Detection of a Cis eQTL Controlling BMCO1 Gene Expression Leads to the Identification of a QTG for Chicken Breast Meat Color

Classical quantitative trait loci (QTL) analysis and gene expression QTL (eQTL) were combined to identify the causal gene (or QTG) underlying a highly significant QTL controlling the variation of breast meat color in a F2 cross between divergent high-growth (HG) and low-growth (LG) chicken lines. Within this meat quality QTL, BCMO1 (Accession number GenBank: {"type":"entrez-nucleotide","attrs":{"text":"AJ271386","term_id":"7799040","term_text":"AJ271386"}}AJ271386), encoding the β-carotene 15, 15′-monooxygenase, a key enzyme in the conversion of β-carotene into colorless retinal, was a good functional candidate. Analysis of the abundance of BCMO1 mRNA in breast muscle of the HG x LG F2 population allowed for the identification of a strong cis eQTL. Moreover, reevaluation of the color QTL taking BCMO1 mRNA levels as a covariate indicated that BCMO1 mRNA levels entirely explained the variations in meat color. Two fully-linked single nucleotide polymorphisms (SNP) located within the proximal promoter of BCMO1 gene were identified. Haplotype substitution resulted in a marked difference in BCMO1 promoter activity in vitro. The association study in the F2 population revealed a three-fold difference in BCMO1 expression leading to a difference of 1 standard deviation in yellow color between the homozygous birds at this haplotype. This difference in meat yellow color was fully consistent with the difference in carotenoid content (i.e. lutein and zeaxanthin) evidenced between the two alternative haplotypes. A significant association between the haplotype, the level of BCMO1 expression and the yellow color of the meat was also recovered in an unrelated commercial broiler population. The mutation could be of economic importance for poultry production by making possible a gene-assisted selection for color, a determining aspect of meat quality. Moreover, this natural genetic diversity constitutes a new model for the study of β-carotene metabolism which may act upon diverse biological processes as precursor of the vitamin A.     

β-Carotene conversion products and their effects on adipose tissue

Recent epidemiological data suggest that β-carotene may be protective against metabolic diseases in which adipose tissue plays a key role. Adipose tissue constitutes the major β-carotene storage tissue and its functions have been shown to be modulated in response to β-carotene breakdown products, especially retinal produced after cleavage by β-carotene 15,15′-monooxygenase (BCMO1), and retinoic acid arising from oxidation of retinal. However, the possibility exists that β-carotene in its intact form can also affect adipocyte function. Development of a knock out model and identification of a loss-of-function mutation have pointed out BCMO1 as being probably the sole enzyme responsible for provitamin A conversion into retinal in mammals. The utilisation of BCMO1^−/−mice should provide insights on β-carotene effect on its own in the future. In humans, intervention studies have highlighted the huge interindividual variation of β-carotene conversion efficiency, possibly due to genetic polymorphisms, which might impact on response to β-carotene. This brief review discusses the processes involved in β-carotene conversion and the effect of cleavage products on body fat and adipose tissue function.     

Characterization of human β,β-carotene-15,15′-monooxygenase (BCMO1) as a soluble monomeric enzyme

The formal first step in in vitamin A metabolism is the conversion of its natural precursor β,β-carotene (C40) to retinaldehyde (C20). This reaction is catalyzed by the enzyme β,β-carotene-15,15′-monooxygenase (BCMO1). BCMO1 has been cloned from several vertebrate species, including humans. However, knowledge about this protein’s enzymatic and structural properties is scant. Here we expressed human BCMO1 in Spodoptera frugiperda 9 insect cells. Recombinant BCMO1 is a soluble protein that displayed Michaelis–Menten kinetics with a K_M of 14 μM for β,β-carotene. Though addition of detergents failed to increase BCMO1 enzymatic activity, short chain aliphatic detergents such as C₈E₄ and C₈E₆ decreased enzymatic activity probably by interacting with the substrate binding site. Thus we purified BCMO1 in the absence of detergent. Purified BCMO1 was a monomeric enzymatically active soluble protein that did not require cofactors and displayed a turnover rate of about 8 molecules of β,β-carotene per second. The aqueous solubility of BCMO1 was confirmed in mouse liver and mammalian cells. Establishment of a protocol that yields highly active homogenous BCMO1 is an important step towards clarifying the lipophilic substrate interaction, reaction mechanism and structure of this vitamin A forming enzyme.     

