Retinyl esters, preparation

In our studies, we have administered tritium-labeled vitamin A in one of its two physiological plasma transport vehicles (associated with either retinol-binding protein or chylomicrons) so that tracer data can be extrapolated to the vitamin A compounds of interest (retinol, retinyl esters, and metabolites). To prepare pH]retinol in its plasma transport complex (Green and Green, 1990b), vitamin A-depleted rats are used as donors to maximize hepatic secretion of the labeled vitamin on acciunulated liver apoRBP. pH]Retinol or pH]retinyl acetate in an emulsion with Tween 40 is administered intravenously to donor rats and blood is harvested 100 min later when plasma radioactivity is maximal. Plasma is isolated and stored under a nitrogen atmosphere at 4°C plasma is used for in vivo studies within 23 days. 

In 1966, Mahadevan et al. (1966) reported that retinyl palmitate hydrolyzing activity was found in the nuclear and mitochondria-Iysosome-rich fractions of rat liver homogenates. The activity was assayed using a reaction mixture containing 0.6 mM retinyl palmitate, 1% sodium taurocholate, and 0.2% Triton X-100. Enzyme activity required the addition of a bile salt and was partially characterized with extracts of acetone powders of rat liver. The pH optimum was 8.6. Enzyme preparations hydrolyzed a variety of long-chain retinyl esters, with the greatest relative activity being seen with retinyl palmitate. 

Hydrolysis of retinyl acetate by highly purified carboxylesterase from liver of several species (pig, ox, man) was reported by Bertram and Krisch (1969). The purified enzyme did not hydrolyze long-chain fatty acid esters of retinol. These findings suggested that previously observed hydrolysis of retinyl acetate by crude liver preparations was due to nonspecific esterases of little or no physiological relevance to hepatic retinyl ester metabolism. 

Analytical SEC has found application for the assay of vitamin E in oils (151,153,183,184), seeds (163), and deodorizer distillates (172), mostly in combination with that of other fat soluble compounds such as retinyl esters, triglycerides, sterols, and fatty acids. This technique has also been used for the determination of a-tocopheryl acetate in pharmaceutical preparations (164,165). SEC can be conducted either on capillary GC columns with EID detection (151,153,163, 172, 184,185) or, alternatively, on EC columns with EID (165) or UV detection (144,164,183). 

Foods are supplemented with vitamin A in the form of standardized preparations of synthetic fatty acyl esters, nowadays chiefly retinyl palmitate. The preparations are available commercially as either dilutions in high-quality vegetable oils containing added vitamin E as an antioxidant or as dry, stabilized beadlets in which the vitamin A is dispersed in a solid matrix of gelatin and sucrose or gum acacia and sucrose. The oily preparations are used to supplement fat-based foods such as margarines the dry preparations are used in dried food products such as milk powder, infant formulas, and dietetic foods (24). 

Free retinol is chemically unstable and does not occur to any significant extent in foods or tissues. Rather, it is present as a variety of esters, mainly retinyl palmitate. Retinyl acetate is generally used as an analytical standard and in pharmaceutical preparations. Dehydroretinol (vitamin A2) is found in freshwater fishes and amphibians it has about half the biological activity of retinol. 

The application of SCF to the extraction of vitamins has been widely reported. Thus, retinyl palmitate and tocopherol acetate have been extracted from a hydrophobic ointment with supercritical CO2 at 40°C and 196 bar for 4 min, the extract analysis being performed by SFC (137). The calibration graphs were linear from 0.5 to 2.5 pg and the recoveries were quantitative. On the other hand, water-soluble vitamins can be extracted mixing them with low substituted hydroxypropil cellulose. Portions were placed in a column to which a reversed micellar extractant was delivered (138). Extraction of vitamins A and E and their esters from tablet preparations prior to FIPLC was performed in the dynamic mode with CO2 at 40°C and 253 bar for 15 min (139). Calibration graphs were linear from 0.02 to 0.8 and from 0.005 to 0.2 mg/mL of vitamins E and A, respectively. The corresponding RSDs (six... 

To prepare pHJvitamin A-labeled chylomicrons (Green et ai, 1993), [ Hjretinol or retinyl acetate is administered intraduodenally to thoracic lymph duct-cannulated donor rats and lymph is collected at 4°C. Chylomicrons containing mainly pHJretinyl esters can be isolated from lymph by preparative ultracentrifugation for administration to recipient rats. Alternatively, aliquots of whole lymph can be injected to minimize handling of the dose (see below). Then the proportions of total lymph radioactivity and vitamin A mass in chylomicrons (typically >8590%) can be determined analytically. In either case, lymph preparations should be used for in vivo studies within 12 days of collection. 

The original work on which subsequent study and synthesis of retinoids are based was carried out by Karrer and Morf (1933) and Heilbron et al, (1932, 1948). In 1931 Karrer et aL were able to determine the structure of retinol (1) using a highly purified vitamin A extract that they had obtained from shark liver oil (von Euler and Karrer, 1931). Using such retinol preparations, the first oily retinol esters [for example retinyl acetate (9)] were prepared (Karrer et al., 1931 Heilbron et al., 1932). 

Because of the inherent instability of labeled retinol, the usual practice is to store this compound as a more stable ester (e.g., retinyl acetate) or to keep it in the retinoic acid stage from which it can be prepared by reduction. From esters like the acetate, retinol can be obtained by hydrolysis with base or even by reduction with lithium aluminum hydride at low temperature. For this conversion, a trans-esterification in methanol is preferred, with catalysis by trace amounts of sodium methoxide followed by purification of the retinol by HPLC (Kaegi and Bupp, 1982). 

Commercial preparations of retinoic acid, retinyl acetate, retinyl palmitate, retinaldehyde, and retinol are pale-yellow-to-yellow crystalline or amorphous solids. Retinol and its esters are low-melting (around 60°C) compounds, and if the room temperature is very high, they may turn to oil. Pure preparations of 3,4-didehydroretinol (vitamin A2 alcohol) can be crystalline or oil. 

Dissolve the appropriate methyl ester in methanol, and saponify as previously described for preparation of retinol from retinyl acetate After saponification, add water, and then acidify with dilute glacial-acetic acid make sure the solution is acidic to litmus paper. (In aqueous-alkaline solution, retinoid carboxylic acids remain as sodium salts, and are not extracted by organic solvents.) Extract the retinoid-carboxylic acid with diethyl ether two or three times (Note that hexane is not a good solvent for these polar retinoids.) Then wash the ether extract with water, and dry it over anhydrous sodium sulfate. Alternatively, if the volume is small, vortex and centrifuge the sample, remove any water, and evaporate the solvent The retinoid-carboxylic acids usually are obtained as yellow solids. Do not add any (not even a trace) HCl to 5,6-epoxy retinoids, because they instantaneously undergo isomerization to 5,8-epoxy retinoids, this change in structure is readily confirmed by the change m absorption spectrum (Table 1). 

---