Retinyl palmitate

Blood (200 ml) was collected from a 29-year-old healthy male volunteer, and EDTA (5 mm) plasma was prepared. Isolation of TG-rich lipoproteins was performed according to a standard protocol. In brief, plasma was adjusted to a density of d = 1.21 using potassium bromide and overlaid with a discontinuous salt gradient (sodium chloride) with densities of d = 1.063, d = 1.019, and d = 1.006. Samples were centrifuged for 24 h at 40,000 rpm and 4 °C using a Beckman Optima L-90K centrifuge and a SW41 rotor (Beckman Instruments Inc., Palo Alto, CA). After centrifugation, TG-rich lipoproteins mostly consisting of chylomicrons and chylomicron remnants were collected from the top of the tube and stored at −20 °C for further use. For RE enrichment, 300 μl of isolated lipoproteins was mixed with 40 μmol of retinyl palmitate (Sigma-Aldrich) and brought to a total volume of 2 ml using 10 mm phosphate-buffered saline. Then the mixture was sonicated three times for 30 s with an amplitude of 1 (Virsonic 475, Virtis).

The following lipids were purchased from Avanti Polar Lipids: dioleoyl-phosphatidic acid, dioleoyl-phosphatidylcholine, dioleoyl-phosphatidylethanolamine, phosphatidylglycerol (PG), dioleoyl-phosphatidylinositol, and dioleoyl-phosphatidylserine and the corresponding lysophospholipids monooleoyl(sn-1)-lysophosphatidic acid, -lysophosphatidylcholine, -lysophosphatidylethanolamine, -lysophosphatidylglycerol, -lysophosphatidylinositol, and -lysophosphatidylserine; dioctanoyl-phosphatidylcholine and dioctanoyl-PG; bis-dioleoyl cardiolipin; bis(dioleoylglycero)phosphate; N-acylphosphatidylethanolamine; plasmanylcholine; sn-3,3′-oleoyl-BMP(S,S and R,R); 1-oleoyl-N-heptadecanoyl-d-erythro-sphingosine; and 1,2-diacyl-3-O-β-d-galactosyl-sn-glycerol. The following lipids were purchased from Sigma-Aldrich: dioleoylglycerol, trioleoylglycerol, glyceryl trioctanoate, monoolein, retinyl palmitate, arachidyl laurate, cholesteryloleate, and oleoyl-carmitin. Methylpalmitate, ethylpalmitate, propylpalmitate, and butylpalmitate as well as sn-2,2′-oleoyl-BMP(S,S) were synthesized according to published procedures (20 , 21 (link)). Labeled BMP was synthesized using oleic acid ¹³C₁₈ (Sigma-Aldrich) according to Refs. 21 (link).

Rat experiments were done in 1995; all procedures were approved at that time by The Pennsylvania State University's Animal Care and Use Committee. Adult male Sprague-Dawley rats (240–260 g) were purchased from Harlan Teklad; rats were individually housed in an environmentally controlled room with a 12-h light cycle; water and food [AIN-93 G diet (15 (link)) with 2.44 nmol retinyl palmitate (Sigma Chemical Co.) added per gram to provide ∼49 nmol/d] were available at all times for 3 wk before and during absorption studies; 2 d before the studies started, rats were transferred to metabolic cages (Nalge Co.).

Zinc and vitamin A solutions were prepared as outlined in the appendix (Appendix A.1). Briefly, zinc sulfate was obtained from Beantown Chemical (Hudson, NH, USA) and retinyl palmitate was acquired from Sigma-Aldrich (CAS No.: 79–81-2; St. Louis, MO, USA). Zinc and vitamin A intra-amniotic administration concentrations were determined according to the National Research Council recommendations for broiler chicks [36 ]. Further, an assumed body weight of 20 g was utilized based on previously unpublished data. Zinc sulfate was diluted with 18 MΩ H₂O to obtain a standard dose of 40 mg/kg and a marginal dose of 20 mg/kg. retinyl palmitate was solubilized in corn oil, diluted with 18 MΩ H₂O to obtain a 0.5% oil in water solution, and vortexed for the final vitamin A doses of 1500 IU/kg and 100 IU/kg. Vitamin A treatments were prepared in semi-dark conditions. Zinc sulfate and retinyl palmitate samples were stored in complete darkness at −20 °C until intra-amniotic administration.

tert-Butyl methyl ether (99%, catalog 34875, MTBE, chromatographic grade), methanol (≥99.9%, catalog 646377, MeOH, chromatographic grade) and potassium hydroxide (ACS reagent, catalog 1050210250) were acquired from Merck Millipore (Burlington, MA, USA). Retinyl acetate (catalog 46958), retinyl palmitate (catalog 46959-U), cholecalciferol (catalog C9774), ergocalciferol (catalog 47768), 3-phytylmenadione catalog (95271), α-tocopherol (catalog 47783), δ-tocopherol (catalog 47784), γ-tocopherol (catalog T1782), tocopherols (mixed, W530066), lutein (catalog 071068), β-carotene (catalog C4582), β-cryptoxanthin (≥97%, catalog C6368), zeaxanthin (catalog 14681), all-trans-astaxanthin (catalog 41659), and lycopene (catalog 75051) from Sigma-Aldrich (unless stated otherwise, all standards were of analytical grade, St. Louis, MO, USA). Chloroform (ACS reagent, catalog 366919), ethanol (200 proof, ACS reagent, ≥99.5%, 459844), 2-propanol (HPLC Plus, 650447), pyrogallol (ACS reagent, ≥99%, catalog 16040), and sodium sulfate (anhydrous, granular, free-flowing, Redi-Dri™, ACS reagent, ≥99%, catalog 798592) were also acquired from Sigma-Aldrich.

Authentic analytical standards, retinol, retinyl palmitate (cis- and trans-isomer), retinyl acetate (cis- and trans-isomer), α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, cis-phylloquinone (cis-K₁), trans-phylloquinone (trans-K₁), menaquinone-4 (K₂-MK4), cholecalciferol (D₃) and ergocalciferol (D₂) were purchased from Sigma-Aldrich (Poznań, Poland). Vitamin D compounds and β-tocopherol were received from Sigma-Aldrich as standard solutions, 1 mg/mL in ethanol and 50 mg/mL in hexane, respectively. According to standard certificates, retinyl acetate as well as retinyl palmitate and phylloquinone (K₁) contain 5% of cis-isomer. As for the materials for separation and analyses, commercially-available, cold-pressed hemp seed oil was used as well as the waste fish oil delivered by the A-SENSE company (Puławy, Poland). Each standard solution was injected alone to measure the corresponding retention time and UV spectrum (Sigma-Aldrich, Poznań, Poland).