Submitted to: United States-Japan Cooperative Program in Natural Resources
Publication Type: Proceedings
Publication Acceptance Date: 10/3/2003
Publication Date: 11/9/2003
Citation: Britz, S.J., Kurilich, A.C., Novotny, J., Clevidence, B.A. 2003. 13C Labeling of Plants to Trace Phytochemical Uptake and Metabolism in Humans. Proceedings of the United States-Japan Cooperative Program in Natural Resources. November 9, 2003. 360-364. Interpretive Summary: Labeling is often required to assess the availability of compounds and to identify metabolites arising from human or microbial biochemistry. Since dietary phytochemicals can be retained in our bodies for days after absorption, it can be difficult to determine the amount of a given compound that derives from a particular feeding unless the compound is labeled. Labeling has also shown that some compounds are completely metabolized by microbial action in the gut and not taken up at all. Labeling has been done typically using radioactive or stable isotopes. Stable isotopes are generally more abundant, so it can be expensive to generate compounds with adequate label to distinguish them above background. Radioisotopes have been used with humans, but restrictions have sometimes resulted in critical information being obtained from a small number of potentially compromised subjects. Accelerator mass spectrometry, an extremely sensitive method to measure rare isotopes, should expand the use of radioisotopes for human study, but the technique is still not widely available. Stable isotopes have often been used to assess nutrient availability and their safety is established (e.g., galactose uptake into babies via breast milk). Therefore, we developed a general method to label phytochemicals with 13C, a stable isotope of carbon, at high specific activity and to apply mass spectroscopic methods for their analysis. The initial results of this work have recently been published. This information will be useful to scientists and nutritionists evaluating human dietary requirements.
Technical Abstract: To determine the bioavailability of phytonutrients from whole foods, a method was developed for the complete and intrinsic labeling of kale (Brassica oleracea acephala cultivar Vates) with 3C, a stable isotope of carbon, based on photosynthetic assimilation of 13C-CO2 (99% enrichment) supplied in a gas-tight acrylic chamber. The plants grew well, indicating that isotope discrimination was not a problem. Furthermore, the kale adapted to continuous illumination, which accelerated growth and eliminated the need to recover respired 13C-CO2. Over 1.5 kg (fresh weight) of labeled material was harvested in each of two separate runs lasting 7 and 9 days, respectively (harvests 15 and 17 days after sowing). Beta-carotene, lutein, and phylloquinone were isolated from the labeled kale and found to contain about 98% 13C. We believe this is the first report of the almost complete labeling of a plant with a stable isotope. Labeled kale was given to a human volunteer in a single bolus feeding who was then bled periodically for 46 days. Labeled phytonutrients were detectable in plasma from the subject for 46 days (beta-carotene), 28 days (lutein) and 7 days (phylloquinone). Peak levels in plasma were detected 8 and 24 h (beta-carotene), 11 h (lutein) and 7 h (phylloquinone) after feeding. Labeled retinol was also measured in plasma, peaking at 24 h (comparable to the second peak of beta-carotene) and detectable to 46 days (also similar to beta-carotene). The retinol presumably derived from beta-carotene, which was the only provitamin A source in the kale. The production of high specific activity phytochemicals greatly improves the analysis of these phytonutrients by mass spectrometry by reducing the number of "isotopomers" with a variable number and positioning of stable isotopic atoms. The ability to track metabolites will also be improved if labeled compounds are isolated and fed in pure form.