The use of p-chloromercuribenzoic acid in the determination of ascorbic acid with 2:6-dichlorophenolindophenol.

Methods based on the ability of ascorbic acid to reduce the dye 2:6-dichlorophenolindophenol (indophenol) to a colourless compound (Tillmans, Hirsch & Reinshagen, 1928) have been widely used in the estimation of ascorbic acid. Many other substances, including thiol compounds, thiosulphates, hydrogen sulphide, polyphenols and reductones (Lugg, 1942; Harris & Mapson, 1947) also decolorize indophenol and may therefore interfere with the determination of ascorbic acid. Most of these interfering substances reduce indophenol more slowly than does ascorbic acid (Harris & Mapson, 1947). The presence of substances which act in this way has been demonstrated in whole serum (Bierring, Mathiessen & Nielsen, 1945), and also in extracts of various mammalian tissues, e.g. kidney (Hopkins & Slater, 1935), brain (Young & Mitolo, 1934), whole blood (Butler & Cushman, 1940) and urine (Evelyn, Malloy & Rosen, 1938; Scarborough & Stewart, 1937). Mann & Leone (1953) reported that about 85% of the indophenol-reducing activity of hog semen was due to the presence of ergothioneine. To increase the specificity of the indophenol method for the estimation of ascorbic acid Van Eekelen & Emmerie (1936) proposed the use of mercuric acetate to precipitate some of the interfering substances. An alternative method, based on the formnation of complexes of interfering substances and formaldehyde, was proposed by Lugg (1942). However, methods based on these principles have certain disadvantages, which are discussed below. In an attempt to devise a specific yet simple method for the determination of ascorbic acid in mammalian tissues we have found that p-chloromercuribenzoic acid successfully suppresses the interference due to thiol compounds. The results obtained by using this reagent in the determination of ascorbic acid in human plasma, erythrocytes and urine are presented. Preliminary conmunications of some of the results have already been given (Owen, Iggo & Horn, 1954; Owen & Iggo, 1955).