A comparison of leukocyte ascorbate levels measured by the 2,4-dinitrophenylhydrazine method with high-performance liquid chromatography using electrochemical detection

In leukocytes a dynamic relationship between the reduced form of ascorbic acid (AA) and its oxidized product dehydro-AA has been described. It is therefore important to know which form of the vitamin predominates when choosing a methodology. The purpose of this study was to find out if the majority of ascorbate in human leukocytes isolated by centrifugation through Percoll is in the reduced AA form by measuring reduced AA by HPLC and comparing the values to those obtained by using the 2,4-dinitrophenylhydrazine (DNPH) method which measures total ascorbate, and quantifying the reduced and oxidized forms of the vitamin in leukocytes using a modification of the DNPH method. There was no significant difference (P greater than .05) between the HPLC and DNPH values for 12 individuals and 87% of the AA was found to be in the reduced form. These results support the assumption that the majority of AA found in a mixed leukocyte population isolated through Percoll is in the reduced form and that both methods can be used for AA measurements.