Carrot solution culture bioproduction of uniformly labeled 13C-lutein and in vivo dosing in non-human primates.

Lutein is a xanthophyll abundant in nature and most commonly present in the human diet through consumption of leafy green vegetables. With zeaxanthin and meso-zeaxanthin, lutein is a component of the macular pigment of the retina, where it protects against photooxidation and age-related macular degeneration. Recent studies have suggested that lutein may positively impact cognition throughout the lifespan, but outside of the retina, the deposition, metabolism, and function(s) of lutein are poorly understood. Using a novel botanical cell culture system ( Daucus carota), the present study aimed to produce a stable isotope lutein tracer for use in future investigations of dietary lutein distribution and metabolism. Carrot cultivars were initiated into liquid solution culture, lutein production conditions optimized, and uniformly labeled 13C-glucose was provided as the sole media carbon source for four serial growth cycles. Lutein yield was 2.58 ± 0.24 µg/g, and mass spectrometry confirmed high enrichment of 13C: 64.9% of lutein was uniformly labeled and 100% of lutein was labeled on at least 37 of 40 possible carbons. Purification of carrot extracts yielded a lutein dose of 1.92 mg with 96.0 ± 0.60% purity. 13C-lutein signals were detectable in hepatic extracts of an adult rhesus macaque monkey ( Macaca mulatta) dosed with 13C-lutein, but not in hepatic samples collected from control animals. This novel botanical biofactory approach can be used to produce sufficient quantities of highly enriched and pure 13C-lutein doses for use in tracer studies investigating lutein distribution, metabolism, and function.

Absorption and Distribution Kinetics of the 13C-Labeled Tomato Carotenoid Phytoene in Healthy Adults.

BACKGROUND: Phytoene is a tomato carotenoid that may contribute to the apparent health benefits of tomato consumption. Although phytoene is a less prominent tomato carotenoid than lycopene, it is a major carotenoid in various human tissues. Phytoene distribution to plasma lipoproteins and tissues differs from lycopene, suggesting the kinetics of phytoene and lycopene differ. OBJECTIVE: The objective of this study was to characterize the kinetic parameters of phytoene absorption, distribution, and excretion in adults, to better understand why biodistribution of phytoene differs from lycopene. METHODS: Four adults (2 males, 2 females) maintained a controlled phytoene diet (1-5 mg/d) for 42 d. On day 14, each consumed 3.2 mg (13)C-phytoene, produced using tomato cell suspension culture technology. Blood samples were collected at 0, 1-15, 17, 21, and 24 h and 2, 3, 4, 7, 10, 14, 17, 21, and 28 d after (13)C-phytoene consumption. Plasma-unlabeled and plasma-labeled phytoene concentrations were determined using ultra-HPLC-quadrupole time-of-flight-mass spectrometry, and data were fit to a 7-compartment carotenoid kinetic model using WinSAAM 3.0.7 software. RESULTS: Subjects were compliant with a controlled phytoene diet, consuming a mean ± SE of 2.5 ± 0.6 mg/d, resulting in a plasma unlabeled phytoene concentration of 71 ± 14 nmol/L. A maximal plasma (13)C-phytoene concentration of 55.6 ± 5.9 nM was achieved 19.8 ± 9.2 h after consumption, and the plasma half-life was 2.3 ± 0.2 d. Compared with previous results for lycopene, phytoene bioavailability was nearly double at 58% ± 19%, the clearance rate from chylomicrons was slower, and the rates of deposition into and utilization by the slow turnover tissue compartment were nearly 3 times greater. CONCLUSIONS: Although only differing from lycopene by 4 double bonds, phytoene exhibits markedly different kinetic characteristics in human plasma, providing insight into metabolic processes contributing to phytoene enrichment in plasma and tissues compared with lycopene. This trial was registered at clinicaltrials.gov as NCT01692340.

Biosynthesis of highly enriched 13C-lycopene for human metabolic studies using repeated batch tomato cell culturing with 13C-glucose.

