RESUMEN
ß-Apocarotenoids are eccentric cleavage products of carotenoids formed by chemical and enzymatic oxidations. They occur in foods containing carotenoids and thus might be directly absorbed from the diet. However, there is limited information about their intestinal absorption. The present research examined the kinetics of uptake and metabolism of ß-apocarotenoids. Caco-2 cells were grown on 6-well plastic plates until a differentiated cell monolayer was achieved. ß-Apocarotenoids were prepared in Tween 40 micelles, delivered to differentiated cells in serum-free medium, and incubated at 37°C for up to 8 h. There was rapid uptake of ß-apo-8'-carotenal into cells, and ß-apo-8'-carotenal was largely converted to ß-apo-8'-carotenoic acid and a minor metabolite that we identified as 5,6-epoxy-ß-apo-8'-carotenol. There was also rapid uptake of ß-apo-10'-carotenal into cells, and ß-apo-10'-carotenal was converted into a major metabolite identified as 5,6-epoxy-ß-apo-10'-carotenol and a minor metabolite that is likely a dihydro-ß-apo-10'-carotenol. Finally, there was rapid cellular uptake of ß-apo-13-carotenone, and this compound was extensively degraded. These results suggest that dietary ß-apocarotenals are extensively metabolized in intestinal cells via pathways similar to the metabolism of retinal. Thus, they are likely not absorbed directly from the diet.
Asunto(s)
Carotenoides/metabolismo , Células CACO-2 , Cromatografía Líquida de Alta Presión , Humanos , Cinética , Espectrometría de Masas , Vitamina A/metabolismo , beta Caroteno/metabolismoRESUMEN
Apocarotenoids are cleavage products of C40 isoprenoid pigments, named carotenoids, synthesized exclusively by plants and microorganisms. The colors of flowers and fruits and the photosynthetic process are examples of the biological properties conferred by carotenoids to these organisms. Mammals do not synthesize carotenoids but obtain them from foods of plant origin. Apocarotenoids are generated upon enzymatic and nonenzymatic cleavage of the parent compounds both in plants and in the tissues of mammals that have ingested carotenoid-containing foods. The best-characterized apocarotenoids are retinoids (vitamin A and its derivatives), generated upon central oxidative cleavage of provitamin A carotenoids, mainly ß-carotene. In addition to the well-known biological actions of vitamin A, it is becoming apparent that nonretinoid apocarotenoids also have the potential to regulate a broad spectrum of critical cellular functions, thus influencing mammalian health. This review discusses the current knowledge about the generation and biological activities of nonretinoid apocarotenoids in mammals.
Asunto(s)
Carotenoides/química , Carotenoides/metabolismo , Animales , Dieta , Análisis de los Alimentos , Humanos , Absorción IntestinalRESUMEN
Consumption of the tomato carotenoid, lycopene, has been associated with favorable health benefits. Some of lycopene's biological activity may be due to metabolites resulting from cleavage of the lycopene molecule. Because of their structural similarity to the retinoic acid receptor (RAR) antagonist, ß-apo-13-carotenone, the "first half" putative oxidative cleavage products of the symmetrical lycopene have been synthesized. All transformations proceed in moderate to good yield and some with high stereochemical integrity allowing ready access to these otherwise difficult to obtain terpenoids. In particular, the methods described allow ready access to the trans isomers of citral (geranial) and pseudoionone, important flavor and fragrance compounds that are not readily available isomerically pure and are building blocks for many of the longer apolycopenoids. In addition, all of the apo-11, apo-13, and apo-15 lycopenals/lycopenones/lycopenoic acids have been prepared. These compounds have been evaluated for their effect on RAR-induced genes in cultured hepatoma cells and, much like ß-apo-13-carotenone, the comparable apo-13-lycopenone and the apo-15-lycopenal behave as RAR antagonists. Furthermore, molecular modeling studies demonstrate that the apo-13-lycopenone efficiently docked into the ligand binding site of RARα. Finally, isothermal titration calorimetry studies reveal that apo-13-lycopenone acts as an antagonist of RAR by inhibiting coactivator recruitment to the receptor.
