The mechanisms as well the genetics underlying the bioavailability and metabolism of carotenoids in humans remain unclear. To begin to address these questions, we used cluster analysis to examine individual temporal responses of plasma carotenoids from a controlled-diet study of subjects who consumed carotenoid-rich beverages. Treatments, given daily for 3 weeks, were watermelon juice at two levels (20-mg lycopene, 2.5-mg β-carotene, n=23 and 40-mg lycopene, 5-mg β-carotene, n=12) and tomato juice (18-mg lycopene, 0.6-mg β-carotene, n=10). Cluster analysis revealed distinct groups of subjects differing in the temporal response of plasma carotenoids and provided the basis for classifying subjects as strong responders or weak responders for β-carotene, lycopene, phytoene and phytofluene. Individuals who were strong or weak responders for one carotenoid were not necessarily strong or weak responders for another carotenoid. Furthermore, individual responsiveness was associated with genetic variants of the carotenoid metabolizing enzyme β-carotene 15,15'-monooxygenase 1. These results support the concept that individuals absorb or metabolize carotenoids differently across time and suggest that bioavailability of carotenoids may involve specific genetic variants of β-carotene 15,15'-monooxygenase 1.

This controlled feeding intervention investigated the effect of dietary blackberries on DNA damage in humans. Healthy adults (n=51, age≥40) consumed a wash‐in diet for 3 d followed by an intervention diet on which they were randomized to receive either 300 g blackberries or an isocaloric gelatin control at breakfast for 6 d. Urinary 8‐oxo‐7′8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) excretion was determined by UHPLC‐MS/MS and values were normalized to urine specific gravity. Mean 8‐oxodG values were 29% lower 3 h after blackberry consumption compared to fasting (14.6±9.6 at 0 h, 11.3±8.7 pmol/mL at 3, P=0.043), and were 24% higher 3 h after gelatin consumption (13.5±10.1 at 0 h, 16.7±13.3 at 3, P=0.013). On d 6 there were no significant differences in 8‐oxodG excretion in either group compared to baseline (11.6±7.1 pmol/mL, P=0.44 control; 14.4±9.3, P=0.67 berries), and 8‐oxodG values increased non‐significantly in both groups 3 h after treatment (15.4±11.4 pmol/mL, P=0.07 control; 18.3±16.9, P=0.29 berries). Age, sex, and BMI were not associated with treatment effects. These findings demonstrate that blackberries may be effective against short‐term DNA damage but this effect appeared to disappear after 6 d. Mechanisms for the long‐term acclimation to berry feeding are currently under investigation.Support: NIH/USDA

Allyl isothiocyanate (AITC) is a dietary component with possible anticancer effects, though much information about AITC and cancer has been obtained from cell studies. To investigate the effect of AITC on DNA integrity in vivo, a crossover study was conducted. Adults (n=46) consumed AITC, AITC-rich vegetables [mustard and cabbage (M/C)] or a control treatment with a controlled diet for 10 days each. On day 11, volunteers provided blood and urine before and after consuming treatments. Volunteers were characterized for genotype for GSTM1 and GSTT1 (glutathione S-transferases) and XPD (DNA repair). DNA integrity in peripheral blood mononuclear cells was assessed by single-cell gel electrophoresis. Urine was analyzed for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and creatinine. Ten-day intake of neither AITC nor M/C resulted in statistically significant differences in DNA strand breaks [least squares mean (LSmean) % DNA in tail±S.E.M.: 4.8±0.6 for control, 5.7±0.7 for AITC, 5.3±0.6 for M/C] or urinary 8-oxodG (LSmean μg 8-oxodG/g creatinine±S.E.M.: 2.95±0.09 for control, 2.88±0.09 for AITC, 3.06±0.09 for M/C). Both AITC and M/C increased DNA strand breaks 3 h postconsumption (LSmean % DNA in tail±S.E.M.: 3.2±0.7 for control, 8.3±1.7 for AITC, 8.0±1.7 for M/C), and this difference disappeared at 6 h (4.2±0.9 for control, 5.7±1.2 for AITC, 5.5±1.2 for M/C). Genotypes for GSTM1, GSTT1 and XPD were not associated with treatment effects. In summary, DNA damage appeared to be induced in the short term by AITC and AITC-rich products, but that damage disappeared quickly, and neither AITC nor AITC-rich products affected DNA base excision repair.