While putative disease-preventing lycopene metabolites are found in both tomato (Solanum lycopersicum) products and in their consumers, mammalian lycopene metabolism is poorly understood. Advances in tomato cell culturing techniques offer an economical tool for generation of highly-enriched (13)C-lycopene for human bioavailability and metabolism studies. To enhance the (13)C-enrichment and yields of labelled lycopene from the hp-1 tomato cell line, cultures were first grown in (13)C-glucose media for three serial batches and produced increasing proportions of uniformly labelled lycopene (14.3±1.2%, 39.6±0.5%, and 48.9±1.5%) with consistent yields (from 5.8 to 9 mg/L). An optimised 9-day-long (13)C-loading and 18-day-long labelling strategy developed based on glucose utilisation and lycopene yields, yielded (13)C-lycopene with 93% (13)C isotopic purity, and 55% of isotopomers were uniformly labelled. Furthermore, an optimised acetone and hexane extraction led to a fourfold increase in lycopene recovery from cultures compared to a standard extraction.

Tracking deposition of a 14C-radiolabeled kudzu hairy root-derived isoflavone-rich fraction into bone.

Hairy roots were induced in four genotypes from three kudzu species (Pueraria montana var. lobata, P. lobata and P. phaseoloides) in vitro using Agrobacterium rhizogenes to stimulate rapid secondary metabolite synthesis. Hairy roots from P. montana var. lobata (United States Department of Agriculture no. PI 434246) yielded the highest puerarin and total isoflavone content and the greatest new biomass per growth cycle among the genotypes evaluated. Hairy roots from this genotype were selected for radiolabeling using (14)C-sucrose as a carbon source. Isoflavones from radiolabeled kudzu hairy root cultures were extracted with 80% methanol, partitioned by solvent extraction, and then subfractionated by Sephadex LH-20 gel filtration. Radiolabeled isoflavones were isolated in a highly enriched fraction, which contained predominantly puerarin, daidzin and malonyl-daidzin and had an average radioactivity of 8.614 MBq/g (232.8 µCi/g) dry fraction. The (14)C-radiolabeled, isoflavone-rich fraction was orally administered at a dose of 60 mg/kg body weight to male Sprague-Dawley rats implanted with a jugular catheter, a subcutaneous ultrafiltrate probe and a brain microdialysate probe. Serum, interstitial fluid, brain microdialysate, urine and feces were collected using a Culex(®) Automated Blood Collection System for 24 h. At the end of this period, rats were sacrificed and major tissues were collected. Analysis by a scintillation counter confirmed that a bolus dose of (14)C-radiolabeled, isoflavone-rich kudzu fraction reached bone tissues, which accumulated 0.011%, 0.09% and 0.003% of the administered dose in femur, tibia and vertebrae, respectively. Femurs extracted with 80% methanol were analyzed by high-performance liquid chromatography with electrospray ionization-mass spectrometry and were found to contain trace quantities of puerarin, daidzein and puerarin glucuronide. This study demonstrates that kudzu isoflavones and metabolites are capable of reaching bone tissues, where they may contribute to the prevention of osteoporosis and the promotion of bone 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.

Pharmacokinetics and tissue distribution of 14C-labeled grape polyphenols in the periphery and the central nervous system following oral administration.

Grape polyphenols confer potential health benefits, including prevention of neurodegenerative diseases. To determine the absorption and tissue distribution of the complex grape polyphenol mixture, (14)C-labeled polyphenols were biosynthesized by grape cell suspension cultures, during co-incubation with radioisotopically labeled sucrose, and fractionated into polyphenolic subfractions. The pharmacokinetics and distribution of grape polyphenols into blood, brain, and peripheral interstitial fluid were determined by tracking the (14)C label. The blood peak (14)C concentration of the fractions ranged from 15 minutes to 4 hours. Absorption and tissue distribution varied greatly between fractions. Concentrations in interstitial fluid were lower than in blood. The amount of residual label in the brain at 24 hours ranged from 0.1% to 1.7% of the dose, depending on the fraction. (14)C label found in the brain tissue and brain microdialysate indicated that grape polyphenols or their metabolites are able to cross the blood-brain barrier. Using (14)C-labeled plant polyphenols it is possible to track the compounds or their metabolic products into any tissue and determine distribution patterns in spite of low concentrations. A central question regarding the potential role of dietary polyphenolics in neurodegenerative research is whether they are bioavailable in the brain. Our observations indicate that some grape-derived polyphenolics do reach the brain, which suggests their potential value for applications in neurodegenerative disorders.

Method for evaluating the potential of C labeled plant polyphenols to cross the blood-brain barrier using accelerator mass spectrometry.

