Factors That Influence the Passage of Ascorbic Acid from Serum to Cells in Human Blood.

Ascorbi acid in serum was determined by the method of Mindlin and Butler (2) with the following modifications. (1) Neither potassium oxalate nor potassium cyanide was used. An anticoagulant was not necessary because the blood was defibrinated by stirring with a glass rod. The possible erroneous influence of added KCN has been demonstrated by other observers (3, 4, 5), as well as in this laboratory. Furthermore, addition of oxalate, cyanide or any other salt seemed undesirable, since changes in cell volume had to be avoided. (2) The strength of the metaphosphoric acid solution is of great significance for the reliability of the colorimetric method. With ascorbic acid a stable color develops; other reducing substances cause progressive fading of the indicator. Mindlin and Butler pointed out, and our own observations agree that, with solutions of pure ascorbic acid, the stability of the reduction of the dye depends on the pH of the final mixture of equal volumes of buffered dye and metaphosphoric acid. The dye solution invariably gave the desired pH of 7.0 when made according to the directions given by the aforementioned authors. Metaphosphoriic acid solutions, however, made up by weight from three different brands, varied widely in strength and were all weaker than theory demanded. Since it is known that metaphosphoric acid is partially converted to the ortho form on standing, the strength of a solution can be adjusted only by standardization. The metaphosphoric acid was, therefore, titrated with 0.1 N sodium hydroxide, using phenolphthalein as an indicator. In agreement with the observations of Mindlin and Butler, stable blanks were obtained when the final pH of the dye-metaphosphoric acid mixture was kept at 4.2 to 4.3. This resulted with solutions of the acid which were 0.21 to 0.22 N. The same normality was attained in