Rachel E. Kopec

Naturally occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors.

Background: Dietary β-carotene can be cleaved centrally to vitamin A, an agonist of retinoic acid receptors, or eccentrically to yield β-apocarotenoids. Results: β-Apocarotenoids antagonize retinoic acid receptors by binding directly to the receptors. Conclusion: β-Apocarotenoids function as naturally occurring retinoid receptor antagonists. Significance: The antagonism of retinoid signaling by these metabolites may explain the negative health effects of large doses of β-carotene. β-Carotene is the major dietary source of provitamin A. Central cleavage of β-carotene catalyzed by β-carotene oxygenase 1 yields two molecules of retinaldehyde. Subsequent oxidation produces all-trans-retinoic acid (ATRA), which functions as a ligand for a family of nuclear transcription factors, the retinoic acid receptors (RARs). Eccentric cleavage of β-carotene at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are β-apocarotenals and β-apocarotenones, whose biological functions in mammals are unknown. We used reporter gene assays to show that none of the β-apocarotenoids significantly activated RARs. Importantly, however, β-apo-14′-carotenal, β-apo-14′-carotenoic acid, and β-apo-13-carotenone antagonized ATRA-induced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that β-apo-13-carotenone can interact directly with the ligand binding site of the retinoid receptors. β-Apo-13-carotenone and the β-apo-14′-carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 cells. Finally, we developed an LC/MS method and found 3–5 nm β-apo-13-carotenone was present in human plasma. These findings suggest that β-apocarotenoids function as naturally occurring retinoid antagonists. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary β-carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer.

Carotenoids are more bioavailable from papaya than from tomato and carrot in humans: a randomised cross-over study

Carrot, tomato and papaya represent important dietary sources of β-carotene and lycopene. The main objective of the present study was to compare the bioavailability of carotenoids from these food sources in healthy human subjects. A total of sixteen participants were recruited for a randomised cross-over study. Test meals containing raw carrots, tomatoes and papayas were adjusted to deliver an equal amount of β-carotene and lycopene. For the evaluation of bioavailability, TAG-rich lipoprotein (TRL) fractions containing newly absorbed carotenoids were analysed over 9·5 h after test meal consumption. The bioavailability of β-carotene from papayas was approximately three times higher than that from carrots and tomatoes, whereas differences in the bioavailability of β-carotene from carrots and tomatoes were insignificant. Retinyl esters appeared in the TRL fractions at a significantly higher concentration after the consumption of the papaya test meal. Similarly, lycopene was approximately 2·6 times more bioavailable from papayas than from tomatoes. Furthermore, the bioavailability of β-cryptoxanthin from papayas was shown to be 2·9 and 2·3 times higher than that of the other papaya carotenoids β-carotene and lycopene, respectively. The morphology of chromoplasts and the physical deposition form of carotenoids were hypothesised to play a major role in the differences observed in the bioavailability of carotenoids from the foods investigated. Particularly, the liquid-crystalline deposition of β-carotene and the storage of lycopene in very small crystalloids in papayas were found to be associated with their high bioavailability. In conclusion, papaya was shown to provide highly bioavailable β-carotene, β-cryptoxanthin and lycopene and may represent a readily available dietary source of provitamin A for reducing the incidence of vitamin A deficiencies in many subtropical and tropical developing countries.

Combined pressure-temperature effects on carotenoid retention and bioaccessibility in tomato juice.

This study highlights the changes in lycopene and β-carotene retention in tomato juice subjected to combined pressure-temperature (P-T) treatments ((high-pressure processing (HPP; 500-700 MPa, 30 °C), pressure-assisted thermal processing (PATP; 500-700 MPa, 100 °C), and thermal processing (TP; 0.1 MPa, 100 °C)) for up to 10 min. Processing treatments utilized raw (untreated) and hot break (∼93 °C, 60 s) tomato juice as controls. Changes in bioaccessibility of these carotenoids as a result of processing were also studied. Microscopy was applied to better understand processing-induced microscopic changes. TP did not alter the lycopene content of the tomato juice. HPP and PATP treatments resulted in up to 12% increases in lycopene extractability. all-trans-β-Carotene showed significant degradation (p < 0.05) as a function of pressure, temperature, and time. Its retention in processed samples varied between 60 and 95% of levels originally present in the control. Regardless of the processing conditions used, <0.5% lycopene appeared in the form of micelles (<0.5% bioaccessibility). Electron microscopy images showed more prominent lycopene crystals in HPP and PATP processed juice than in thermally processed juice. However, lycopene crystals did appear to be enveloped regardless of the processing conditions used. The processed juice (HPP, PATP, TP) showed significantly higher (p < 0.05) all-trans-β-carotene micellarization as compared to the raw unprocessed juice (control). Interestingly, hot break juice subjected to combined P-T treatments showed 15-30% more all-trans-β-carotene micellarization than the raw juice subjected to combined P-T treatments. This study demonstrates that combined pressure-heat treatments increase lycopene extractability. However, the in vitro bioaccessibility of carotenoids was not significantly different among the treatments (TP, PATP, HPP) investigated.

