Figure 5.

Oxidized ferryl hemoglobins disrupt pulmonary endothelial bioenergetics. (A) HPAEC were grown on 24 well XF-plates. Mitochondrial oxygen consumption rates (OCR) were measured for 2 h using XF24 extracellular flux analyzer with direct infusion of ferryl forms of HbA, HbS, or HbE at equimolar concentration (100 μM) (N = 3). (B) Basal OCR following 2 h incubation with ferryl Hbs (N = 3). (C) Average basal OCR values from a similar set of experiments in which HPAEC were exposed to either ferryl HbS (100 μM) or ferryl HbE (100 μM) for 2 h co-incubated with or without equimolar Hp or Hpx or Asc or NAC (N = 3). Statistical significance between means was calculated by paired Student's t-test. ^*P < 0.05 vs. untreated control, ^#P < 0.05 vs. respective ferryl Hb. (D) Complex I activity (E) complex II-III and (F) complex IV activities were measured in isolated mitochondrial fractions incubated with different ferryl Hb proteins for 2 h at 37°C (N = 4). Differences between individual treated groups and respective untreated controls were compared using Mann-Whitney U-test. ^*P < 0.05 vs. controls.