An LC/MS method for d8-ß-carotene and d4-retinyl esters: ß-carotene absorption and its conversion to vitamin A in humans.

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.

Hepatic stellate cells are an important cellular site for ß-carotene conversion to retinoid.

Hepatic stellate cells (HSCs) are responsible for storing 90-95% of the retinoid present in the liver. These cells have been reported in the literature also to accumulate dietary ß-carotene, but the ability of HSCs to metabolize ß-carotene in situ has not been explored. To gain understanding of this, we investigated whether ß-carotene-15,15'-monooxygenase (Bcmo1) and ß-carotene-9',10'-monooxygenase (Bcmo2) are expressed in HSCs. Using primary HSCs and hepatocytes purified from wild type and Bcmo1-deficient mice, we establish that Bcmo1 is highly expressed in HSCs; whereas Bcmo2 is expressed primarily in hepatocytes. We also confirmed that HSCs are an important cellular site within the liver for accumulation of dietary ß-carotene. Bcmo2 expression was found to be significantly elevated for livers and hepatocytes isolated from Bcmo1-deficient compared to wild type mice. This elevation in Bcmo2 expression was accompanied by a statistically significant increase in hepatic apo-12'-carotenal levels of Bcmo1-deficient mice. Although apo-10'-carotenal, like apo-12'-carotenal, was readily detectable in livers and serum from both wild type and Bcmo1-deficient mice, we were unable to detect either apo-8'- or apo-14'-carotenals in livers or serum from the two strains. We further observed that hepatic triglyceride levels were significantly elevated in livers of Bcmo1-deficient mice fed a ß-carotene-containing diet compared to mice receiving no ß-carotene. Collectively, our data establish that HSCs are an important cellular site for ß-carotene accumulation and metabolism within the liver.

The formation, occurrence, and function of ß-apocarotenoids: ß-carotene metabolites that may modulate nuclear receptor signaling.

ß-Carotene is the major dietary source of provitamin A. Central cleavage of ß-carotene yields 2 molecules of retinal followed by further oxidation to retinoic acid. Eccentric cleavage of ß-carotene occurs at double bonds other than the central double bond, and the products of these reactions are ß-apocarotenals and ß-apocarotenones. We reviewed recent developments in 3 areas: 1): the enzymatic production of ß-apocarotenoids in higher animals; 2) the occurrence of ß-apocarotenoids in foods and animal tissues; and 3) the biological activity of ß-apocarotenoids, particularly on retinoid receptors. HPLC-mass spectrometry techniques were developed to quantify these compounds in mouse serum and tissues and in foods. ß-Apo-10'- and -12'-carotenals were detected in mouse serum and liver. ß-Apo-8'-, ß-apo-10'-, ß-apo-12'-, and ß-apo-14'-carotenals and ß-apo-13-carotenone were detected in orange-fleshed melons. Transactivation assays were performed to see whether apocarotenoids activate or antagonize retinoid X receptor (RXR) α. Reporter gene constructs and retinoid receptor (RXRα) were transfected into cells, which were used to perform quantitative assays for the activation of this ligand-dependent transcription factor. None of the ß-apocarotenoids significantly activated RXRα. However, ß-apo-13-carotenone antagonized the 9-cis-retinoic acid activation of RXRα. Competitive radioligand binding assays showed that this antagonist competes directly with the agonist for binding to purified receptor, a finding confirmed by molecular modeling studies. These findings suggest that a possible biological function of ß-apocarotenoids is their ability to interfere with nuclear receptor signaling. Recent work showed that ß-apo-13-carotenone is also a high-affinity antagonist of all 3 retinoic acid receptors (RARα, RARß, and RARγ).

Carotene and novel apocarotenoid concentrations in orange-fleshed Cucumis melo melons: determinations of ß-carotene bioaccessibility and bioavailability.

Muskmelons, both cantaloupe (Cucumis melo Reticulatus Group) and orange-fleshed honeydew (C. melo Inodorus Group), a cross between orange-fleshed cantaloupe and green-fleshed honeydew, are excellent sources of ß-carotene. Although ß-carotene from melon is an important dietary antioxidant and precursor of vitamin A, its bioaccessibility/bioavailability is unknown. We compared ß-carotene concentrations from previously frozen orange-fleshed honeydew and cantaloupe melons grown under the same glasshouse conditions, and from freshly harvested field-grown, orange-fleshed honeydew melon to determine ß-carotene bioaccessibility/bioavailability, concentrations of novel ß-apocarotenals, and chromoplast structure of orange-fleshed honeydew melon. ß-Carotene and ß-apocarotenal concentrations were determined by HPLC and/or HPLC-MS, ß-carotene bioaccessibility/bioavailability was determined by in vitro digestion and Caco-2 cell uptake, and chromoplast structure was determined by electron microscopy. The average ß-carotene concentrations (µg/g dry weight) for the orange-fleshed honeydew and cantaloupe were 242.8 and 176.3 respectively. The average dry weights per gram of wet weight of orange-fleshed honeydew and cantaloupe were 0.094 g and 0.071 g, respectively. The bioaccessibility of field-grown orange-fleshed honeydew melons was determined to be 3.2 ± 0.3%, bioavailability in Caco-2 cells was about 11%, and chromoplast structure from orange-fleshed honeydew melons was globular (as opposed to crystalline) in nature. We detected ß-apo-8'-, ß-apo-10', ß-apo-12'-, and ß-apo-14'-carotenals and ß-apo-13-carotenone in orange-fleshed melons (at a level of 1-2% of total ß-carotene). Orange-fleshed honeydew melon fruit had higher amounts of ß-carotene than cantaloupe. The bioaccessibility/bioavailability of ß-carotene from orange-fleshed melons was comparable to that from carrot (Daucus carota).