Avocado Consumption Enhances Human Postprandial Provitamin A Absorption and Conversion from a Novel High–β-Carotene Tomato Sauce and from Carrots

Dietary lipids have been shown to increase bioavailability of provitamin A carotenoids from a single meal, but the effects of dietary lipids on conversion to vitamin A during absorption are essentially unknown. Based on previous animal studies, we hypothesized that the consumption of provitamin A carotenoids with dietary lipid would enhance conversion to vitamin A during absorption compared with the consumption of provitamin A carotenoids alone. Two separate sets of 12 healthy men and women were recruited for 2 randomized, 2-way crossover studies. One meal was served with fresh avocado (Persea americana Mill), cultivated variety Hass (delivering 23 g of lipid), and a second meal was served without avocado. In study 1, the source of provitamin A carotenoids was a tomato sauce made from a novel, high–β-carotene variety of tomatoes (delivering 33.7 mg of β-carotene). In study 2, the source of provitamin A carotenoids was raw carrots (delivering 27.3 mg of β-carotene and 18.7 mg of α-carotene). Postprandial blood samples were taken over 12 h, and provitamin A carotenoids and vitamin A were quantified in triglyceride-rich lipoprotein fractions to determine baseline-corrected area under the concentration-vs.-time curve. Consumption of lipid-rich avocado enhanced the absorption of β-carotene from study 1 by 2.4-fold (P < 0.0001). In study 2, the absorption of β-carotene and α-carotene increased by 6.6- and 4.8-fold, respectively (P < 0.0001 for both). Most notably, consumption of avocado enhanced the efficiency of conversion to vitamin A (as measured by retinyl esters) by 4.6-fold in study 1 (P < 0.0001) and 12.6-fold in study 2 (P = 0.0013). These observations highlight the importance of provitamin A carotenoid consumption with a lipid-rich food such as avocado for maximum absorption and conversion to vitamin A, especially in populations in which vitamin A deficiency is prevalent. This trial was registered at clinicaltrials.gov as NCT01432210.

Comparison of high‐performance liquid chromatography/tandem mass spectrometry and high‐performance liquid chromatography/photo‐diode array detection for the quantitation of carotenoids, retinyl esters, α‐tocopherol and phylloquinone in chylomicron‐rich fractions of human plasma

RATIONALE Bioavailability of essential lipophilic micronutrients and carotenoids is of utmost interest for human health, as the consumption of these compounds may help alleviate major nutritional deficiencies, cardiovascular disease, and cancer. High-performance liquid chromatography/photo-diode array detection (HPLC-PDA) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) were compared for the quantitative analysis of α- and β-carotene, β-cryptoxanthin, lutein, lycopene, α-tocopherol, phylloquinone, and several retinyl esters from chylomicron-containing triglyceride rich lipoprotein (TRL) fractions of human plasma obtained from two clinical trials. METHODS After selecting an efficient extraction method for the analytes, both the HPLC/PDA and the HPLC/MS/MS methods were developed and several parameters validated using an HP 1200 series HPLC system interfaced with a HP 1200 series diode-array detector (Agilent Technologies, Santa Clara, CA, USA) and a QTRAP 5500 (AB Sciex, Foster City, CA, USA) via an atmospheric pressure chemical ionization (APCI) probe operated in positive ion mode. RESULTS For lycopene, α- and β-carotene, HPLC/MS/MS was up to 37 times more sensitive than HPLC-PDA. PDA detection was shown to be up to 8 times more sensitive for lutein. MS/MS signals were enhanced by matrix components for lutein and β-cryptoxanthin, as determined by referencing to the matrix-independent PDA signal. In contrast, matrix suppression was observed for retinyl palmitate, α-carotene, and β-carotene. Both detectors showed similar suitability for α-tocopherol, lycopene and retinyl palmitate (representing ~73% of total retinyl esters). MS/MS exclusively allowed the quantitation of minor retinyl esters, phylloquinone, and (Z)-lycopene isomers. CONCLUSIONS HPLC/MS/MS was more sensitive than HPLC-PDA for six of the eight analytes and represents a powerful tool for the analysis of chylomicron samples and potentially other biological samples of limited sample size. When internal standards are available for the target carotenoid, employing MS/MS detection may reduce the necessary blood sample volume, which is particularly advantageous for minimizing risk and discomfort to human subjects during clinical studies.