The kinetics of anthocyanin metabolism was investigated in a human feeding trial. Volunteers (n 12) consumed purple carrots containing five anthocyanin forms: cyanidin-3-(xylose-glucose-galactoside), cyanidin-3-(xylose-galactoside), cyanidin-3-(xylose-sinapoyl-glucose-galactoside), cyanidin-3-(xylose-feruloyl-glucose-galactoside) and cyanidin-3-(xylose-coumuroyl-glucose-galactoside). The purple carrots were served as three different treatments in a crossover design with a 3-week washout between treatments. Purple carrot treatments were 250 g raw carrots, 250 g cooked carrots and 500 g cooked carrots. Serial blood and urine samples were collected for 8 and 24 h after the dose, respectively, and analysed for anthocyanins. Of the anthocyanin forms ingested, four were detected in plasma and urine: cyanidin-3-(xylose-glucose-galactoside), cyanidin-3-(xylose-galactoside), cyanidin-3-(xylose-sinapoyl-glucose-galactoside) and cyanidin-3-(xylose-feruloyl-glucose-galactoside). The time courses of plasma and urine anthocyanin contents were evaluated with compartmental modelling. Results showed that absorption, gastrointestinal transit and plasma elimination are dependent on anthocyanin structure. Absorption efficiencies of acylated compounds (cyanidin-3-(xylose-sinapoyl-glucose-galactoside) and cyanidin-3-(xylose-feruloyl-glucose-galactoside)) were less than those for non-acylated anthocyanins (cyanidin-3-(xylose-glucose-galactoside) and cyanidin-3-(xylose-galactoside)). The acylated anthocyanins exhibited a shorter half-life for gastrointestinal absorption than the non-acylated anthocyanins. Fractional elimination of non-acylated compounds was slower than that for acylated anthocyanins. These results provide the first information about the kinetics of individual anthocyanins in human beings.

Healthy adults (n=51) over the age of 40 consumed a wash‐in diet for 3 d followed by an intervention diet with 300 g blackberries or an isocaloric gelatin control at breakfast for 6 d. Metabolite profiling was conducted by Metabolon using GC/MS and LC/MS/MS platforms using fasting plasma samples from d 9. Following log transformation and imputation with minimum observed values, Welch's two‐sample t‐test was used to identify biochemicals that differed significantly between experimental groups. Blackberry intake compared to control revealed metabolite changes that reflect likely changes in gut metabolism (phenol sulfate), TCA cycle energetics (α‐ketoglutarate), the pentose phosphate pathway (threitol), and metabolism of benzoate (phenol sulfate), isoleucine (3‐hydroxy‐2‐ethylpropionate), medium‐chain fatty acids (undecanoate), collagen (trans‐4‐hydroxyproline), choline (betaine), and xanthine (3‐methylxanthine) and decreased fucose. Methyl‐beta‐glucopyranoside, a likely anthocyanidin metabolite, and one unknown compound were identified as potential markers of compliance for blackberry intake. Levels of these markers, however, did not correlate with levels of other altered metabolites. Support: NIH/USDA

Allyl isothiocyanate (AITC), which is derived from Brassica vegetables, is associated with anti‐cancer effects, though much evidence is from cell studies. A feeding study was conducted to investigate the effect of AITC on DNA integrity in vivo. Adults (n=46) consumed AITC, AITC‐rich foods (cabbage and mustard (C/M)), or a control diet for 10 d each in a crossover design. Volunteers were genotyped for the deleted or functional alleles of GSTM1 and GSTT1, and SNPs Asp312Asn and Lys751Gln in the DNA repair gene XPD. Ten day intake of neither AITC nor C/M resulted in significant differences in DNA strand breaks in peripheral blood mononuclear cells (LSmean % DNA in tail ± SEM: 4.8±0.6 for control, 5.7±0.7 for AITC, 5.3±0.6 for C/M). However, both AITC and C/M increased DNA strand breaks 3 h post‐consumption (%DNA in tail: 3.2±0.7 for control, 8.3±1.7 for AITC, 8.0±1.7 for C/M), and this difference disappeared at 6 h (4.2±0.9 for control, 5.7±1.2 for AITC, 5.5±1.2 for C/M). Genotypes for GSTM1, GSTT1, and XPD were not associated with treatment effects. In summary, DNA damage appeared to be induced in the short term by AITC and AITC‐rich products, but that damage disappeared quickly. Funded by USDA and NCI.

