uniformly labeled 13C‐Lut and 13C‐αT. Seven carrot (Daucus carota) cultivars were germinated, separated into tissue subtypes, dedifferentiated into solid callus, and initiated in liquid suspension culture. Conditions for production of Lut and αT in suspension culture were optimized and uniformly labeled 13C‐glucose was provided as the sole media carbon source for four serial growth cycles. Lut and αT yields were 2.6 and 3.3 μg/g cells, respectively, as measured by reverse‐phase HPLC. Cell extracts were fractionated by preparatory reverse‐phase HPLC and analyzed for 13C enrichment by mass spectrometry. 58% of Lut and 50% of αT in carrot extract fractions were uniformly labeled with 13C. These tracers can be utilized in studies of Lut and αT in the brain, enhancing our understanding of their function in cognition.