Determination of human plasma and leukocyte ascorbic acid by microtiter plate assay

Abstract

Methods for analyzing ascorbic acid (AA) in human blood are often laborious, difficult, expensive, and require the use of large sample volumes. The lack of an inexpensive reliable method is particularly disconcerting for leukocyte analysis. We have overcome these concerns by developing a microtiter plate assay (MPA) to measure the AA dinitrophenylhydrazine (DNPH) derivative (515 nm and 562 nm) in plasma or leukocytes. Plasma and leukocytes were isolated from whole blood by gradient sedimentation using Histopaque 1077 and 1119. The AA contribution from red blood cells was < 0.01%. The incubation time for AA DNPH derivative formation 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 subjects agreed with published values or when compared with standard spectophotometic assays. The advantages of the MPA assay over traditional methods are that it can handle small sample volumes and multiple samples simultaneously. In addition, the MPA method requires using smaller volumes of hazardous reagents and can be done in less time with less blood.