A Mediterranean-style low-glycemic-load diet increases plasma carotenoids and decreases LDL oxidation in women with metabolic syndrome ☆

Thirty-five women with metabolic syndrome and high plasma low-density lipoprotein (LDL) cholesterol (≥100 mg/dl) participated in a dietary intervention consisting of a Mediterranean-style low-glycemic-load diet for 12 weeks. Participants were randomly allocated to consume diet only (n=15) or diet plus a medical food containing soy protein and plant sterols (n=20). Plasma concentrations of carotenoids, lipoprotein subfractions and oxidized LDL (OxLDL) were measured. Independent of treatment, women had a significant increase in plasma lutein (P<.0001) and β-carotene (P<.0001), while plasma lycopene was reduced (P<.05) after 12 weeks. Low-density lipoprotein cholesterol was reduced from 138±35 to 114±33 mg/dl (P<.0001). In addition, decreases were observed in the atherogenic subfractions: large very low-density lipoprotein (P<.05), small LDL (P<.00001) and medium high-density lipoprotein (P<.05). Oxidized LDL was significantly reduced by 12% in both groups (P<.01). Changes in OxLDL were inversely correlated with plasma lutein (r=-.478, P<.0001). The data indicate that women complied with the dietary regimen by increasing fruits and vegetable intake. Decreased consumption of high-glycemic foods frequently co-consumed with lycopene-rich tomato sauce such as pasta and pizza may be responsible for the lowering of this carotenoid in plasma after 12 weeks. These results also suggest that plasma lutein concentrations may protect against oxidative stress by reducing the concentrations of OxLDL.

Concentration of pro-vitamin A carotenoids in common beef cattle feedstuffs

Quantification of the pro-vitamin A carotenoids in feedstuffs commonly fed to livestock has been ignored for many years. A greater dietary concentration of vitamin A has the potential to limit adipogenesis in cattle, thereby reducing carcass quality and value. A survey of 18 feedstuffs commonly fed to beef cattle was conducted for determination of vitamin A equivalents based on analysis of carotenoids. The pro-vitamin A carotenoids of interest were β-carotene, α-carotene, and β-cryptoxanthin. Collaborators in 5 states collected the feedstuffs and then shipped them to The Ohio State University for compilation and analysis. Carotenoids were extracted from the feedstuffs and then quantified using HPLC with photodiode array analysis. Fresh fescue pasture contained approximately 10 times more vitamin A equivalents than hay and 5 times more than corn silage (39,865, 2,750, and 6,900 IU of vitamin A/kg of DM for fresh pasture, hay, and corn silage, respectively). Beta-cryptoxanthin and α-carotene could not be detected in any forage samples. Hay and corn silage vitamin A equivalents decreased over extended periods of time from harvest to sample collection. Corn was the only feedstuff to have appreciable concentrations of all 3 pro-vitamin A carotenoids quantified. Corn processing had a minimal impact on the vitamin A equivalents. High-moisture corn contained 54% more vitamin A equivalents than whole shelled corn (378 and 174 IU of vitamin A/kg of DM, respectively). Pro-vitamin A carotenoids were more concentrated in corn coproducts than in whole shelled corn. The drying of distillers grains with solubles may significantly degrade β-carotene (800 and 480 IU/kg of DM for wet and dry distillers grains, respectively). Soybean-based feedstuffs contain a small concentration of pro-vitamin A carotenoids, at 55 and 45 IU of vitamin A/kg of DM for soybean meal and soybean hulls, respectively. Overall, there was considerable variation in the pro-vitamin A content of feedstuffs based on location and storage conditions. An extensive analysis of feedstuffs would need to be conducted for an accurate estimation of the vitamin A content of feedlot cattle diets.

Lycopene dietary intervention: a pilot study in patients with heart failure.

