The determination of vitamin A in animal tissues and its presence in the liver of the vitamin A-deficient rat.

Our attention was directed to this problem during a study of the effect of vitamin A deficiency on ubiquinone concentration in the rat, during which it was necessary to determine relatively small amounts of vitamin A in certain tissues (Edwin, Runyan, Green & Diplock, 1962). The results of that work suggested that the amounts of vitamin A found by us were often considerably higher than expected. Accordingly, several methods for measuring vitamin A in animal tissues were investigated. In the usual methods for determining vitamin A, the tissue is first digested with alkali and the lipid fraction (saponified or partially saponified) is then extracted with a solvent (nearly always diethyl ether) and finally dissolved in chloroform before measurement of the vitamin A by use of the antimony trichloride reagent. Most methods published are only simple modifications of this sequence. Liver, which usually contains many times more vitamin A than other tissues, is occasionally treated differently and assayed without preliminary alkali treatment. Thompson, Ganguly & Kon (1949) have already criticized the use of total tissue saponification and preferred to use a direct-extraction method for determining vitamin A in either intestine or liver. We have selected for study three exemplary methods that seem to cover the range used by most workers and compared them with the method used by us. The methods were compared for normal rat liver and also for rat kidney, which is a suitable tissue containing only small amounts of vitamin A. Alkali-digestion method. This was the method of Davies ( I 933), who described it in some detail, although it had essentially been used for some years by Moore (1930) and was originally due to Rosenheim & Webster (1927). The tissue is heated on a steam-bath with 5 yo aqueous KOH for between 10 and 90 min, depending on the time taken to complete digestion. After addition of a measured amount of alcohol, the digest, which may not be totally saponified, is extracted with diethyl ether. Total-saponijication method. This was the method of Heaton, Lowe & Morton (1957), who used it to examine the vitamin A content of a range of rat tissues, including liver, kidney and intestine. The main difference from Davies’s method is that much stronger alkali (60 ”/, w/w, KOH) is used and some ethanol is added during a second period of heating under reflux. The tissue and the fat are more or less completely saponified by this treatment. Direct-extraction method. In this procedure, due to Ames, Risley & Harris (1954), liver is ground with anhydrous Na,SO,, and the dry powder is then extracted in the