Asunto(s)
Carotenoides/síntesis química , Carotenoides/farmacología , Receptores de Ácido Retinoico/antagonistas & inhibidores , Carotenoides/química , Carotenoides/metabolismo , Técnicas de Química Sintética , Regulación de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Licopeno , Simulación del Acoplamiento Molecular , Conformación Proteica , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismoRESUMEN
Provitamin A carotenoids are oxidatively cleaved by ß-carotene 15,15'-dioxygenase (BCO1) at the central 15-15' double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, ß-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10' bond to yield an ionone and an apo-10'-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids ß-carotene, α-carotene, and ß-cryptoxanthin. Its catalytic activity with ß-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-ß-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and ß-apocarotenoids. Apo-10'-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-ß-apo-8'-carotenal were also consistently detected in BCO2-ß-cryptoxanthin reaction mixtures. With the exception of this activity with ß-cryptoxanthin, BCO2 cleaves specifically at the 9'-10' bond to produce apo-10'-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this.
Asunto(s)
Pollos/metabolismo , Dioxigenasas/metabolismo , beta Caroteno/metabolismo , Secuencia de Aminoácidos , Animales , Carotenoides/química , Carotenoides/metabolismo , Pollos/genética , Criptoxantinas/química , Criptoxantinas/metabolismo , Dioxigenasas/química , Dioxigenasas/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , beta Caroteno/químicaRESUMEN
ß-Carotene is an important source of vitamin A for the mammalian embryo, which depends on its adequate supply to achieve proper organogenesis. In mammalian tissues, ß-carotene 15,15'-oxygenase (BCO1) converts ß-carotene to retinaldehyde, which is then oxidized to retinoic acid, the biologically active form of vitamin A that acts as a transcription factor ligand to regulate gene expression. ß-Carotene can also be cleaved by ß-carotene 9',10'-oxygenase (BCO2) to form ß-apo-10'-carotenal, a precursor of retinoic acid and a transcriptional regulator per se The mammalian embryo obtains ß-carotene from the maternal circulation. However, the molecular mechanisms that enable its transfer across the maternal-fetal barrier are not understood. Given that ß-carotene is transported in the adult bloodstream by lipoproteins and that the placenta acquires, assembles, and secretes lipoproteins, we hypothesized that the aforementioned process requires placental lipoprotein biosynthesis. Here we show that ß-carotene availability regulates transcription and activity of placental microsomal triglyceride transfer protein as well as expression of placental apolipoprotein B, two key players in lipoprotein biosynthesis. We also show that ß-apo-10'-carotenal mediates the transcriptional regulation of microsomal triglyceride transfer protein via hepatic nuclear factor 4α and chicken ovalbumin upstream promoter transcription factor I/II. Our data provide the first in vivo evidence of the transcriptional regulatory activity of ß-apocarotenoids and identify microsomal triglyceride transfer protein and its transcription factors as the targets of their action. This study demonstrates that ß-carotene induces a feed-forward mechanism in the placenta to enhance the assimilation of ß-carotene for proper embryogenesis.
Asunto(s)
Proteínas Portadoras/biosíntesis , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas Gestacionales/biosíntesis , Embarazo/metabolismo , beta Caroteno/metabolismo , Animales , Transporte Biológico Activo/fisiología , Proteínas Portadoras/genética , Dioxigenasas/genética , Dioxigenasas/metabolismo , Femenino , Ratones , Ratones Noqueados , Embarazo/genética , Proteínas Gestacionales/genética , beta-Caroteno 15,15'-Monooxigenasa/genética , beta-Caroteno 15,15'-Monooxigenasa/metabolismoRESUMEN
Carotenoids are polyenes synthesized in plants and certain microorganisms and are pigments used by plants and animals in various physiological processes. Some of the over 600 known carotenoids are capable of metabolic conversion to the essential nutrient vitamin A (retinol) in higher animals. Vitamin A also gives rise to a number of other metabolites which, along with their analogs, are known as retinoids. To facilitate discussion about these important molecules, a nomenclature is required to identify specific substances. The generally accepted rules for naming these important molecules have been agreed to by various Commissions of the International Union of Pure and Applied Chemistry and International Union of Biochemistry. These naming conventions are explained along with comparisons to more systematic naming rules that apply for these organic chemicals. Identification of the carotenoids and retinoids has been advanced by their chemical syntheses, and here, both classical and modern methods for synthesis of these molecules, as well as their analogs, are described. Because of their importance in biological systems, sensitive methods for the detection and quantification of these compounds from various sources have been essential. Early analyses that relied on liquid adsorption and partition chromatography have given way to high-performance liquid chromatography (HPLC) coupled with various detection methods. The development of HPLC coupled to mass spectrometry, particularly LC/MS-MS with Multiple Reaction Monitoring, has resulted in the greatest sensitivity and specificity in these analyses.
