Strong and weak plasma response to dietary carotenoids identified by cluster analysis and linked to beta-carotene 15,15'-monooxygenase 1 single nucleotide polymorphisms.

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 ß-carotene, n=23 and 40-mg lycopene, 5-mg ß-carotene, n=12) and tomato juice (18-mg lycopene, 0.6-mg ß-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 ß-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 ß-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 ß-carotene 15,15'-monooxygenase 1.

Assessment of DNA damage and repair in adults consuming allyl isothiocyanate or Brassica vegetables.

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.

Anthocyanin kinetics are dependent on anthocyanin structure.

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.

Whey protein but not soy protein supplementation alters body weight and composition in free-living overweight and obese adults.

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.

Vitamin K absorption and kinetics in human subjects after consumption of 13C-labelled phylloquinone from kale.

The absorption and plasma disappearance of vitamin K were investigated by uniformly labelling phylloquinone in kale with carbon-13, and by feeding the kale to study subjects. Seven healthy volunteers ingested a single 400 g serving of kale with 30 g vegetable oil. The kale provided 156 nmol of phylloquinone. Serial plasma samples were collected and analysed for the appearance of 13C-phylloquinone by HPLC-MS. Six of the subjects showed significant amounts of labelled phylloquinone in plasma, though one subject's plasma was not consistently enriched above the detection limit, and this subject's baseline plasma phylloquinone level was the lowest in the group. After ingestion of the labelled kale, plasma 13C-phylloquinone concentration increased rapidly to a peak between 6 and 10 h, and then rapidly decreased. Average peak plasma concentration for the six subjects with detectable 13C-phylloquinone was 2.1 nmol/l. Plasma concentration-time data were analysed by compartmental modelling. Modelling results demonstrated a mean (n 6) bioavailability of phylloquinone from kale to be 4.7%. Plasma and tissue half-times for phylloquinone were found to be 8.8 and 215 h, respectively.

Phytosterol intake and dietary fat reduction are independent and additive in their ability to reduce plasma LDL cholesterol.

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

Bioavailability of anthocyanins from purple carrot juice: effects of acylation and plant matrix.

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.

Anthocyanin excretion by humans increases linearly with increasing strawberry dose.

A clinical study was conducted to investigate the dose response and metabolism of strawberry anthocyanins. In a crossover study design, 12 healthy adults consumed each of 3 strawberry treatments. The treatments were 100 g, 200 g, and 400 g of pureed strawberries, delivering 15 micromol, 30 micromol, and 60 micromol anthocyanin, respectively. Urine samples were collected for 24 h after each dose and samples were analyzed by HPLC with diode array detection and ion trap MS. Pelargonidin 3-glucoside was the major anthocyanin form in the treatments, and pelargonidin 3-glucoside and 3 metabolites of pelargonidin 3-glucoside (detected as monoglucuronides) were excreted in urine after ingestion. One predominant monoglucuronide form was detected in urine in masses 10-fold higher than the other 2 monoglucuronide forms. Increasing dose resulted in increasing appearance of anthocyanins in urine, and the mass of each pelargonidin monoglucuronide increased in urine with increasing dose. These results suggest that pelargonidin 3-glucoside absorption and metabolism are not saturated at masses < or = 60 micromol, thus showing that more strawberry anthocyanin can be absorbed with increasing dose.

Effects of anthocyanin and carotenoid combinations on foliage and immature fruit color of Capsicum annuum L.

Shades ranging from violet to black pigmentation in pepper (Capsicum annuum L.) are attributed to anthocyanin accumulation. High-performance liquid chromatography and mass spectrometry analysis of violet and black fruit tissue identified a single anthocyanin that was determined to be delphinidin-3-p-coumaroyl-rutinoside-5-glucoside. Leaf tissue of a black-pigmented foliage genotype contained the same anthocyanin found in fruit but at a considerably higher concentration in comparison to violet and black fruit tissue. Fruit chlorophyll concentration was approximately 14-fold higher in black fruit in comparison to violet fruit that contained relatively little chlorophyll. Beta-carotene, lutein, violaxanthin, and neoxanthin carotenoid concentrations in black fruit were also significantly greater in comparison to violet fruit. High concentrations of delphinidin in combination with chlorophyll and accessory carotenoid pigments produced the characteristic black pigmentation observed in fruits and leaves of selected genotypes. Anthocyanins were accumulated in the outer mesocarp of violet and black fruit and in the palisade and mesophyll cells of black leaves. Consistent with chlorophyll content of respective genotypes, chloroplast density was greater in cells of black fruits. Utilizing Capsicum pigment variants, we determine the biochemical factors responsible for violet versus black-pigmented pepper tissue in the context of described pepper color genes.

