Retinol preparation from retinyl acetate

Several different quantification procedures for vitamin A have been described in the literature, some using retinol directly as a standard and some using retinyl acetate, which is converted to retinol by saponification. The latter approach is generally preferred, because crystalline all-trans-retinyl acetate is commercially available in high purity and is free from cis isomers. Commercial sources of retinol are oily preparations and are at best only about 70% pure. There are two ways of preparing a retinol standard from retinyl acetate. 

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). 

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). 

For the synthesis of retinaldehyde (2), a large number of oxidation processes have been worked out that permit (2) to be prepared in a very simple manner from commercially available retinyl acetate (9). Thus, when manganese(lV) oxide precipitated in alkaline medium was used and the reaction was carried out in petroleum ether, retinol (1) was converted to retinaldehyde (2), without unde-... 

Retinol-11,12- H2 of low specific activity has been prepared (Isler et al., 1960) based on the work of Isler et al. (1947) on the synthesis of retinyl acetate. The same sequence of reactions as shown in Fig. 8, with several improvements in technique because of the small scale, was applied by Perry et al. (1982) to the preparation of retinoic acid tritiated at very high specific activity. Pure, recrystallized diol (XXVI) was partially hydrogenated with tritium over Lindlar catalyst in the presence of quinoline and the dihydro compound (XXVII), acety-lated in such a way as to afford mainly the monoacetate (XXVIII). Exposure of the acetate at low temperature for a very short time to very dilute hydrogen bromide in methylene chloride gave, after chromatography, pure retinyl-11,12- H2 acetate (XXIXa). Simultaneous hydrolysis and oxidation of the retinyl acetate by silver oxide in aqueous methanolic sodium hydroxide then yielded all-fran -reti-noic-11,12- H2 acid (Vllh). The specific activities obtained ranged from 25 to 40 Ci/mmol. 

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). 

Retinol or its solutions do not keep well even when kept under an inert gas at low temperature. Commercial retinol often contains substantial amounts of impurities because of its instability. Retinal and retinyl acetate are commercially readily available and are quite stable when kept under an inert gas and at low temperatures If a pure standard of retinol is not available, it can be prepared easily from either retinal or retinyl acetate as follows. 

Retmyl acetate is readily available commercially and is relatively inexpensive. Dissolve a few crystals (or a drop, if oily), 1-10 mg as appropnate, of retinyl acetate in methanol (about 1-2 mL). Add about 100 iL of NaOH solution (prepared by adding a drop of water to 1 or 2 pellets of NaOH, and then adding about 1 mL methanol shake until clear) to the retinyl-acetate solution. Make sure that the solution is alkaline to litmus paper. If necessary, add more NaOH solution (excess will not be harmful). Reflux this solution at 60°C for 15-30 min, or keep warm at 50 C for 1-2 h. Analyze the product by TLC, developing the plate as previously described, and look for the fluorescent retinol spot under a UV lamp (366 nm). When no more retinyl acetate is seen, extract the retinol from the solution by adding water (about 1 mL), and hexane... 

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. 

---