A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates

Abstract

Fluorescence emission of free protoporphyrin IX (PPIX, em.∼626 nm), zinc protoporphyrin IX (ZPP, em.∼594 nm) and fluorescent heme degradation product (FHDP, em.∼466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F365/594) correlates with the ZPP fluorescence obtained from acetone/water extraction (R2 = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R2 = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.