Asunto(s)
Carotenoides , Animales , Carotenoides/análisis , Carotenoides/química , Carotenoides/clasificación , Cromatografía/métodos , Predicción , Humanos , Estructura Molecular , Plantas/química , Retinoides/análisis , Retinoides/química , Retinoides/clasificación , Terminología como Asunto , Vitamina A/química , Vitamina A/metabolismoRESUMEN
The xanthophylls, lutein and zeaxanthin, are dietary carotenoids that selectively accumulate in the macula of the eye providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the retinal pigment epithelium (RPE). Xanthophylls and ß-carotene mostly associate with HDL and LDL, respectively. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism, while LDL binds via the LDL receptor. Using an in-vitro, human RPE cell model (ARPE-19), we studied the mechanisms of carotenoid uptake into the RPE by evaluating kinetics of cell uptake when delivered in serum or isolated LDL or HDL. For lutein and ß-carotene, LDL delivery resulted in the highest rates and extents of uptake. In contrast, HDL was more effective in delivering zeaxanthin and meso-zeaxanthin leading to the highest rates and extents of uptake of all four carotenoids. Inhibitors of SR-B1 suppressed zeaxanthin delivery via HDL. Results show a selective HDL-mediated uptake of zeaxanthin and meso-zeaxanthin via SR-B1 and a LDL-mediated uptake of lutein. This demonstrates a plausible mechanism for the selective accumulation of zeaxanthin greater than lutein and xanthophylls over ß-carotene in the retina. We found no evidence of xanthophyll metabolism to apocarotenoids or lutein conversion to meso-zeaxanthin.
Asunto(s)
Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Luteína/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Receptores Depuradores de Clase B/metabolismo , Zeaxantinas/metabolismo , Transporte Biológico Activo/fisiología , Línea Celular , Humanos , Epitelio Pigmentado de la Retina/citología , beta Caroteno/metabolismoRESUMEN
Retinoid X receptor (RXRα) is activated by 9-cis-retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other nuclear receptors. We have previously demonstrated that ß-apo-13-carotenone, an eccentric cleavage product of ß-carotene, antagonizes the activation of RXRα by 9cRA in mammalian cells overexpressing this receptor. However, the molecular mechanism of ß-apo-13-carotenone's modulation on the transcriptional activity of RXRα is not understood and is the subject of this report. We performed transactivation assays using full-length RXRα and reporter gene constructs (RXRE-Luc) transfected into COS-7 cells, and luciferase activity was examined. ß-Apo-13-carotenone was compared with the RXRα antagonist UVI3003. The results showed that both ß-apo-13-carotenone and UVI3003 shifted the dose-dependent RXRα activation by 9cRA. In contrast, the results of assays using a hybrid Gal4-DBD:RXRαLBD receptor reporter cell assay that detects 9cRA-induced coactivator binding to the ligand binding domain demonstrated that UVI3003 significantly inhibited 9cRA-induced coactivator binding to RXRαLBD, but ß-apo-13-carotenone did not. However, both ß-apo-13-carotenone and UVI3003 inhibited 9-cRA induction of caspase 9 gene expression in the mammary carcinoma cell line MCF-7. To resolve this apparent contradiction, we investigated the effect of ß-apo-13-carotenone on the oligomeric state of purified recombinant RXRαLBD. ß-Apo-13-carotenone induces tetramerization of the RXRαLBD, although UVI3003 had no effect on the oligomeric state. These observations suggest that ß-apo-13-carotenone regulates RXRα transcriptional activity by inducing the formation of the "transcriptionally silent" RXRα tetramer.