Anthocyanins present in selected tropical fruits: acerola, jambolão, jussara, and guajiru.

Many tropical fruits are rich in anthocyanins, though limited information is available about the characterization and quantification of these anthocyanins. The identification and quantification of anthocyanin pigments in four tropical fruits were determined by HPLC-MS/MS. Fruits studied included acerola (Malphigia emarginata), jussara (Euterpe edulis), jambolão (Syzygium cumini), and guajiru (Chrysobalanus icaco). All four fruits were found to contain anthocyanin pigments. Anthocyanidin backbones included cyanidin, delphinidin, peonidin, pelargonidin, petunidin, and malvidin. Guajiru contained several acylated forms, while acerola, jussara, and jambolão contained only nonacylated glycosides. These results demonstrate that these tropical fruits are rich in anthocyanins and that the anthocyanins are widely ranging in anthocyanidin backbone, glycosylation, and acylation.

effect of dose size on bioavailability of acylated and nonacylated anthocyanins from red cabbage (Brassica oleracea L. Var. capitata).

Recent studies indicate that anthocyanin intake conveys a variety of health benefits, which depend on absorption and metabolic mechanisms that deliver anthocyanins and their bioactive metabolites to responsive tissues. The anthocyanin bioavailability of red cabbage (Brassica oleracea L. var. capitata) was evaluated as reflected by urinary excretion of anthocyanins and anthocyanin metabolites. Twelve volunteers consumed 100, 200, and 300 g of steamed red cabbage (containing 1.38 micromol of anthocyanins/g of cabbage) in a crossover design. Anthocyanin concentration in cabbage extract and urine was measured by HPLC-MS/MS. Six nonacylated and 30 acylated anthocyanins were detected in red cabbage, and 3 nonacylated anthocyanins, 8 acylated anthocyanins, and 4 metabolites were present in urine. Mean 24 h excretion of intact anthocyanins increased linearly from 45 (100 g dose) to 65 nmol (300 g dose) for acylated anthocyanins and from 52 (100 g dose) to 79 nmol (300 g dose) for nonacylated anthocyanins. Urinary recovery of intact anthocyanins (percent of anthocyanin intake) decreased linearly from 0.041% (100 g dose) to 0.020% (300 g dose) for acylated anthocyanins and from 0.18% (100 g dose) to 0.09% (300 g dose) for nonacylated anthocyanins. Anthocyanin metabolites consisted of glucuronidated and methylated anthocyanins. The results show that red cabbage anthocyanins were excreted in both intact and metabolized forms and that recovery of nonacylated anthocyanins in urine was >4-fold that of acylated anthocyanins.

Watermelon consumption increases plasma arginine concentrations in adults.

OBJECTIVE: Watermelon is a rich source of citrulline, an amino acid that can be metabolized to arginine, a conditionally essential amino acid for humans. Arginine is the nitrogenous substrate used in the synthesis of nitric oxide and plays an essential role in cardiovascular and immune functions. No detailed studies have been conducted to evaluate plasma arginine response in humans after long-term feeding of citrulline from natural plant sources. This study investigated if watermelon juice consumption increases fasting concentrations of plasma arginine, ornithine, and citrulline in healthy adult humans. METHODS: Subjects (n = 12-23/treatment) consumed a controlled diet and 0 (control), 780, or 1560 g of watermelon juice per day for 3 wk in a crossover design. The treatments provided 1 and 2 g of citrulline per day. Treatment periods were preceded by washout periods of 2 to 4 wk. RESULTS: Compared with the baseline, fasting plasma arginine concentrations increased 12% after 3 wk of the lower-dose watermelon treatment; arginine and ornithine concentrations increased 22% and 18%, respectively, after 3 wk of the higher-dose watermelon treatment. Fasting citrulline concentrations did not increase relative to the control but remained stable throughout the study. CONCLUSION: The increased fasting plasma concentrations of arginine and ornithine and stable concentrations of plasma citrulline in response to watermelon juice consumption indicated that the citrulline from this plant origin was effectively converted into arginine. These results demonstrate that plasma concentration of arginine can be increased through intake of citrulline from watermelon.

