ARS | Publication request: STRUCTURE DETERMINATION OF PARTIALLY DEUTERATED CAROTENOIDS FROM INTRINSICALLY LABELED VEGETABLES BY HPLC-MS AND PROTON-NMR

Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 5, 2004
Publication Date: February 5, 2005
Citation: Putzbach, K., Krucker, M., Albert, K., Grusak, M.A., Tang, G., Dolnikowski, G.G. 2005. Structure determination of partially deuterated carotenoids from intrinsically labeled vegetables by hplc-ms and proton-nmr. Journal of Agriculture and Food Chemistry. 53 (3), 671 -677. Available at http://pubs.acs.org/cgi-bin/article.cgi/jafcau/2005/53/i03/pdf/jf0487506.pdf

Interpretive Summary: We have grown vegetables hydroponically in heavy water to create vegetables labeled with deuterium. These vegetables can be fed to human subjects because they are not radioactive. When they are fed to human subjects all the nutrients in the vegetables can be tracked as they are absorbed, metabolized and excreted by the body because they are all labeled with deuterium. Until now nobody knew the position of the deuterium labels on the nutrient molecules. The position of these deuterium labels is important information, because as the nutrient molecules in the vegetables are changed by the body, we want to be certain that the deuterium labels are not lost. We have employed two sophisticated analytical scientific instruments (a mass spectrometer and a nuclear magnetic resonance spectrometer) to determine the positions of the deuterium labels on lutein and beta-carotene from spinach, and alpha-carotene and beta-carotene from carrots. We determined that the deuterium labels were located randomly throughout all the nutrient molecules that we studied. This is important information, because we are now certain that even if some of the deuterium labels are lost from a labeled nutrient by metabolism in the body, there will be other labels in different positions that will not be lost. This will enable us to track the labeled nutrients as they move through the human body.

Technical Abstract: The structures of biosynthetic deuterated carotenoids in labeled vegetables were investigated: (all-E)-lutein and (all-E)-beta-carotene from spinach, and (all-E)-beta-carotene and (all-E)-alpha-carotene from carrots. The vegetables were grown hydroponically using a nutrient solution enriched with deuterium oxide (D2O) and were extracted using matrix solid-phase dispersion (MSPD). Deuterium enrichment in the carotenoid molecules was determined by liquid chromatography-mass spectrometry (LC-MS). (All-E)-lutein and (all-E)-beta-carotene in spinach showed partial deuteration from D1 to D12, with the abundance maximum at D5. (All-E)-beta-carotene and (all-E)-alpha-carotene from carrots showed partial deuteration from D1 to D17, with the abundance maximum at D17. The proton-NMR spectra of the four deuterated carotenoids showed additional signals for all methyl groups and decreased signal intensity for the olefinic protons and the methylene protons in the ring. These differences are due to isotopic effects and are based on the substitution of protons by deuterium atoms. The deuteration was distributed randomly throughout the carotenoid molecules.