Flavanols may provide protection against insulin resistance, but little is known about the amounts and types of flavanols that may be efficacious. This study was designed to determine whether cocoa flavanols, over a range of intakes, improve biomarkers of glucose regulation, inflammation and hemostasis in obese adults at risk for insulin resistance. As an adjunct, green tea and cocoa flavanols were compared for their ability to modulate these biomarkers. In a randomized crossover design, 20 adults consumed a controlled diet for 5 days along with four cocoa beverages containing 30–900 mg flavanol per day, or tea matched to a cocoa beverage for monomeric flavanol content. Cocoa beverages produced no significant changes in glucose, insulin, total area under the concentration–time curve (AUC) for glucose or total insulin AUC. As the dose of cocoa flavanols increased, total 8-isoprostane concentrations were lowered (linear contrast, P=0.02), as were C-reactive protein (CRP) concentrations (linear contrast, P=0.01). The relationship between cocoa flavanol levels and interleukin-6 (IL-6) concentrations was quadratic, suggesting that a maximum effective dose was achieved (quadratic contrast, P=0.01). There were no significant effects on measured indices of glucose regulation, nor on those of total 8-isoprostane, CRP and IL-6 concentrations, when cocoa and green tea were compared. However, relative to cocoa, green tea lowered fibrinogen concentrations (P=0.0003). Short-term intake of cocoa and green tea flavanols does not appear to improve glucose metabolism; they do affect selected markers of one or more measures of oxidative stress, inflammation or hemostasis in obese adults at risk for insulin resistance.

A double-blind, randomized clinical trial was conducted to determine the effect of consumption of supplemental whey protein (WP), soy protein (SP), and an isoenergetic amount of carbohydrate (CHO) on body weight and composition in free-living overweight and obese but otherwise healthy participants. Ninety overweight and obese participants were randomly assigned to 1 of 3 treatment groups for 23 wk: 1) WP; 2) SP (each providing ~56 g/d of protein and 1670 kJ/d); or 3) an isoenergetic amount of CHO. Supplements were consumed as a beverage twice daily. Participants were provided no dietary advice and continued to consume their free-choice diets. Participants' body weight and composition data were obtained monthly. Dietary intake was determined by 24-h dietary recalls collected every 10 d. After 23 wk, body weight and composition did not differ between the groups consuming the SP and WP or between SP and CHO; however, body weight and fat mass of the group consuming the WP were lower by 1.8 kg (P < 0.006) and 2.3 kg (P < 0.005), respectively, than the group consuming CHO. Lean body mass did not differ among any of the groups. Waist circumference was smaller in the participants consuming WP than in the other groups (P < 0.05). Fasting ghrelin was lower in participants consuming WP compared with SP or CHO. Through yet-unknown mechanisms, different sources of dietary protein may differentially facilitate weight loss and affect body composition. Dietary recommendations, especially those that emphasize the role of dietary protein in facilitating weight change, should also address the demonstrated clinical potential of supplemental WP.

Carotenoids have a wide range of human health benefits. Yellow-fleshed tetraploid potato (Solanum tuberosum) cultivars have more than twice the concentration of carotenoids as white-fleshed cultivars. However, carotenoid concentrations in some diploid potatoes have been reported to be up to 13 times higher than in 'Yukon Gold', the most popular yellow-fleshed potato cultivar grown in the United States, and up to 22 times higher than in white-fleshed potatoes. The purpose of this study was to determine the feasibility of using these high-carotenoid diploids to develop high-carotenoid tetraploid germplasm. Three diploid clones with high (dark yellow-flesh), moderate (moderate yellow-flesh), and low (white cream-flesh) carotenoid levels that produced 2n pollen were crossed with a light yellow-fleshed tetraploid advanced breeding selection to determine the inheritance of carotenoid content. Twenty-six to 43 progeny from these three 4x-2x families were grown in a replicated field experiment in Presque Isle, ME, for 2 years. After harvest, carotenoids were extracted and quantified by high-performance liquid chromatography in 13 to 14 randomly selected clones from each family; however, flesh color was scored as white or yellow in all progeny. A continuous distribution of carotenoid concentration with high- and low-carotenoid segregants was observed in all three families. There were no significant differences among these three families for individual or total carotenoid concentrations; however, there were significant differences among clones within families. Broad-sense heritability estimates were high for total carotenoid (0.81), lutein (0.77), zeaxanthin (0.73), and the lycopene beta-cyclase pathway carotenoids (0.73); moderate for neoxanthin (0.42); and low for violaxanthin (0.21) and antheraxanthin (0.13). Based on flesh color segregation, the two yellow-fleshed diploid parents were heterozygous for the Chy2 allele governing yellow-flesh and produced 2n gametes by a second division restitution mechanism. It appears that selection for high-carotenoid tetraploid germplasm can be made from within any family with at least one yellow-fleshed parent. Selections will have to be made on an individual clonal basis rather than on a family basis.