Background/Objectives:Heart failure (HF) is a condition of chronic exacerbations and injury resulting from an intricate relationship between biochemical and biological mechanisms. Inflammation can be a significant contributor in the pathophysiology of HF. Antioxidants may slow the progression of HF because of their ability to inhibit damaging inflammatory processes. The purpose of this study was to test a dietary intervention in patients with HF to assess the impact of lycopene on biomarkers of inflammation. Subjects/Methods:Forty participants with HF were randomly assigned to 1 of 2 groups: lycopene intervention and usual care. The lycopene intervention group received 29.4 mg of lycopene intake per day by drinking an 11.5 oz serving of V8 100% vegetable juice for 30 days. We obtained serum lycopene, uric acid, C-reactive protein (CRP), and b-type natriuretic peptide to determine the impact of the intervention. Results:Plasma lycopene levels increased in the intervention group compared with the usual care group (0.51 &mgr;mol/L to 0.76 &mgr;mol/L, P = .002; 0.56 &mgr;mol/L to 0.58 &mgr;mol/L). C-reactive protein levels decreased significantly in the intervention group in women and but not in men (P = .04). The preintervention CRP level for women was 5.9 ± 3.7 mg/dL and for men was 2.2 ± 2.1 mg/dL. The postintervention CRP level for women was 4.5 ± 3.6 mg/dL and for men was 2.4 ± 2.1 mg/dL. Conclusions:These findings suggest that the antioxidants in a 30-day intervention of V8 juice affect CRP levels in a sample of female patients with HF.

Sex differences in skin carotenoid deposition and acute UVB‐induced skin damage in SKH‐1 hairless mice after consumption of tangerine tomatoes

SCOPE UVB exposure, a major factor in the development of skin cancer, has differential sex effects. Tomato product consumption reduces the intensity of UVB-induced erythema in humans, but the mechanisms are unknown. METHODS AND RESULTS Four-week-old SKH-1 hairless mice (40 females, 40 males) were divided into two feeding groups (control or with 10% tangerine tomatoes naturally rich in UV-absorbing phytoene and phytofluene) and two UV exposure groups (with or without UV). After 10 weeks of feeding, the UV group was exposed to a single UV dose and sacrificed 48 h later. Blood and dorsal skin samples were taken for carotenoid analysis. Dorsal skin was harvested to assess sex and UV effects on carotenoid deposition, inflammation (skinfold thickness, myeloperoxidase levels), and DNA damage (cyclobutane pyrimidine dimers, p53). Females had significantly higher levels of both skin and blood carotenoids relative to males. UV exposure significantly reduced skin carotenoid levels in females but not males. Tomato consumption attenuated acute UV-induced increases in CPD in both sexes, and reduced myeloperoxidase activity and percent p53 positive epidermal cells in males. CONCLUSION Tangerine tomatoes mediate acute UV-induced skin damage in SKH-1 mice via reduced DNA damage in both sexes, and through reduced inflammation in males.

Relative contribution of α-carotene to postprandial vitamin A concentrations in healthy humans after carrot consumption

Background: Asymmetric α-carotene, a provitamin A carotenoid, is cleaved to produce retinol (vitamin A) and α-retinol (with negligible vitamin A activity). The vitamin A activity of α-carotene-containing foods is likely overestimated because traditional analytic methods do not separate α-retinol derivatives from active retinol.Objective: This study aimed to accurately characterize intestinal α-carotene cleavage and its relative contribution to postprandial vitamin A in humans after consumption of raw carrots.Design: Healthy adults (n = 12) consumed a meal containing 300 g raw carrot (providing 27.3 mg β-carotene and 18.7 mg α-carotene). Triglyceride-rich lipoprotein fractions of plasma were isolated and extracted, and α-retinyl palmitate (αRP) and retinyl palmitate were measured over 12 h postprandially via high-performance liquid chromatography-tandem mass spectrometry. The complete profile of all α-retinyl esters and retinyl esters was measured at 6 h, and total absorption of α- and β-carotene was calculated.Results: αRP was identified and quantified in every subject. No difference in preference for absorption of β- over α-carotene was observed (adjusting for dose, 28% higher, P = 0.103). After absorption, β-carotene trended toward preferential cleavage compared with α-carotene (22% higher, P = 0.084). A large range of provitamin A carotenoid conversion efficiencies was observed, with α-carotene contributing 12-35% of newly converted vitamin A (predicted contribution = 25.5%). In all subjects, a majority of α-retinol was esterified to palmitic acid (as compared with other fatty acids).Conclusions: α-Retinol is esterified in the enterocyte and transported in the blood analogous to retinol. The percentage of absorption of α-carotene from raw carrots was not significantly different from β-carotene when adjusting for dose, although a trend toward higher cleavage of β-carotene was observed. The results demonstrate large interindividual variability in α-carotene conversion. The contribution of newly absorbed α-carotene to postprandial vitamin A should not be estimated but should be measured directly to accurately assess the vitamin A capacity of α-carotene-containing foods. This trial was registered at clinicaltrials.gov as NCT01432210.