Body Mass Index, percent body fat, and regional body fat distribution in relation to leptin concentrations in healthy, non-smoking postmenopausal women in a feeding study.

BACKGROUND: The relationship between BMI and leptin has been studied extensively in the past, but previous reports in postmenopausal women have not been conducted under carefully controlled dietary conditions of weight maintenance using precise measures of body fat distribution. The aim of the present study was to examine the association between serum leptin concentration and adiposity as estimated by BMI and dual energy x-ray absorptiometry (DEXA) measures (percent body fat, central and peripheral fat, and lean mass) in postmenopausal women. METHODS: This study was conducted as a cross-sectional analysis within the control segment of a randomized, crossover trial in which postmenopausal women (n = 51) consumed 0 (control), 15 (one drink), and 30 (two drinks) g alcohol (ethanol)/d for 8 weeks as part of a controlled diet. BMIs were determined and DEXA scans were administered to the women during the 0 g alcohol treatment, and a blood sample was collected at baseline and week 8 of each study period for leptin analysis. RESULTS AND DISCUSSION: In multivariate analysis, women who were overweight (BMI > 25 to < or = 30 kg/m2) had a 2-fold increase, and obese women (BMI > 30 kg/m2) had more than a 3-fold increase in serum leptin concentrations compared to normal weight (BMI < or =25 kg/m2) women. When the models for the different measures of adiposity were assessed by multiple R2, models which included percent body fat explained the highest proportion (approximately 80%) of the serum leptin variance. CONCLUSION: Under carefully controlled dietary conditions, we confirm that higher levels of adiposity were associated with higher concentrations of serum leptin. It appears that percent body fat in postmenopausal women may be the best adiposity-related predictor of serum leptin.

Usefulness of body mass index as a sufficient adiposity measurement for sex hormone concentration associations in postmenopausal women.

BACKGROUND: Both obesity and sex hormones are known risk factors for postmenopausal breast cancer. Although adiposity and sex hormones have been studied in the past, previous reports in postmenopausal women have not been conducted under carefully controlled dietary conditions. In this study, we investigated the usefulness of body mass index (BMI) as a sufficient adiposity measurement to assess associations with sex hormone levels. METHODS: This study was conducted as a cross-sectional analysis within the control segment (0 g alcohol group) of a randomized, crossover design, in which 51 postmenopausal women consumed 0 (control), 15 (one drink), and 30 (two drinks) g alcohol (ethanol)/d for 8 weeks each as part of a controlled diet. Dual-energy X-ray absorptiometry scans were administered to the women during the control (0 g alcohol) segment, and a blood sample was drawn at the end of that diet period for hormone analysis. RESULTS: In multivariate analysis (adjusted for age, race, family history of breast cancer, parity, and menarche <12 years), women who were overweight or obese had significantly higher serum concentrations of estradiol, bioavailable estradiol, estrone, and estrone sulfate and lower sex hormone-binding globulin than normal weight women (all P < 0.05). In models adjusted for BMI and the covariates above, none of the dual-energy X-ray absorptiometry adiposity measures added further information (all P > 0.10) for these five analytes beyond that of BMI alone. CONCLUSIONS: In this population of postmenopausal women, under carefully controlled dietary conditions, we confirmed previous findings that higher levels of adiposity were associated with higher concentrations of estrogens and lower sex hormone-binding globulin, and we found that the use of the epidemiology-friendly BMI seems sufficient to assess associations with these hormone levels.

Comparison of energy expenditure estimates from 4 physical activity questionnaires with doubly labeled water estimates in postmenopausal women.

