Mechanistic insights into cell-free hemoglobin-induced injury during septic shock

Abstract

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.

Keywords: cell-free hemoglobin; iron; septic shock.

Figure 1.

Effects of CFH on survival and pulmonary injury in both septic and normal (nonseptic) animals. A: survival in septic animals receiving CFH (solid line) is compared with septic controls not receiving CFH (dotted line). B: survival in nonseptic animals receiving CFH (solid line) is compared with nonseptic control animals not receiving CFH (dotted line). C: serial mean (± SE) AaO₂ in septic animals that received CFH (filled circles) is compared with septic controls not receiving CFH (open circles). D: serial mean (± SE) AaO₂ in nonseptic animals that received CFH (filled circles) is compared with nonseptic control animals not receiving CFH (open circles). E: serial mean (± SE) AaO₂ is compared in survivors vs. nonsurvivors for animals receiving both bacteria and CFH. F: the format is similar to (E) except that animals represented here received CFH alone. G: serial mean (± SE) $\text{F}{\mathrm{I}}_{{\text{O}}_2}$ is compared in survivors vs. nonsurvivors for animals receiving both bacteria and CFH. H: the format is similar to (G) except that animals represented here received CFH alone. On the x-axis, CFH represents duration of CFH infusion in animals receiving CFH and duration of placebo infusion in animals receiving placebo. AaO₂, alveolar-arterial gradient; CFH, cell-free hemoglobin; $\text{F}{\mathrm{I}}_{{\text{O}}_2}$, fraction of inspired oxygen.

Figure 2.

The format is similar to Fig. 1 (C and D), except now serial mean (± SE) pulmonary artery pressure (mPAP) (A and B), pulmonary vascular resistance index (PVRI) (C and D), and stroke volume index (SVI) (E and F) are shown.

Figure 3.

For the panels on the left, the format is similar to Fig. 1 (E), except now serial mean (± SE) mPAP (A), PVRI (B), SVI (C), shock score (D), and norepinephrine levels (E) are shown. For the panels on the right, the format is similar to Fig. 1 (F), except now serial mean (± SE) mPAP (F), PVRI (G), SVI (H), shock score (I), and norepinephrine levels (J) are shown. mPAP, mean pulmonary artery pressure; PVRI, pulmonary vascular resistance index; SVI, stroke volume index.

Figure 4.

The format is similar to Fig. 1 (C and D), except now serial mean (± SE) serum creatinine in logarithmic scale (A and B), alanine aminotransferase (ALT) in logarithmic scale (C and D), creatine phosphokinase (CPK) (E and F), white blood cell (WBC) count (G and H), lactate in logarithmic scale (I and J), and pH (K and L) are shown.

Figure 5.

The format is similar to Fig. 1 (C and D), except now serial mean (± SE) cell-free hemoglobin (CFH) levels in logarithmic scale (A and B) are shown. C and D: the serial mean percent (± SE) oxyhemoglobin and methemoglobin levels are shown in animals receiving CFH with bacteria (C) and CFH without bacteria (D).

Figure 6.

The format on the top is similar to Fig. 1 (C and D), except now mean (± SE) nontransferrin bound iron (NTBI) levels in logarithmic scale (A and B) are shown. In the lower panels, interaction figures are shown demonstrating the differential effect of cell-free hemoglobin (CFH) on mean NTBI levels in the presence or absence of bacterial challenge at 48 h (C), 72 h (D), and 96 h (E). Please note that in the absence of bacteria (dotted line) at 48, 72, and 96 h, NTBI levels rise with the addition of CFH. However, in the setting of bacterial infection (solid line) at these same time points, these rises are attenuated.