Asunto(s)
Carotenoides/farmacología , Multimerización de Proteína/efectos de los fármacos , Receptor alfa X Retinoide/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Células COS , Caspasa 9/biosíntesis , Caspasa 9/genética , Línea Celular Tumoral , Chlorocebus aethiops , Ácidos Cumáricos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Ratones , Multimerización de Proteína/fisiología , Receptor alfa X Retinoide/antagonistas & inhibidores , Receptor alfa X Retinoide/genética , Tetrahidronaftalenos/farmacología , Transcripción Genética/fisiologíaRESUMEN
ß-Carotene 15-15'-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O2 and H2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and ß-carotene in either (16)O2-H2(18)O or (18)O2-H2(16)O medium for 15 min at 37 °C, and the relative amounts of (18)O-retinal and (16)O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O2 gas used. Together with the data from (18)O-retinal-H2(16)O and (16)O-retinal-H2(18)O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O2 into retinal. Thus, BCO1 is a dioxygenase.
Asunto(s)
Dioxigenasas/química , Oxígeno/química , Retinaldehído/química , Vitamina A/biosíntesis , Dioxigenasas/genética , Dioxigenasas/metabolismo , Humanos , Oxígeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinaldehído/genética , Retinaldehído/metabolismo , Vitamina A/química , Vitamina A/genéticaRESUMEN
The intestine and liver are crucial organs for vitamin A uptake and storage. Liver accounts for 70% of total body retinoid stores. Vitamin A deficiency (VAD) is a major micronutrient deficiency around the world. The provitamin A carotenoid, ß-carotene, is a significant source of vitamin A in the diet. ß-Carotene 15,15' oxygenase-1 (BCO1) and ß-carotene 9',10' oxygenase-2 (BCO2) are the two known carotenoid cleavage enzymes in humans. BCO1 and BCO2 are highly expressed in liver and intestine. Hepatocytes and hepatic stellate cells are two main cell types involved in the hepatic metabolism of retinoids. Stellate-like cells in the intestine also show ability to store vitamin A. Liver is also known to accumulate carotenoids, however, their uptake, retention and metabolism in specific liver and intestinal cell types is still unknown. Hence, we studied the cellular and subcellular expression and localization of BCO1 and BCO2 proteins in rat liver and intestine. We demonstrate that both BCO1 and BCO2 proteins are localized in hepatocytes and mucosal epithelium. We also show that BCO1 is also highly expressed in hepatic stellate cells (HSC) and portal endothelial cells in liver. At the subcellular level in liver, BCO1 is found in cytosol, while BCO2 is found in mitochondria. In intestine, immunohistochemistry showed strong BCO1 immunoreactivity in the duodenum, particularly in Brunner's glands. Both BCO1 and BCO2 showed diffuse presence along epithelia with strong immunoreactivity in endothelial cells and in certain epithelial cells which warrant further investigation as possible intestinal retinoid storage cells.
Asunto(s)
Ácido Graso Desaturasas/metabolismo , Intestinos/enzimología , Hígado/enzimología , beta-Caroteno 15,15'-Monooxigenasa/metabolismo , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Células Epiteliales/enzimología , Intestinos/citología , Espacio Intracelular/metabolismo , Hígado/citología , Transporte de Proteínas , RatasRESUMEN
ß-Apo-carotenoids, including ß-apo-13-carotenone and ß-apo-14'-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of ß-apo-13-carotenone and ß-apo-14'-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all-trans-retinoic acid. Treatment of P19 cells with either compound failed to block all-trans-retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these ß-apo-carotenoids accumulates in P19 cells. All-trans-retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of ß-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two ß-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either ß-apo-13-carotenone or ß-apo-10'-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all-trans-retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some ß-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation.
