DETERMINATION OF HUMAN PLASMA AND LEUKOCYTE ASCORBIC-ACID BY MICROTITER PLATE ASSAY

Methods for analyzing ascorbic acid (AA) in human blood are often labo rious, difficult expensive, and require the use of large sample volume s. The lack of an inexpensive reliable method is particularly disconce rting for leukocyte analysis. We have overcome these concerns by devel oping a microtiter plate assay (MPA) to measure the AA dinitrophenylhy drazine (DNPH) derivative (515 nm and 562 nm) in plasma or leukocytes. Plasma and leukocytes were isolated from whole blood by gradient sedi mentation using Histopaque 1077 and 1119. The AA contribution from red blood cells was < 0.01%. The incubation time for AA DNPH derivative f ormation was reduced to 50% of the time used by investigators cited in previously published papers with no significant loss in recovery (for 2 hr incubation, > 90%). AA values by our MPA method using human subj ects agreed with published values or when compared with standard spect ophotometic assays. The advantages of the MPA assay over traditional m ethods are that it can handle small sample volumes and multiple sample s simultaneously. In addition, the MPA method requires using smaller v olumes of hazardous reagents and can be done in less time with less bl ood.