Methods for assessing the interaction of apocarotenoids with vertebrate nuclear receptors.

Apocarotenoids occur in the diets of human and other vertebrates and form from the metabolism of intact carotenoids. The metabolism of the provitamin A carotenoid, ß-carotene, produces ß-apo-15-carotenoic acid or retinoic acid. Retinoic acid functions as a hormone by binding to and activating nuclear retinoic acid receptors to modulate gene transcription. It is likely that other apocarotenoids may similarly modulate other ligand-activated nuclear receptors. This chapter describes in vitro biochemical and biophysical methods to characterize the direct binding of apocarotenoids to nuclear receptors and to assess their effect on binding of transcriptional coactivators to the receptor. It also provides a few widely used methods to study the consequences of nuclear receptor activation in intact cells. The effects of ß-apocarotenoids on retinoid receptors provide examples.

Carotenoids, ß-Apocarotenoids, and Retinoids: The Long and the Short of It.

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.

Enzymology of vertebrate carotenoid oxygenases.

Mammals and higher vertebrates including humans have only three members of the carotenoid cleavage dioxygenase family of enzymes. This review focuses on the two that function as carotenoid oxygenases. ß-Carotene 15,15'-dioxygenase (BCO1) catalyzes the oxidative cleavage of the central 15,15' carbon-carbon double of ß-carotene bond by addition of molecular oxygen. The product of the reaction is retinaldehyde (retinal or ß-apo-15-carotenal). Thus, BCO1 is the enzyme responsible for the conversion of provitamin A carotenoids to vitamin A. It also cleaves the 15,15' bond of ß-apocarotenals to yield retinal and of lycopene to yield apo-15-lycopenal. ß-Carotene 9',10'-dioxygenase (BCO2) catalyzes the cleavage of the 9,10 and 9',10' double bonds of a wider variety of carotenoids, including both provitamin A and non-provitamin A carotenoids, as well as the xanthophylls, lutein and zeaxanthin. Indeed, the enzyme shows a marked preference for utilization of these xanthophylls and other substrates with hydroxylated terminal rings. Studies of the phenotypes of BCO1 null, BCO2 null, and BCO1/2 double knockout mice and of humans with polymorphisms in the enzymes, has clarified the role of these enzymes in whole body carotenoid and vitamin A homeostasis. These studies also demonstrate the relationship between enzyme expression and whole body lipid and energy metabolism and oxidative stress. In addition, relationships between BCO1 and BCO2 and the development or risk of metabolic diseases, eye diseases and cancer have been observed. While the precise roles of the enzymes in the pathophysiology of most of these diseases is not presently clear, these gaps in knowledge provide fertile ground for rigorous future investigations. This article is part of a Special Issue entitled Carotenoids: Recent Advances in Cell and Molecular Biology edited by Johannes von Lintig and Loredana Quadro.

Uptake and metabolism of ß-apo-8'-carotenal, ß-apo-10'-carotenal, and ß-apo-13-carotenone in Caco-2 cells.

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.

Mechanisms of Transport and Delivery of Vitamin A and Carotenoids to the Retinal Pigment Epithelium.

Vision depends on the delivery of vitamin A (retinol) to the retina. Retinol in blood is bound to retinol-binding protein (RBP). Retinal pigment epithelia (RPE) cells express the RBP receptor, STRA6, that facilitates uptake of retinol. The retinol is then converted to retinyl esters by the enzyme lecithin:retinol acyltransferase. The esters are the substrate for RPE65, an enzyme that produces 11-cis retinol, which is converted to 11-cis retinaldehyde for transport to the photoreceptors to form rhodopsin. The dietary xanthophylls, lutein (LUT) and zeaxanthin (ZEA), accumulate in the macula of the eye, providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the RPE. In blood, xanthophylls and ß-carotene mostly associate with high-density lipoprotein (HDL) and low-density lipoprotein (LDL), respectively. Studies using a human RPE cell model evaluate the kinetics of cell uptake when carotenoids are delivered in LDL or HDL. For LUT and ß-carotene, LDL delivery result in the highest rate of uptake. HDL is more effective in delivering ZEA (and meso-ZEA). This selective HDL-mediated uptake of ZEA, via a scavenger receptor and LDL-mediated uptake of LUT and ß-carotene provides a mechanism for the selective accumulation of ZEA > LUT and xanthophylls over ß-carotene in the macula.

Limited appearance of apocarotenoids is observed in plasma after consumption of tomato juices: a randomized human clinical trial.

