Echinenone

Lutein, β-cryptoxanthin, β-carotene, lycopene, ammonium acetate, butylatedhydroxytoluene (BHT), sodium hydroxide, potassium hydroxide, L-ascorbic acid, Na₂-ethylenediaminetetraacetic acid, ethyl gallate, and HPLC-grade denatured ethanol were from Sigma-Aldrich (St. Louis, MO). Solvents including ethyl acetate, methanol, isopropyl alcohol, acetone, petroleum ether, hexanes and HCl were purchased from Mallinckrodt Baker (Phillipsburg, NJ, USA). Echinenone, α-cryptoxanthin and α-carotene were from CaroteNature (Lupsingen, Switzerland). Zeaxanthin was from IndoFine (Hillsborough, NJ).

Reverse-phase HPLC analysis was performed to measure serum and tissue retinoid and β-carotene concentrations, as previously described [6 (link),21 (link)]. Briefly, tissues (100–200 mg) were homogenized in PBS. Half of the tissue homogenate, or 100 µL in the case of serum, were used to extract retinoids [21 (link)]. The other half of the tissue homogenate, or 100 µL of serum or lipoprotein fraction, were used to extract β-carotene. Retinol, retinyl ester, and β-carotene were separated on a C18 column (Hichrom Limited, Reading, UK) using acetonitrile, methanol, and methylene chloride (70:15:15, v/v) as the mobile phase. A Dionex Ultimate 3000 HPLC system and a computerized data analysis workstation with Chromeleon software were used. Retinol, retinyl esters and β-carotene were identified by comparing retention times and spectral data of experimental compounds with those of authentic standards. Retinyl acetate (Sigma, St. Louis, MO, USA; for retinoids) and echinenone (CaroteNature, Ostermundigen, Switzerland; for β-carotene) were added as internal standards. Detection limits are as follows: for retinoids-serum <0.1 ng/dL and tissues <1 ng/g; for β-carotene-serum <1 ng/dL and tissues 10 ng/g).