Production and utilization of phytoene, phytofluene, and lycopene tracers for bioavailability and biodistribution studies
Tomato product consumption is epidemiologically associated with a decreased risk of cardiovascular disease as well as several cancers, and is most strongly associated with decreased prostate cancer risk. Tomatoes contain a complex milieu of nutrients and bioactive compounds including the carotenoids lycopene (LYC), phytoene (PE), and phytofluene (PF). Much emphasis has been placed on the bioactivity of LYC, but emerging research suggests that the colorless carotenoids, PE and PF, are bioactive and significantly accumulate in human and animal model tissues. To elucidate the absorption and metabolism of these tomato carotenoids, an in vitro plant cell culture system for production of isotopically labeled carotenoids was developed, and absorption and biodistribution studies were performed in a relevant animal model, the Mongolian gerbil (Meriones unguiculatus). To enhance 14C-carotenoid production from tomato (Solanum lycopersicum cv. VFNT cherry) in vitro cell suspension cultures were treated with two bleaching herbicides during the culture incubation, 2-(4-chlorophenyl-thio) triethylamine and norflurazon separately or in combination, to produce varying ratios of PE, PF, and LYC (CPTA, 1:2:18; norflurazon, 11:5:1; combination, 6:1:4, respectively). Treatment with both herbicides resulted in optimal production of all three carotenoids (1.24 mg LYC/L, 1.74 mg PE/L, and 0.31 mg PF/L). Subsequently, cultures were incubated in [14C]-sucrose-containing media to produce labeled LYC, PE, and PF. The impact of the timing of14C-sucrose addition was evaluated in norflurazon-treated cultures, and adding [14C]-sucrose on day 1 of the 14-d culture incubation cycle to led to a small increase in labeling efficiency compared to adding it on day 7. In short, for optimal label incorporation, cultures should be grown with labeled carbohydrate for the duration of the growth cycle. Further, if primarily PE and PF are needed, norflurazon treatment should be used, for LYC accumulation CPTA should be administered, and to obtain a mixture of PE, PF, and LYC, cultures should be grown with a combination of CPTA and norflurazon. A second in vitro tomato cell culture study identified high LYC-producing cell line for [13C]-carotenoid production. Different Solanum lycopersicum allelic variants for high LYC and varying herbicide treatments were compared for carotenoid accumulation in callus and suspension culture. The gh tomato cell cultures produced more PE (3.5 mg/L) without the addition of norflurazon than that previously reported for VFNT cultures treated with norflurazon (2.1 mg/L), making it a preferred culture system for labeled PE production. When the hp-1cell cultures were treated with herbicides, they produced greater total carotenoids (3.6-5.2 mg/L) than the previously used VFNT cherry tomato cell line, and therefore hp-1 cell suspension culture system was chosen for isotopic labeling. When grown with [U]-[13C]-glucose and treated with CPTA for LYC production, hp-1 suspensions yielded highly enriched 13C-LYC, with 45% LYC in the M+40 form, and 88% in the M+35 to M+40 isotopomer range. In conclusion, the hp-1 cell line is preferable to other allelic variants or the VFNT cherry cell line for labeled tomato carotenoid production. Lastly, the gerbil was utilized as a model of tomato carotenoid absorption and bioavailability. The tomato carotenoids PE, PF, LYC, and zeta-carotene (ZC) were all absorbed from a 10% tomato powder diet and were differentially distributed to tissues at physiologically relevant levels compared to those previously reported for human tissues. Further, although LYC was much more concentrated in the tomato powder diet, the tomato carotenoids accumulated in tissues at similar concentrations. When unlabeled PE and LYC doses were provided to tomato powder-fed gerbils (LYC dose, 1.31 ± 0.05 mg in 206 ± 3 μL cottonseed oil; PE dose, 1.00 ± 0.01 mg in 202 ± 3 μL cottonseed oil), PE was more readily absorbed (serum PE increased by 570% versus serum Z-LYC 90% and E-LYC 40% at 6 hr post-dosing). PE dosing led to greater increases in liver PE at 6, 12, and 24 hours postdosing (81, 68, and 113%, respectively) compared to the respective increase in liver LYC after LYC dosing (-12, 20, and 0%, respectively). In conclusion, although the prominent tomato carotenoids structurally differ only by number of double bonds, absorption and bioaccumulation of these carotenoids differs substantially. These results warrant future studies into the differential bioavailability and metabolism of tomato carotenoids in mammals by utilizing isotopically labeled tracers.
Carotenoids, Phytoene, Phytofluene, lycopene, Tracers, Plant cell culture, Bioavailability, Absorption, Meriones unguiculatus
University of Illinois - Urbana-Champaign
Networked Digital Library of Theses and Dissertations
University of Illinois at Urbana-Champaign