The Inhibition of Macrophage Foam Cell Formation by 9-Cis β-Carotene Is Driven by BCMO1 Activity

Atherosclerosis is a major cause of morbidity and mortality in developed societies, and begins when activated endothelial cells recruit monocytes and T-cells from the bloodstream into the arterial wall. Macrophages that accumulate cholesterol and other fatty materials are transformed into foam cells. Several epidemiological studies have demonstrated that a diet rich in carotenoids is associated with a reduced risk of heart disease; while previous work in our laboratory has shown that the 9-cis β-carotene rich alga Dunaliella inhibits atherogenesis in mice. The effect of 9-cis β-carotene on macrophage foam cell formation has not yet been investigated. In the present work, we sought to study whether the 9-cis β-carotene isomer, isolated from the alga Dunaliella, can inhibit macrophage foam cell formation upon its conversion to retinoids. The 9-cis β-carotene and Dunaliella lipid extract inhibited foam cell formation in the RAW264.7 cell line, similar to 9-cis retinoic acid. Furthermore, dietary enrichment with the algal powder in mice resulted in carotenoid accumulation in the peritoneal macrophages and in the inhibition of foam cell formation ex-vivo and in-vivo. We also found that the β-carotene cleavage enzyme β-carotene 15,15’-monooxygenase (BCMO1) is expressed and active in macrophages. Finally, 9-cis β-carotene, as well as the Dunaliella extract, activated the nuclear receptor RXR in hepa1-6 cells. These results indicate that dietary carotenoids, such as 9-cis β-carotene, accumulate in macrophages and can be locally cleaved by endogenous BCMO1 to form 9-cis retinoic acid and other retinoids. Subsequently, these retinoids activate the nuclear receptor RXR that, along with additional nuclear receptors, can affect various metabolic pathways, including those involved in foam cell formation and atherosclerosis.     

Conversion of carotenoids into vitamins A(1) and A(2) in two species of freshwater fish

1. Examination of two zooplankton species predominating in fish ponds, Daphnia magna and Chironomus larvae, revealed the presence of α- and β-carotene, echinenone, canthaxanthin and 3-hydroxy-4-oxo-β-carotene in Daphnia, and β-carotene and cryptoxanthin ester in Chironomus. No specific provitamins A₂ (containing a 3,4-dehydro-β-ionone ring) were detected. 2. Guppies (Lebistes reticulatus) and platies (Xiphophorus variatus) were found to form vitamin A from β-carotene and from its oxygen-containing derivatives isozeaxanthin, canthaxanthin and astaxanthin. Slight conversion into vitamin A₂ seemed to occur simultaneously. 3,4-Dehydro-3′-hydroxy-β-carotene formed little vitamin A, and the latter was mainly of the A₂ type. Lutein was devoid of provitamin A properties. 3. In addition to vitamin A, β-carotene was detected in fish receiving the 4-oxo- and 4-hydroxy-carotenoids. A reaction scheme for the conversion of carotenoids into retinal and and 3,4-dehydroretinal is presented. 4. It is concluded that natural 4-oxo derivatives of β-carotene may play a significant role as vitamin A precursors for fish.     

Cooperation between MEF2 and PPARγ in human intestinal β,β-carotene 15,15'-monooxygenase gene expression

Background  

Vitamin A and its derivatives, the retinoids, are essential for normal embryonic development and maintenance of cell differentiation. β, β-carotene 15,15'-monooxygenase 1 (BCMO1) catalyzes the central cleavage of β-carotene to all-trans retinal and is the key enzyme in the intestinal metabolism of carotenes to vitamin A. However, human and various rodent species show markedly different efficiencies in intestinal BCMO1-mediated carotene to retinoid conversion. The aim of this study is to identify potentially human-specific regulatory control mechanisms of BCMO1 gene expression.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-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 (k_cat/K_m). 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 β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-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-β-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.     