Asunto(s)
Adipocitos/citología , Adipocitos/efectos de los fármacos , Carotenoides/farmacología , Diferenciación Celular/efectos de los fármacos , Células 3T3-L1 , Animales , Ratones , Receptores de Ácido Retinoico/antagonistas & inhibidores , Tretinoina/farmacologíaRESUMEN
Humans cannot synthesize vitamin A and thus must obtain it from their diet. ß-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of ß-carotene with a Vmax = 197.2 nmol retinal/mg BCO1 × h, Km = 17.2 µM and catalytic efficiency kcat/Km = 6098 M(-1) min(-1). The enzyme also catalyzed the oxidative cleavage of α-carotene, ß-cryptoxanthin, and ß-apo-8'-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of ß-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of ß-carotene. The shorter ß-apocarotenals (ß-apo-10'-carotenal, ß-apo-12'-carotenal, ß-apo-14'-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-ß-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.
Asunto(s)
Carotenoides/química , beta-Caroteno 15,15'-Monooxigenasa/química , Carotenoides/metabolismo , Catálisis , Humanos , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , beta-Caroteno 15,15'-Monooxigenasa/genética , beta-Caroteno 15,15'-Monooxigenasa/aislamiento & purificación , beta-Caroteno 15,15'-Monooxigenasa/metabolismoRESUMEN
Dietary carotenoids like ß-carotene are converted within the body either to retinoid, via ß-carotene-15,15'-dioxygenase (BCO1), or to ß-apo-carotenoids, via ß-carotene-9',10'-oxygenase 2. Some ß-apo-carotenoids are potent antagonists of retinoic acid receptor (RAR)-mediated transcriptional regulation, which is required to ensure normal heart development and functions. We established liquid chromatography tandem mass spectrometery methods for measuring concentrations of 10 ß-apo-carotenoids in mouse plasma, liver, and heart and assessed how these are influenced by Bco1 deficiency and ß-carotene intake. Surprisingly, Bco1(-/-) mice had an increase in heart levels of retinol, nonesterified fatty acids, and ceramides and a decrease in heart triglycerides. These lipid changes were accompanied by elevations in levels of genes important to retinoid metabolism, specifically retinol dehydrogenase 10 and retinol-binding protein 4, as well as genes involved in lipid metabolism, including peroxisome proliferator-activated receptor-γ, lipoprotein lipase, Cd36, stearoyl-CoA desaturase 1, and fatty acid synthase. We also obtained evidence of compromised heart function, as assessed by two-dimensional echocardiography, in Bco1(-/-) mice. However, the total absence of Bco1 did not substantially affect ß-apo-carotenoid concentrations in the heart. ß-Carotene administration to matched Bco1(-/-) and wild-type mice elevated total ß-apo-carotenal levels in the heart, liver, and plasma and total ß-apo-carotenoic acid levels in the liver. Thus, BCO1 modulates heart metabolism and function, possibly by altering levels of cofactors required for the actions of nuclear hormone receptors.
Asunto(s)
Cardiopatías/genética , Metabolismo de los Lípidos/genética , Retinoides/metabolismo , beta-Caroteno 15,15'-Monooxigenasa/deficiencia , beta-Caroteno 15,15'-Monooxigenasa/genética , Animales , Carotenoides/metabolismo , Cardiopatías/enzimología , Cardiopatías/metabolismo , Homeostasis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismoRESUMEN
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-ß-carotene variety of tomatoes (delivering 33.7 mg of ß-carotene). In study 2, the source of provitamin A carotenoids was raw carrots (delivering 27.3 mg of ß-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 ß-carotene from study 1 by 2.4-fold (P < 0.0001). In study 2, the absorption of ß-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.
Asunto(s)
Daucus carota/química , Persea , Periodo Posprandial/fisiología , Solanum lycopersicum/química , Vitamina A/farmacocinética , Adulto , Disponibilidad Biológica , Carotenoides/farmacocinética , Estudios Cruzados , Dieta , Femenino , Voluntarios Sanos , Humanos , Estilo de Vida , Lipoproteínas/metabolismo , Masculino , Encuestas y Cuestionarios , Triglicéridos/metabolismo , Adulto Joven , beta Caroteno/farmacocinéticaRESUMEN
Vitamin A was recognized as an essential nutrient 100 years ago. In the 1930s, it became clear that dietary ß-carotene was cleaved at its central double to yield vitamin A (retinal or ß-apo-15'-carotenal). Thus a great deal of research has focused on the central cleavage of provitamin A carotenoids to form vitamin A (retinoids). The mechanisms of formation and the physiological role(s) of noncentral (eccentric) cleavage of both provitamin A carotenoids and nonprovitamin A carotenoids has been less clear. It is becoming apparent that the apocarotenoids exert unique biological activities themselves. These compounds are found in the diet and thus may be absorbed in the intestine, or they may form from enzymatic or nonenzymatic cleavage of the parent carotenoids. The mechanism of action of apocarotenoids in mammals is not fully worked out. However, as detailed in this review, they have profound effects on gene expression and work, at least in part, through the modulation of ligand-activated nuclear receptors. Understanding the interactions of apocarotenoids with other lipid-binding proteins, chaperones, and metabolizing enzymes will undoubtedly increase our understanding of the biological roles of these carotenoid metabolites.