The objective of this study was to investigate the differences in kinetics of individual cyanidin-based anthocyanins in a human feeding trial. Volunteers (n=12) consumed purple carrots containing five different anthocyanin forms. The carrots were administered as three treatments in a crossover design with a 3 week washout between treatments. Carrot treatments were 250 g raw carrots, 250 g cooked carrots, and 500 g cooked carrots. Serial blood samples were collected for 8 h, and urine samples were collected for 24 h after the dose. Anthocyanins were extracted from plasma and urine by SPE, then separated and identified by LC-MS. The resulting plasma concentration time curves and urine accumulation curves were analyzed by compartmental modeling using the WinSAAM modeling package. Model results showed that absorption, gastrointestinal transit, and plasma elimination were dependent on anthocyanin structure. The absorption efficiency of acylated forms was less than that for non-acylated anthocyanins. The acylated anthocyanins exhibited a shorter half-time for gastrointestinal absorption than the non-acylated forms. Non-acylated anthocyanins were eliminated more slowly than acylated anthocyanins. These results provide the first information about differences in the kinetics of individual anthocyanins in humans. This work was supported by the US Department of Agriculture.

Consumption of carotenoid-containing foods can promote human health. Although yellow-fleshed potatoes ( Solanum tuberosum ) have a higher carotenoid content than white-fleshed potatoes, little is known about how growing environments may affect individual and total carotenoid content in different potato clones. The purposes of this study were to estimate the amount of genetic variability in potato for five xanthophyll carotenoids, their concentration, and to determine the stability of these carotenoids across environments. Nine white- or yellow-fleshed tetraploid clones were grown in Maine and Florida for 2 years. Carotenoids were extracted in acetone and analyzed by high-performance liquid chromatography. There were significant differences among clones for zeaxanthin, antheraxanthin, lutein, and total carotenoid content. There were significant clone × environment interactions for zeaxanthin, antheraxanthin, violaxanthin, neoxanthin, lutein, and total carotenoid. Broad-sense heritabilities (and their 95% confidence intervals) were 0.89 (0.79–0.98) for zeaxanthin, 0.93 (0.87–0.99) for antheraxanthin, 0.68 (0.14–0.92) for violaxanthin, 0.51 (0.00–0.88) for neoxanthin, 0.85 (0.70–0.97) for lutein, and 0.96 (0.89–0.99) for total carotenoid. Clonal mean total carotenoid content ranged from 101 to 511 μg/100 g fresh weight. A higher proportion of carotenoids were produced by the lycopene epsilon cyclase branch of the carotenoid biosynthetic pathway in white-fleshed than yellow-fleshed clones. Total carotenoid content in B2333-5 was significantly greater than in ‘Yukon Gold’. With genetic variation for individual and total carotenoid content in potatoes, improving the levels of carotenoids has been and should continue to be feasible; however, concentrations are likely to vary in different environments.

Tropical fruits are rich sources of phytonutrients, and these compounds hold promise for preventing disease and extending productive, active lifespans. Phytonutrients can be classified as isoprenoids (e.g., carotenoids, limonoids), flavonoids (e.g., anthocyanins, flavanones) and other phenolic compounds, and amino acid-based (e.g., capsaicinoids) compounds. Although phytochemicals are best known as antioxidants, protection may also be provided by phytonutrient-induced upregulation of detoxification enzymes, communication among adjacent cells, apoptosis, and control of angiogenesis. The body of knowledge linking individual phytonutrients to human health is intriguing, but efficacy is uncertain and dietary recommendations for individual phytonutrients are premature. There is, however, a strong scientific base of evidence for recommending increased consumption of phytonutrient-rich whole foods for health promotion and disease prevention.

Absorption of cyanidin-based anthocyanins is not fully understood with respect to dose or anthocyanin structure. In feeding studies using whole foods, nonacylated anthocyanins are more bioavailable than their acylated counterparts, but the extent to which plant matrix determines relative bioavailability of anthocyanins is unknown. Using juice of purple carrots to circumvent matrix effects, a feeding trial was conducted to determine relative bioavailability of acylated and nonacylated anthocyanins and to assess dose-response effects. Appearance of anthocyanins in plasma was measured in 10 healthy adults for 8 h following consumption of purple carrot juice. Each subject consumed 50, 150, and 250 mL of juice containing 76 micromol (65 mg), 228 micromol (194 mg), and 380 micromol (323 mg) of total anthocyanins, respectively. Acylated anthocyanins comprised 76% of total anthocyanins in the juice, yet their bioavailability was found to be significantly less than that of nonacylated anthocyanins. Peak plasma concentrations of nonacylated anthocyanins were 4-fold higher than that for acylated anthocyanins. Absorption efficiency declined across the doses administered. Because the treatments were consumed as juice, it could be discerned that the difference in bioavailability of acylated versus nonacylated anthocyanins was not primarily caused by interactions with the plant matrix.