Bioactive compounds in botanicals may be beneficial in preventing age-related neurodegenerative diseases, but for many compounds conventional methods may be inadequate to detect if these compounds cross the blood brain barrier or to track the pharmacokinetics in the brain. By combining a number of unique technologies it has been possible to utilize the power of AMS to study the pharmacokinetics of bioactive compounds in the brain at very low concentrations. (14)C-labeled compounds can be biosynthesized by plant cell suspension cultures co-incubated with radioisotopically-labeled sucrose and isolated and separated into a series of bioactive fractions.To study the pharmacokinetics and tissue distribution of (14)C labeled plant polyphenols, rats were implanted with jugular catheters, subcutaneous ultrafiltration probes and brain microdialysis probes. Labeled fractions were dosed orally. Interstitial fluid (ISF) and brain microdialysate samples were taken in tandem with blood samples. It was often possible to determine (14)C in blood and ISF with a ß-counter. However, brain microdialysate samples (14)C levels on the order of 10(7) atoms/sample required AMS technology. The Brain Microdialysate(AUC)/Serum(AUC) ranged from .021- to .029, with the higher values for the glycoside fractions. By using AMS in combination with traditional methods, it is possible to study uptake by blood, distribution to ISF and determine the amount of a dose which can reach the brain and follow the pharmacokinetics in the brain.

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.

In vivo metabolic tracking of 14C-radiolabelled isoflavones in kudzu (Pueraria lobata) and red clover (Trifolium pratense) extracts.

Absorption, distribution and elimination of 14C-labelled isoflavone-containing extracts from kudzu (Pueraria lobata) root culture and red clover (Trifolium pratense) cell culture were investigated in an in vivo rat model. The predominant isoflavones in the kudzu extract were the glycosides puerarin, daidzin and malonyl daidzin, while in the red clover extract, the major isoflavones were formononetin and its derivatives, genistein and biochanin A, with radioactivities of 3.770 and 7.256 MBq/g, respectively. Male Sprague-Dawley rats, implanted with a jugular catheter and a subcutaneous ultrafiltrate probe, were orally administered with 14C-labelled isoflavone extracts from either kudzu or clover cell cultures. Serum, interstitial fluid (ISF), urine and faeces were collected using a Culex Automated Blood Collection System for 24 h. Analysis of bone tissues revealed that radiolabel accumulated in the femur, tibia and vertebrae at 0.04, 0.03 and 0.01 % of the administered dose, respectively, in both kudzu and red clover treatments. The liver accumulated the greatest concentration of radiolabel among the tissues tested, at 1.99 and 1.54 % of the administered kudzu and red clover extracts, respectively. Serum and ISF analysis showed that both extracts were rapidly absorbed, distributed in various tissues, and largely eliminated in the urine and faeces. Urine and faeces contained 8.53 and 9.06 % of the kudzu dose, respectively, and 3.60 and 5.64 % of the red clover dose, respectively. Serum pharmacokinetics suggest that extracts from kudzu may undergo enterohepatic circulation.

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.

Isolation of radiolabeled isoflavones from kudzu (Pueraria lobata) root cultures.

Isoflavones have potential for preventing and treating several chronic health conditions, such as osteoporosis, cardiovascular disease, and cancer. In this study, radiolabeled isoflavones were recovered from kudzu (Pueraria lobata) root cultures after incubation with uniformly labeled (14)C-sucrose in the culture medium for 21 days. Approximately 19% of administered label was recovered in the isoflavone-rich dried extracts of kudzu root cultures (90.2 microCi/g or 3.3 MBq/g extract). HPLC-PDA analysis revealed the predominant isoflavones isolated from kudzu root cultures to be puerarin, daidzin, and malonyl-daidzin. The average concentration of the major isoflavone puerarin in kudzu root cultures was 33.6 mg/g extract, with a specific activity of 63.5 microCi/g (2.3 MBq/g). The isolated isoflavones were sufficiently (14)C-labeled to permit utilization for subsequent in vivo metabolic tracking studies.

Phytochemical composition and metabolic performance-enhancing activity of dietary berries traditionally used by Native North Americans.