Asunto(s)
Carotenoides/metabolismo , Animales , Humanos , MasculinoRESUMEN
ß-Carotene is the major dietary source of provitamin A. Central cleavage of ß-carotene catalyzed by ß-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 ß-carotene at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are ß-apocarotenals and ß-apocarotenones, whose biological functions in mammals are unknown. We used reporter gene assays to show that none of the ß-apocarotenoids significantly activated RARs. Importantly, however, ß-apo-14'-carotenal, ß-apo-14'-carotenoic acid, and ß-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 ß-apo-13-carotenone can interact directly with the ligand binding site of the retinoid receptors. ß-Apo-13-carotenone and the ß-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 ß-apo-13-carotenone was present in human plasma. These findings suggest that ß-apocarotenoids function as naturally occurring retinoid antagonists. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary ß-carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer.
Asunto(s)
Carotenoides/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , beta Caroteno/metabolismo , Animales , Unión Competitiva , Células COS , Carotenoides/química , Carotenoides/farmacología , Chlorocebus aethiops , Sistema Enzimático del Citocromo P-450 , Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Modelos Moleculares , Estructura Molecular , Ensayo de Unión Radioligante , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/genética , Ácido Retinoico 4-Hidroxilasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Activación Transcripcional/efectos de los fármacos , Tretinoina/farmacología , Tritio , beta Caroteno/químicaRESUMEN
Vitamin A is an essential nutrient for humans and is converted to the visual chromophore, 11-cis-retinal, and to the hormone, retinoic acid. Vitamin A in animal-derived foods is found as long chain acyl esters of retinol and these are digested to free fatty acids and retinol before uptake by the intestinal mucosal cell. The retinol is then reesterified to retinyl esters for incorporation into chlylomicrons and absorbed via the lymphatics or effluxed into the portal circulation facilitated by the lipid transporter, ABCA1. Provitamin A carotenoids such as ß-carotene are found in plant-derived foods. These and other carotenoids are transported into the mucosal cell by scavenger receptor class B type I (SR-BI). Provitamin A carotenoids are partly converted to retinol by oxygenase and reductase enzymes and the retinol so produced is available for absorption via the two pathways described above. The efficiency of vitamin A and carotenoid intestinal absorption is determined by the regulation of a number of proteins involved in the process. Polymorphisms in genes for these proteins lead to individual variability in the metabolism and transport of vitamin A and carotenoids. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.
Asunto(s)
Carotenoides/metabolismo , Absorción Intestinal , Mucosa Intestinal/metabolismo , Vitamina A/metabolismo , Animales , Transporte Biológico , Humanos , Absorción Intestinal/genética , Ratones , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Ratas , Retinaldehído/metabolismo , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Tretinoina/metabolismoRESUMEN
The intestinal absorption and metabolism of ß-carotene is of vital importance in humans, especially in populations that obtain the majority of their vitamin A from provitamin A carotenoids. MS has provided a better understanding of the absorption of ß-carotene, the most potent provitamin A carotenoid, through the use of stable isotopes of ß-carotene. We report here an HPLC-MS method that eliminates the need for complicated sample preparation and allows us to detect and quantify newly absorbed d8-ß-carotene as well as its d4-retinyl ester metabolites in human plasma and chylomicron fractions. Both retinoids and ß-carotene were recovered in a single simple extraction that did not involve saponification, thus allowing subsequent quantitation of individual fatty acyl esters of retinol. Separation of d8-ß-carotene and its d4-retinyl ester metabolites was achieved using the same C30 reversed-phase liquid chromatography followed by mass spectrometry in selected ion monitoring and negative atmospheric pressure chemical ionization modes, respectively. Total time for the two successive runs was 30 min. This HPLC-MS method allowed us to quantify the absorption of intact d8-ß-carotene as well as its extent of conversion to d4-retinyl esters in humans after consumption of a single 5 mg dose of d8-ß-carotene.
