THE DETERMINATION OF DIKETO-l-GULONIC ACID, DEHYDRO-l-ASCORBIC ACID, AND l-ASCORBIC ACID IN THE SAME TISSUE EXTRACT BY THE 2,4-DINITROPHENYLHYDRAZINE METHOD

Abstract

By the Roe and Kuether (1) method for the determination of vitamin C dehydro-Z-ascorbic (DHA) and diketo-Z-gulonic acid (DKA) are not differentiated. The basic principle of this method, however., is adaptable to the determination of Z-ascorbic acid (AsA), DHA, and DKA in the presence of each other. It is the purpose of this paper to report methods for such determinations. The first problem to overcome in this work was the extraction of the three compounds from tissues. This must be done by techniques that will not permit the oxidation of AsA to DHA and will keep at a minimum the ‘change of DHA into DKA. Roe and Oesterling (2) prevented the oxidation of AsA in plant tissues by extracting with a metaphosphoric acid solution containing 1 per cent of thiourea. However, in animal tissues thiourea will not adequately prevent the oxidation of AsA during extraction because of the powerful oxidant effect of oxyhemoglobin. The problem was solved by adding SnCla in 10 per cent concentration to the HP03 solution; SnClz effectively reduces the oxyhemoglobin of animal tissues. Furthermore SnCh is a more convenient reagent, since it can be removed later in the procedure. The procedure developed consists of grinding the tissue under 10 per cent solution of stannous chloride in 5 per cent HPOI, after which enough 5 per cent metaphosphoric acid solution is added to make the SnCh. concentration 0.5 per cent. From data on melting points, nitrogen content, and chromatographic adsorption Penney and Zilva (3) came to the conclusion that the 2,4-dinitrophenylhydrazine derivatives of AsA, DHA, and DKA are the same compound. Absorption curves of the derivatives of DHA and DKA, prepared by ourselves, also indicate that these derivatives are identical substances. Penney and Zilva (3) have postulated that 2,4-dinitrophenylhydrazine couples only with DKA and, therefore, DHA gives rise to a derivative in