Figure 8. COCl₂ exposure increases RBC fragility in mice.

Equal number of male and female mice were sacrificed 24 hours post exposure to either air or COCl₂ (20 ppm for 10 min). Blood was withdrawn from the left ventricle and plasma separated. RBCs from these mice were washed thoroughly to remove free heme; RBC suspensions of 1.0% hematocrit along with 4mm solid glass beads (Pyrex) in DPBS were rotated 360° for 2 hours at 24 rpm at 37°C. The hemoglobin released from the RBCs during rotation was transferred into a new tube and centrifuged at 13,400g for 4 min and the absorbance of the supernatant were recorded at 540nm. Means ± SEM; n=8 for each group. One hundred percent hemolysis of RBCs was achieved by treating them with 1% Titon x-100 solution. The percent hemolysis of the sample was then obtained by dividing the optical density of the sample from the optical density of the 100% hemolyzed sample. (A). Next, total heme levels was measured in the plasma of air and COCl₂ exposed mice using the QuantiChrom heme assay kit, according to the manufacturer’s instructions. Means ± 1 SEM; n=16 air and 20 for COCl₂. (B). in the second series of experiments total (C) and free (D) heme levels in plasma were measured using an absorbance spectrum deconvolution (46). Means ± 1 SEM; n=33 air and 19 for COCl₂. Total heme levels determined by these two methods were very similar.