Common Variation in the β-Carotene 15,15′-Monooxygenase 1 Gene Affects Circulating Levels of Carotenoids: A Genome-wide Association Study

Low plasma levels of carotenoids and tocopherols are associated with increased risk of chronic disease and disability. Because dietary intake of these lipid-soluble antioxidant vitamins is only poorly correlated with plasma levels, we hypothesized that circulating carotenoids (vitamin A-related compounds) and tocopherols (vitamin E-related compounds) are affected by common genetic variation. By conducting a genome-wide association study in a sample of Italians (n = 1190), we identified novel common variants associated with circulating carotenoid levels and known lipid variants associated with α-tocopherol levels. Effects were replicated in the Women's Health and Aging Study (n = 615) and in the α-Tocopherol, β-Carotene Cancer Prevention (ATBC) study (n = 2136). In meta-analyses including all three studies, the G allele at rs6564851, near the β-carotene 15,15′-monooxygenase 1 (BCMO1) gene, was associated with higher β-carotene (p = 1.6 × 10⁻²⁴) and α-carotene (p = 0.0001) levels and lower lycopene (0.003), zeaxanthin (p = 1.3 × 10⁻⁵), and lutein (p = 7.3 × 10⁻¹⁵) levels, with effect sizes ranging from 0.10–0.28 SDs per allele. Interestingly, this genetic variant had no significant effect on plasma retinol (p > 0.05). The SNP rs12272004, in linkage disequilibrium with the S19W variant in the APOA5 gene, was associated with α-tocopherol (meta-analysis p = 7.8 × 10⁻¹⁰) levels, and this association was substantially weaker when we adjusted for triglyceride levels (p = 0.002). Our findings might shed light on the controversial relationship between lipid-soluble anti-oxidant nutrients and human health.     

Development of β-Carotene Rich Maize Hybrids through Marker-Assisted Introgression of β-carotene hydroxylase Allele

Development of vitamin A-rich cereals can help in alleviating the widespread problem of vitamin A deficiency. We report here significant enhancement of kernel β-carotene in elite maize genotypes through accelerated marker-assisted backcross breeding. A favourable allele (543 bp) of the β-carotene hydroxylase (crtRB1) gene was introgressed in the seven elite inbred parents, which were low (1.4 µg/g) in kernel β-carotene, by using a crtRB1-specific DNA marker for foreground selection. About 90% of the recurrent parent genome was recovered in the selected progenies within two backcross generations. Concentration of β-carotene among the crtRB1-introgressed inbreds varied from 8.6 to 17.5 µg/g - a maximum increase up to 12.6-fold over recurrent parent. The reconstituted hybrids developed from improved parental inbreds also showed enhanced kernel β-carotene as high as 21.7 µg/g, compared to 2.6 µg/g in the original hybrid. The reconstituted hybrids evaluated at two locations possessed similar grain yield to that of original hybrids. These β-carotene enriched high yielding hybrids can be effectively utilized in the maize biofortification programs across the globe.     

An LC/MS/MS method for stable isotope dilution studies of β-carotene bioavailability,bioconversion, and vitamin A status in humans

Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as β-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [¹³C₁₀]β-carotene and 1 mg [¹³C₁₀]retinyl acetate in human subjects over a 2 week period. A reverse phase C₁₈ column and binary mobile phase solvent system separated β-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [¹²C]β-carotene and [¹²C] retinoids; m/z 547→330 and m/z 274→98 for [¹³C₁₀]β-carotene and [¹³C₅] cleavage products; and m/z 279→100 for metabolites of [¹³C₁₀]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [¹³C₁₀]retinyl acetate with [¹³C₁₀]β-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual''s vitamin A status.