Four wild berry species, Amelanchier alnifolia, Viburnum trilobum, Prunus virginiana, and Shepherdia argentea, all integral to the traditional subsistence diet of Native American tribal communities, were evaluated to elucidate phytochemical composition and bioactive properties related to performance and human health. Biological activity was screened using a range of bioassays that assessed the potential for these little-known dietary berries to affect diabetic microvascular complications, hyperglycemia, pro-inflammatory gene expression, and metabolic syndrome symptoms. Nonpolar constituents from berries, including carotenoids, were potent inhibitors of aldose reductase (an enzyme involved in the etiology of diabetic microvascular complications), whereas the polar constituents, mainly phenolic acids, anthocyanins, and proanthocyanidins, were hypoglycemic agents and strong inhibitors of IL-1beta and COX-2 gene expression. Berry samples also showed the ability to modulate lipid metabolism and energy expenditure in a manner consistent with improving metabolic syndrome. The results demonstrate that these berries traditionally consumed by tribal cultures contain a rich array of phytochemicals that have the capacity to promote health and protect against chronic diseases, such as diabetes.

Biosynthesis of 14C-phytoene from tomato cell suspension cultures (Lycopersicon esculentum) for utilization in prostate cancer cell culture studies.

This work describes the development and utilization of a plant cell culture production approach to biosynthesize and radiolabel phytoene and phytofluene for prostate cancer cell culture studies. The herbicide norflurazon was added to established cell suspension cultures of tomato (Lycopersicon esculentum cv. VFNT cherry), to induce the biosynthesis and accumulation of the lycopene precursors, phytoene and phytofluene, in their natural isomeric forms (15-cis-phytoene and two cis-phytofluene isomers). Norflurazon concentrations, solvent carrier type and concentration, and duration of culture exposure to norflurazon were screened to optimize phytoene and phytofluene synthesis. Maximum yields of both phytoene and phytofluene were achieved after 7 days of treatment with 0.03 mg norflurazon/40 mL fresh medium, provided in 0.07% solvent carrier. Introduction of 14C-sucrose to the tomato cell culture medium enabled the production of 14C-labeled phytoene for subsequent prostate tumor cell uptake studies. In DU 145 prostate tumor cells, it was determined that 15-cis-phytoene and an oxidized product of phytoene were taken up and partially metabolized by the cells. The ability to biosynthesize, radiolabel, and isolate these carotenoids from tomato cell cultures is a novel, valuable methodology for further in vitro and in vivo investigations into the roles of phytoene and phytofluene in cancer chemoprevention.

Biosynthesis and characterization of 14C-enriched flavonoid fractions from plant cell suspension cultures.

A range of radiolabeled anthocyanins, proanthocyanidins, and other flavonoids were accumulated by cell suspension cultures of two plant species, ohelo (Vaccinium pahalae) and grape (a Vitis hybrid, Bailey Alicant A), after providing uniformly labeled [(14)C]sucrose to the medium. Approximately 15% of administered label was recovered in a series of flavonoid-rich fractions varying in composition. Anthocyanins, and monomers to oligomers of proanthocyanidins, were labeled effectively and characterized from both species. Most of the proanthocyanidin oligomers were based on the flavan-3-ols (+)-catechin and (-)-epicatechin. Cyanidin and peonidin glycosides were the dominant forms of anthocyanins in both species. Whereas the predominant form of flavonoids identified from ohelo cell cultures was proanthocyanidins, grape cell cultures produced mostly anthocyanins. The labeled phytochemicals were produced for use in subsequent in vivo animal feeding studies to gauge their bioavailability and accumulation in target organs.

Composition of a chemopreventive proanthocyanidin-rich fraction from cranberry fruits responsible for the inhibition of 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity.

Phenolics from the American cranberry (Vaccinium macrocarpon) were fractionated into a series of proanthocyanidins and other flavonoid compounds by vacuum chromatography on a hydrophilic, porous polyvinylic gel permeation polymer. Antioxidant activity was not restricted to a particular class of components in the extract but was found in a wide range of the fractions. Significant chemopreventive activity, as indicated by an ornithine decarboxylase assay, was localized in one particular proanthocyanidin-rich fraction from the initial fractionation procedure. Further fractionation of the active anticarcinogenic fraction revealed the following components: seven flavonoids, mainly quercetin, myricetin, the corresponding 3-O-glycosides, (-)-epicatechin, (+)-catechin, and dimers of both gallocatechin and epigallocatechin types, and a series of oligomeric proanthocyanidins.