Nutritional aspects of phytoene and phytofluene, carotenoid precursors to lycopene.

Epidemiological studies suggest an inverse relationship between tomato consumption and serum and tissue lycopene (LYC) levels with risk of some chronic diseases, including several cancers and cardiovascular disease. LYC, the red carotenoid found in tomatoes, is often considered to be the primary bioactive carotenoid in tomatoes that mediates health benefits, but other colorless precursor carotenoids, phytoene (PE) and phytofluene (PF), are also present in substantial quantities. PE and PF are readily absorbed from tomato foods and tomato extracts by humans. Animal models of carotenoid absorption suggest preferential accumulation of PE and PF in some tissues. The reasonably high concentrations of PE and PF detected in serum and tissues relative to the concentrations in foods suggest that absorption or metabolism of these compounds may be different from that of LYC. Experimental studies, both in vitro and in vivo, suggest that PE and PF exhibit bioactivity but little is known about their impact in humans. Methods for producing isotopically labeled PE, PF, and LYC tracers from tomato plant cell culture offer a unique tool for further understanding the differential bioavailability and metabolism of these 3 prominent tomato carotenoids and how they may affect health.

Screening and selection of high carotenoid producing in vitro tomato cell culture lines for [13C]-carotenoid production.

Isotopically labeled tomato carotenoids, phytoene, phytofluene, and lycopene, are needed for mammalian bioavailability and metabolism research but are currently commercially unavailable. The goals of this work were to establish and screen multiple in vitro tomato cell lines for carotenoid production, test the best producers with or without the bleaching herbicides, norflurazon and 2-(4-chlorophenyl-thio)triethylamine (CPTA), and to use the greatest carotenoid accumulator for in vitro 13C-labeling. Different Solanum lycopersicum allelic variants for high lycopene and varying herbicide treatments were compared for carotenoid accumulation in callus and suspension culture, and cell suspension cultures of the hp-1 line were chosen for isotopic labeling. When grown with [U]-13C-glucose and treated with CPTA, hp-1 suspensions yielded highly enriched 13C-lycopene with 45% of lycopene in the M+40 form and 88% in the M+35 to M+40 isotopomer range. To the authors' knowledge this is the first report of highly enriched 13C-carotenoid production from in vitro plant cell culture.

In Vitro Production of Radiolabeled Red Clover (Trifolium pratense) Isoflavones.

Red clover isoflavones are increasingly used in dietary supplements for their purported estrogenic effects. However, little is known about their metabolism in animals due to a lack of commercially available isotopically-labeled tracers. The goal of this research was to establish red clover cell culturing methodology for (14)C-biolabeling of isoflavones. When root, leaf, and petiole-derived suspension cultures were grown in darkness or light, dark-grown, petiole-derived solution cultures produced the highest concentrations of the two major red clover isoflavones, formononetin (0.67 mg/g FM inoculum) and biochanin A (0.13 mg/g FM inoculum). Varying levels and timing of copper chloride elicitor did not significantly affect isoflavone accumulation. Approximately 38% of the (14)C-sucrose dose accumulated in the cells. Eighteen percent of the initial labeled dose was detected in the isoflavone-rich methanolic extract and of that, 22% accumulated in isoflavones.

Herbicide treatments alter carotenoid profiles for 14C tracer production from tomato ( Solanum lycopersicum cv. VFNT cherry) cell cultures.

Progress in learning about underlying carotenoid bioactivity mechanisms has been limited because of the lack of commercially available radiolabeled lycopene (LYC), phytoene (PE), and phytofluene (PF). Tomato ( Solanum lycopersicum cv. VFNT cherry) cell cultures have been treated to produce [(14)C]-PE and PF but with relatively low yields. To increase carotenoid production, two bleaching herbicides were administered during the culture incubation, 2-(4-chlorophenyl-thio)triethylamine and norflurazon, separately or in combination to produce varying ratios of PE, PF, and LYC. Treatment with both herbicides resulted in optimal production of all three carotenoids. Subsequently, cultures were incubated in [(14)C]-sucrose-containing media to produce labeled LYC, PE, and PF. Adding [(14)C]-sucrose on day 1 of the 14 day culture incubation cycle to norflurazon-treated cultures led to a small increase in labeling efficiency compared to adding it on day 7. Improved culture conditions efficiently provided sufficient (14)C-carotenoids for future cell culture and animal metabolic tracking studies.

Optimization of lycopene extraction from tomato cell suspension culture by response surface methodology.

Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.

Phytoene, Phytofluene, and Lycopene from Tomato Powder Differentially Accumulate in Tissues of Male Fisher 344 Rats.

Tomato product consumption is inversely related to prostate cancer incidence, and lycopene (LYC) has been implicated in reduced prostate cancer risk. The contribution of other tomato carotenoids, phytoene (PE) and phytofluene (PF), towards prostate cancer risk has not been adequately studied. The relative uptake and tissue distribution of tomato carotenoids are not known. We hypothesize that PE and PF are bioavailable from a tomato powder diet or from a purified source and accumulate in androgen-sensitive tissues. In this study, 4 wk old male Fisher 344 rats were pre-fed an AIN-93G powder diet composed of 10% tomato powder containing PE, PF, and LYC (0.015, 0.012, and 0.011 g/kg diet, respectively). After 30 d tomato powder feeding, hepatic PF concentrations (168 ± 20 nmol/g) were higher than PE or LYC (104 ± 13 and 104 ± 13 nmol/g, respectively). In contrast, LYC, followed by PF, had the highest accumulation of the measured carotenoids in the prostate lobes and seminal vesicles. When tomato powder-fed rats received a single oral dose of either ∼2.7 mg PE or PF, an increase in the dosed carotenoid concentration was observed in all measured tissues, except the adrenal. Percent increases of PF were greater than that of PE in liver, serum, and adipose (37, 287 and 49% versus 16, 179 and 23%, respectively). Results indicate that the relative tomato carotenoid biodistribution differs in liver and androgen-sensitive tissues, suggesting that minor changes in the number of sequential double bonds in carotenoid structures alter absorption and/or metabolism of tomato carotenoids.