Background: Nonvitamin A apocarotenoids occur in foods. Some function as retinoic acid receptor antagonists in vitro, though it is unclear if apocarotenoids are absorbed or accumulate to levels needed to elicit biological function. Objective: The aim of this study was to quantify carotenoids and apocarotenoids (ß-apo-8'-, -10'-, -12'-, and -14'-carotenal, apo-6'-, -8'-, -10'-, -12'-, and -14'-lycopenal, retinal, acycloretinal, ß-apo-13-carotenone, and apo-13-lycopenone) in human plasma after controlled consumption of carotenoid-rich tomato juices. Design: Healthy subjects (n = 35) consumed a low-carotenoid diet for 2 wk, then consumed 360 mL of high-ß-carotene tomato juice (30.4 mg of ß-carotene, 34.5 µg total ß-apocarotenoids/d), high-lycopene tomato juice (42.5 mg of lycopene, 119.2 µg total apolycopenoids/d), or a carotenoid-free control (cucumber juice) per day for 4 wk. Plasma was sampled at baseline (after washout) and after 2 and 4 wk, and analyzed for carotenoids and apocarotenoids using high-pressure liquid chromatography (HPLC) and HPLC-tandem mass spectrometry, respectively. The methods used to analyze the apocarotenoids had limits of detection of ∼ 100 pmol/L. Results: Apocarotenoids are present in tomato juices at 0.1-0.5% of the parent carotenoids. Plasma lycopene and ß-carotene increased (P < 0.001) after consuming high-lycopene and ß-carotene tomato juices, respectively, while retinol remained unchanged. ß-Apo-13-carotenone was found in the blood of all subjects at every visit, although elevated (P < 0.001) after consuming ß-carotene tomato juice for 4 wk (1.01 ± 0.27 nmol/L) compared with both baseline (0.37 ± 0.17 nmol/L) and control (0.46 ± 0.11 nmol/L). Apo-6'-lycopenal was detected or quantifiable in 29 subjects, while ß-apo-10'- and 12'-carotenal were detected in 6 and 2 subjects, respectively. No other apolycopenoids or apocarotenoids were detected. Conclusions: ß-Apo-13-carotenone was the only apocarotenoid that was quantifiable in all subjects, and was elevated in those consuming high-ß-carotene tomato juice. Levels were similar to previous reports of all-trans-retinoic acid. Other apocarotenoids are either poorly absorbed or rapidly metabolized or cleared, and so are absent or limited in blood. ß-Apo-13-carotenone may form from vitamin A and its presence warrants further investigation. This trial was registered at clinicaltrials.gov as NCT02550483.

ß-apo-10'-carotenoids support normal embryonic development during vitamin A deficiency.

Vitamin A deficiency is still a public health concern affecting millions of pregnant women and children. Retinoic acid, the active form of vitamin A, is critical for proper mammalian embryonic development. Embryos can generate retinoic acid from maternal circulating ß-carotene upon oxidation of retinaldehyde produced via the symmetric cleavage enzyme ß-carotene 15,15'-oxygenase (BCO1). Another cleavage enzyme, ß-carotene 9',10'-oxygenase (BCO2), asymmetrically cleaves ß-carotene in adult tissues to prevent its mitochondrial toxicity, generating ß-apo-10'-carotenal, which can be converted to retinoids (vitamin A and its metabolites) by BCO1. However, the role of BCO2 during mammalian embryogenesis is unknown. We found that mice lacking BCO2 on a vitamin A deficiency-susceptible genetic background (Rbp4-/-) generated severely malformed vitamin A-deficient embryos. Maternal ß-carotene supplementation impaired fertility and did not restore normal embryonic development in the Bco2-/-Rbp4-/- mice, despite the expression of BCO1. These data demonstrate that BCO2 prevents ß-carotene toxicity during embryogenesis under severe vitamin A deficiency. In contrast, ß-apo-10'-carotenal dose-dependently restored normal embryonic development in Bco2-/-Rbp4-/- but not Bco1-/-Bco2-/-Rbp4-/- mice, suggesting that ß-apo-10'-carotenal facilitates embryogenesis as a substrate for BCO1-catalyzed retinoid formation. These findings provide a proof of principle for the important role of BCO2 in embryonic development and invite consideration of ß-apo-10'-carotenal as a nutritional supplement to sustain normal embryonic development in vitamin A-deprived pregnant women.

Apocarotenoids: Emerging Roles in Mammals.

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.

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 ß-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 ß-carotene was calculated.Results: αRP was identified and quantified in every subject. No difference in preference for absorption of ß- over α-carotene was observed (adjusting for dose, 28% higher, P = 0.103). After absorption, ß-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 ß-carotene when adjusting for dose, although a trend toward higher cleavage of ß-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.

Synthesis of apo-13- and apo-15-lycopenoids, cleavage products of lycopene that are retinoic acid antagonists.

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.

Carotenoids and Retinoids: Nomenclature, Chemistry, and Analysis.

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.

Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins.

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.

Complementary shifts in photoreceptor spectral tuning unlock the full adaptive potential of ultraviolet vision in birds.

Color vision in birds is mediated by four types of cone photoreceptors whose maximal sensitivities (λmax) are evenly spaced across the light spectrum. In the course of avian evolution, the λmax of the most shortwave-sensitive cone, SWS1, has switched between violet (λmax > 400 nm) and ultraviolet (λmax < 380 nm) multiple times. This shift of the SWS1 opsin is accompanied by a corresponding short-wavelength shift in the spectrally adjacent SWS2 cone. Here, we show that SWS2 cone spectral tuning is mediated by modulating the ratio of two apocarotenoids, galloxanthin and 11',12'-dihydrogalloxanthin, which act as intracellular spectral filters in this cell type. We propose an enzymatic pathway that mediates the differential production of these apocarotenoids in the avian retina, and we use color vision modeling to demonstrate how correlated evolution of spectral tuning is necessary to achieve even sampling of the light spectrum and thereby maintain near-optimal color discrimination.

ß-Apo-10'-carotenoids Modulate Placental Microsomal Triglyceride Transfer Protein Expression and Function to Optimize Transport of Intact ß-Carotene to the Embryo.

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.

Substrate Specificity of Purified Recombinant Chicken ß-Carotene 9',10'-Oxygenase (BCO2).

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.

Cellular localization of ß-carotene 15,15' oxygenase-1 (BCO1) and ß-carotene 9',10' oxygenase-2 (BCO2) in rat liver and intestine.

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.

Actions of ß-apo-carotenoids in differentiating cells: differential effects in P19 cells and 3T3-L1 adipocytes.

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.

ß-Apo-13-carotenone regulates retinoid X receptor transcriptional activity through tetramerization of the receptor.

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.

Cardiac dysfunction in ß-carotene-15,15'-dioxygenase-deficient mice is associated with altered retinoid and lipid metabolism.

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.

Avocado consumption enhances human postprandial provitamin A absorption and conversion from a novel high-ß-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-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.