Figure 4

Globin cross-linking and free heme in urine of dogs with hemoglobinuria. (a) Hb-cross-linking products in dog urine. Urine samples of four dogs before (ctrl) and during infusion (Hb) were analyzed by western blotting. The most right lane is from an identical western blot that was performed with hydrogen peroxide (H₂O₂)-reacted purified Hb, which shows the typical peroxidation-induced Hb-cross-linking pattern. (b) Free heme concentrations in dog urine (n=8 Hb infused animals) were estimated with a hemopexin capture assay. Post-infusion urine samples were incubated with an excess of hemopexin for 1 min (1 min heme release) or 60 min (total heme). The concentration of heme–hemopexin was measured at the end of the incubation time by spectrophotometry. The lower panels indicate heme concentrations that were normalized to an estimated creatinine concentration of 90 μM in the proximal tubule