Spondence must be addressed. e-mail: [email protected] The on the internet version of this article (accessible at http://jlr.org) consists of supplementary information within the type of two figures.Journal of Lipid Study Volume 55,of carotenoids/retinoids in the plasma matrix for MS evaluation frequently requires in depth and time-consuming extraction/purification procedures which have incorporated: saponification, solid-phase extraction, preparative HPLC, and, in the case of GC-MS evaluation, additional conversion to tert-butyl-dimethylsilyl derivatives (11?8). The aim was to develop an analytical system that involved a simplified extraction procedure, sensitive MS/MS for detection of physiological doses of steady isotopes, and brief LC runtimes so as to become appropriate for high-throughput of samples from human intervention research.Synthesis of retinyl estersRetinyl esters have been synthesized via an enzyme-catalyzed transesterification (19) as follows.Formula of 113451-59-5 Into a dry Schlenk flask, retinyl acetate (33 mg, 0.10 mmol), Novozyme 435 (120 mg), and Aberlyst A-21 (50 mg) were suspended in dry toluene (5 ml). The reaction mixture was stirred below an atmosphere of N2, and five equivalents (0.50 mmol) of the proper acid (palmitic, stearic, linoleic, or oleic) were added. Just after 20 h at room temperature, the reaction mixture was filtered plus the solvent was removed under decreased pressure to offer a mixture (around 1:4) of the preferred retinyl ester and unreacted acid. The resulting mixtures had been used without further purification as LC/MS/MS standards for the corresponding retinyl esters.Subjects and blood collectionMATERIALS AND METHODSChemicalsThe following carotenoid and retinoid (95 all-trans) standards have been purchased from Sigma (St. Louis, MO): -carotene, lycopene, retinol, retinyl acetate, and retinyl palmitate. The [12-, 12-, 13-, 13-, 14-, 14-, 15-, 15-, 20-, 20-13C10] -carotene and [8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 19-, 20-13C10]retinyl acetate (Fig. 1) to be administered to human subjects were custom synthesized by Buchem BV (Apeldoorn, The Netherlands) and certified match for human consumption. Similarly, the [8-, 8-, 9-, 9-, 10-, 10-, 11-, 11-, 13 12-, 12-, 13-, 13-, 14-, 14-, 15-, 15-, 19-, 19-, 20-, 20- C20] carotene, [12-, 13-, 14-, 15-, 20-13C5]retinol, and [10-, 19-, 19-, 19-d4]retinyl palmitate steady isotopes (Fig. 1) have been also bought from Buchem BV. Methanol, propan-2-ol, chloroform, ethanol, ethyl acetate, toluene, and acetic acid were all of HPLC grade and bought from Fisher Scientific (Loughborough, UK).7-Bromo-1H-indole-6-carbonitrile web Butylated hydroxytoluene (BHT), Novozyme 435, and ammonium acetate were obtained from Sigma-Aldrich (St.PMID:23672196 Louis, MO). Aberlyst A-21, linoleic acid, oleic acid, and stearic acid for synthesis of retinyl esters were obtained from Alfa Aesar (Heysham, Lancashire, UK).Healthful male and female volunteers with an age range of 18?5 years have been recruited into the “BetaSNP” dietary intervention study where written informed consent was obtained. Exclusion criteria had been: pregnancy, smoking, higher blood pressure, diabetes, BMI 30, liver/kidney/gastrointestinal disease, lipid metabolic problems, and consumption of multivitamins (containing vitamins A, C, E) or -carotene supplements 3 months before the study begin. The study was conducted in line with the recommendations set forth inside the Declaration of Helsinki, and all procedures involving human subjects have been authorized by the National Investigation Ethics Service (NRES), North East – Sunderland Committee (REC 09/H090.