Asunto(s)
Cromatografía de Fase Inversa/métodos , Espectrometría de Masas/métodos , Retinoides/química , Vitamina A/metabolismo , beta Caroteno/metabolismo , Absorción , Presión Atmosférica , Carotenoides/metabolismo , Colesterol/metabolismo , Cromatografía de Fase Inversa/normas , Humanos , Licopeno , Masculino , Espectrometría de Masas/normas , Estructura Molecular , Retinoides/sangre , Fumar , Factores de Tiempo , beta Caroteno/administración & dosificación , beta Caroteno/sangreRESUMEN
Although esterification of free cholesterol to cholesteryl ester in the liver is known to be catalyzed by the enzyme acyl-coenzyme A:cholesterol acyltransferase, ACAT, the neutral cholesteryl ester hydrolase (nCEH) that catalyzes the reverse reaction has remained elusive. Because cholesterol undergoes continuous cycling between free and esterified forms, the steady-state concentrations in the liver of the two species and their metabolic availability for pathways, such as lipoprotein assembly and bile acid synthesis, depend upon nCEH activity. On the basis of the general characteristics of the family of rat carboxylesterases, we hypothesized that one member, ES-4, was a promising candidate as a hepatic nCEH. Using under- and overexpression approaches, we provide multiple lines of evidence that establish ES-4 as a bona fide endogenous nCEH that can account for the majority of cholesteryl ester hydrolysis in transformed rat hepatic cells and primary rat hepatocytes.
Asunto(s)
Carboxilesterasa/metabolismo , Colesterol/metabolismo , Hepatocitos/enzimología , Hígado/enzimología , Esterol Esterasa/metabolismo , Animales , Carboxilesterasa/genética , Línea Celular Tumoral , Colesterol/genética , Hidrólisis , Ratones , Ratas , Ratas Sprague-Dawley , Esterol Esterasa/genéticaRESUMEN
Naturally occurring retinoids (retinol, retinal, retinoic acid, retinyl esters) are a subclass of ß-apocarotenoids, defined by the length of the polyene side chain. Provitamin A carotenoids are metabolically converted to retinal (ß-apo-15-carotenal) by the enzyme ß-carotene-15,15'-dioxygenase (BCO1) that catalyzes the oxidative cleavage of the central C=C double bond. A second enzyme ß-carotene-9'-10'-dioxygenase cleaves the 9',10' bond to yield ß-apo-10'-carotenal and ß-ionone. Chemical oxidation of the other double bonds leads to the generation of other ß-apocarotenals. Like retinal, some of these ß-apocarotenals are metabolically oxidized to the corresponding ß-apocarotenoic acids or reduced to the ß-apocarotenols, which in turn are esterified to ß-apocarotenyl esters. Other metabolic fates such as 5,6-epoxidation also occur as for retinoids. Whether the same enzymes are involved remains to be understood. ß-Apocarotenoids occur naturally in plant-derived foods and, therefore, are present in the diet of animals and humans. However, the levels of apocarotenoids are relatively low, compared with those of the parent carotenoids. Moreover, human studies show that there is little intestinal absorption of intact ß-apocarotenoids. It is possible that they are generated in vivo under conditions of oxidative stress. The ß-apocarotenoids are structural analogs of the naturally occurring retinoids. As such, they may modulate retinoid metabolism and signaling. In deed, those closest in size to the C-20 retinoids-namely, ß-apo-14'-carotenoids (C-22) and ß-apo-13-carotenone (C-18) bind with high affinity to purified retinoid receptors and function as retinoic acid antagonists in transactivation assays and in retinoic acid induction of target genes. The possible pathophysiologic relevance in human health remains to be determined.