BACKGROUND: Physical activity energy expenditure (EE) is an important determinant of health, and epidemiologists have used various methods, such as physical activity and energy intake recalls and records, to estimate energy cost. However, most epidemiologic studies have not validated these methods against the doubly labeled water (DLW) technique for measuring EE. OBJECTIVE: The aim was to compare EE estimated by 4 physical activity questionnaires with that obtained with the DLW technique in free-living postmenopausal women. DESIGN: We measured EE in kcal/d using the DLW method, the Harvard Alumni questionnaire, the Five City Project questionnaire, the Cross-Cultural Activity Participation Study (CAPS) Four Week Activity Recall, and the CAPS Typical Week Activity Survey in 65 healthy postmenopausal women. RESULTS: Compared with DLW, the Harvard Alumni questionnaire, the Five City Project questionnaire, and the CAPS Four Week Activity Recall overestimated (P < 0.05) daily EE by 62%, 16%, and 11%, respectively, whereas the CAPS Typical Week Activity Recall underestimated (P < 0.05) EE by 31%. Both the Harvard Alumni and Five City Project questionnaires overestimated EE in obese and overweight women. CONCLUSIONS: When using 3 of the 4 questionnaire methods, postmenopausal women overestimated EEs. Of all women, obese women overestimated daily EE the most.

Plasma and urine responses are lower for acylated vs nonacylated anthocyanins from raw and cooked purple carrots.

The bioavailability of acylated vs nonacylated anthocyanins and the effect of cooking and dose on the comparative bioavailability were investigated in a clinical feeding study using purple carrots as the anthocyanin source. Treatments were purple carrots as follows: 250 g raw (463 micromol of anthocyanins: 400 micromol acylated, 63 micromol nonacylated), 250 g cooked (357 micromol of anthocyanins: 308.5 micromol acylated, 48.5 micromol nonacylated), and 500 g cooked (714 micromol of anthocyanins: 617 micromol acylated, 97 micromol nonacylated). Four of the five carrot anthocyanins were found intact in plasma by 30 min after carrot consumption and peaked between 1.5 and 2.5 h. Acylation of anthocyanins resulted in an 11-14-fold decrease in anthocyanin recovery in urine and an 8-10-fold decrease in anthocyanin recovery in plasma. Cooking increased the recovery of nonacylated anthocyanins but not acylated anthocyanins. Large dose size significantly reduced recovery of both acylated and nonacylated anthocyanins, suggesting saturation of absorption mechanisms.

Plasma appearance of labeled beta-carotene, lutein, and retinol in humans after consumption of isotopically labeled kale.

The bioavailability of carotenoids from kale was investigated by labeling nutrients in kale with 13C, feeding the kale to seven adult volunteers, and analyzing serial plasma samples for labeled lutein, beta-carotene, and retinol. Ingested doses of labeled carotenoids were 34 micromol for beta-carotene and 33 micromol for lutein. Peak plasma concentrations, areas under the plasma concentration-time curves (AUCs), and percentages of dose recovered at peak plasma concentrations were calculated. Average peak plasma concentrations were 0.38, 0.068, and 0.079 microM for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Average AUC values (over 28 days) were 42.8, 13.6, 13.2 microM h for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. Percentages of dose recovered at peak plasma concentrations were 3.6, 0.7, and 0.7% for [13C]lutein, [13C]beta-carotene, and [13C]retinol, respectively. A positive relationship was observed between baseline plasma retinol levels and [13C]retinol plasma response. It is possible that this relationship was mediated either through some aspect of beta-carotene absorption or via the common pathways of metabolism for postdose and endogenous retinoid.

Effects of alcohol and menstrual cycle on insulin-like growth factor-I and insulin-like growth factor binding protein-3.

Alcohol ingestion and insulin-like growth factor-I (IGF-I) have been associated with increased breast cancer risk, the latter primarily in premenopausal women. We investigated whether alcohol ingestion altered IGF-I or its major binding protein (BP), IGFBP-3, in a controlled feeding study in premenopausal women. We also determined whether IGF-I or IGFBP-3 was affected by menstrual cycle phase. Serum was collected from 31 individuals who were randomly assigned to consume either 0 or 30 g (two drinks) of alcohol daily for three menstrual cycles and who then crossed over to the other alcohol level for three cycles. All calories were provided and weight was maintained during the study. For both alcohol levels, serum was collected during the final cycle at early follicular, periovulatory, and luteal phases. Relative to the follicular phase, IGF-I levels increased by 3.3% and 7.6% in the periovulatory and luteal phases, respectively (P for trend = 0.004). Although alcohol ingestion did not affect this increase, it significantly reduced IGF-I concentrations at all phases (9.5%; P < 0.001), whereas IGFBP-3 was unaffected by either menstrual phase or alcohol. This is the first controlled diet study to show that alcohol decreases serum IGF-I in premenopausal women and that IGF-I significantly increases over the course of the menstrual cycle whether or not alcohol is present.