Oolong tea, as well as the polyphenols it contains, has been shown to alter physiological processes related to glucose regulation. As part of a double blinded randomized crossover design study, 19 overweight and obese men (BMI 25‐34, ages 25‐64 years) consumed a controlled diet at weight maintenance along with 5 treatment beverages: 1) oolong tea, 2) oolong tea with added catechins, 3) oolong tea with added oolong tea polyphenols, 4) water with caffeine (matched for caffeine content of oolong tea) and 5) water. Each subject consumed four 350 ml servings (1400 ml/day) of the treatment beverages per day with breakfast, lunch, mid‐afternoon and dinner for four days. On the fifth day, after a 12 hour fast, subjects consumed a mixed meal tolerance test breakfast (450 kcals, 85 g carbohydrate, 10 g fat, 8 g protein) along with the treatment beverage. Biomarkers, glucose, insulin, triglycerides and the homeostasis model assessment of insulin resistance (HOMA‐IR), were measured every 30 minutes during a four hour period. Results showed no significant postprandial changes in glucose, insulin, triglycerides and HOMA‐IR after consumption of the treatment beverages. Consumption of the oolong tea treatments had no adverse effects on glucoregulatory biomarkers in overweight and obese men.Supported by US Department of Agriculture and Suntory Corporation, Osaka, Japan

We studied the interrelationship of diet and plant sterols (PS) on plasma lipids, lipoproteins and carotenoids. Mildly hypercholesterolemic men (n = 13) and postmenopausal women (n = 9) underwent four randomized, crossover, double-blind, controlled feeding periods of 23 days each. The design consisted of two levels of PS (0 and 3.3 g/day) and two background diets having fat content either typical of the American diet (total and saturated fat at 33.5 and 13.2% of energy, respectively), or a Step 1 type of diet (total and saturated fat at 26.4 and 7.7% of energy, respectively). Plasma total cholesterol (TC), high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, Apo A1 and Apo B were 4.3, 5.3, 4.5, 2.8 and 2.5% lower, respectively (P ≤ 0.0001; <0.0001, 0.0016, 0.0006, and 0.0069), with the Step 1 diet than with the typical American diet. Diet had no effect on TC/HDL cholesterol (P = 0.1062). Plant sterol intake lowered TC, LDL cholesterol, and Apo B by 9.0, 12.4 and 6.1% and TC/HDLC by 9.6% (P ≤ 0.0001 for all), respectively, without affecting HDL cholesterol and Apo A1 (P = 0.2831 and 0.732). The PS effect in lowering plasma TC and LDL cholesterol was independent of and additive to the effect due to dietary fat reduction. Responses of plasma carotenoids to PS intake were consistent with the literature.

Although health benefits of dietary anthocyanins (ACN) have received significant attention, scientific progress has been slowed by an incomplete understanding of ACN absorption and metabolism. Thus, we developed a system to produce carbon‐13 labeled ACNs in red cabbage to enable study of ACN fate after ingestion. Twenty‐eight ‘Super Red’ red cabbage seedlings were grown in an inorganic growth medium in 10.1 cm pots. Upon appearance of the first true leaves, pots were placed in a gas‐tight acrylic box located in a walk‐in growth chamber. Nutrient solution was delivered hydroponically. Temperature was maintained at 22.0 ± 0.5 °C until day 26, then at 10.5 ± 1.5 °C until plants were harvested on day 34. 13CO2 concentration in the box was monitored with an infrared gas analyzer, and 13CO2 (99.3% pure) was injected when concentration was < 390 μL L−1. ACNs were analyzed by HPLC‐MS/MS. Carbon sites of the predominant nonacylated ACN, cyanidin 3‐diglucoside‐5‐glucoside, and the predominant acylated ACN, cyanidin 3‐(feruloyl)(sinapoyl) diglucoside‐5‐glucoside, were > 98% labeled. This high incorporation rate of 13C favors precise tracing of ACNs and ACN metabolites in upcoming feeding studies. Funded by the USDA/ARS.