Cell-free
hemoglobin (CFH) levels are elevated in
septic shock and are higher in nonsurvivors. Whether CFH is only a marker of
sepsis severity or is involved in pathogenesis is unknown. This study aimed to investigate whether CFH worsens
sepsis-associated
injuries and to determine potential mechanisms of harm. Fifty-one, 10-12 kg purpose-bred beagles were randomized to receive Staphylococcus aureus intrapulmonary challenges or saline followed by CFH infusions (
oxyhemoglobin >80%) or placebo. Animals received
antibiotics and
intensive care support for 96 h. CFH significantly increased mean pulmonary arterial pressures and right ventricular afterload in both septic and nonseptic animals, effects that were significantly greater in nonsurvivors. These findings are consistent with CFH-associated
nitric oxide (NO) scavenging and were associated with significantly depressed cardiac function, and worsened
shock,
lactate levels,
metabolic acidosis, and multiorgan failure. In septic animals only, CFH administration significantly increased mean alveolar-arterial oxygenation gradients, also to a significantly greater degree in nonsurvivors. CFH-associated
iron levels were significantly suppressed in infected animals, suggesting that bacterial
iron uptake worsened
pneumonia. Notably,
cytokine levels were similar in survivors and nonsurvivors and were not predictive of outcome. In the absence and presence of
infection, CFH infusions resulted in
pulmonary hypertension,
cardiogenic shock, and multiorgan failure, likely through NO scavenging. In the presence of
infection alone, CFH infusions worsened
oxygen exchange and
lung injury, presumably by supplying
iron that promoted bacterial growth. CFH elevation, a known consequence of clinical
septic shock, adversely impacts
sepsis outcomes through more than one mechanism, and is a biologically plausible, nonantibiotic, noncytokine target for therapeutic intervention.NEW & NOTEWORTHY Cell-free
hemoglobin (CFH) elevations are a known consequence of clinical
sepsis. Using a two-by-two factorial design and extensive physiological and biochemical evidence, we found a direct mechanism of injury related to
nitric oxide scavenging leading to
pulmonary hypertension increasing right heart afterload, depressed cardiac function, worsening
circulatory failure, and death, as well as an indirect mechanism related to
iron toxicity. These discoveries alter conventional thinking about
septic shock pathogenesis and provide novel therapeutic approaches.