BIO18: Hemoglobin and Heme Scavenger Proteins Reduce Free Hemoglobin During Venovenous Extracorporeal Circulation and Preserves Microvascular Function.

Abstract

Extracorporeal circulation procedures often utilize a blood pump to drive flow through an external circuit, which can cause hemolysis due to high shear conditions and foreign surface contact. The purpose of this study was to determine whether priming fluids containing proteins to sequester hemoglobin (Hb) and heme could be used in venovenous extracorporeal circulation (VV-ECC) to reduce their toxicity. A day prior to the experiment, male Golden Syrian hamsters were instrumented with a dorsal window chamber to monitor the microcirculation. The day of the experiment, both jugular veins were then cannulated under isoflurane anesthesia. Baseline (BL) microvascular oxygenation was measured using a hyperspectral camera. The animals were then heparinized (2.5 IU/g) and connected to the VV-ECC circuit, consisting of a peristaltic pump and bubble trap. The ECC circuit was primed with either 5% human serum albumin (HSA), ApoHb-Hp (62.01 mg/mL), or a protein solution containing hemopexin, transferrin, and Hp (protein cocktail, 84.50 mg/mL). Haptoglobin (Hp), hemopexin, and transferrin are native proteins that bind to Hb, heme, and iron respectively. Apohemoglobin (apoHb) is a synthetic scavenger produced by removing heme from Hb. ApoHb binds to haptoglobin (Hp) forming the apoHb-Hp complex that retains its ability to bind heme and scavenge free Hb. The experiment lasted a total of 2 hrs (Table 1), in which first the animals were hemodiluted, and the flow rate through the circuit was ramped up to 50% of the animal’s cardiac output. This max flow rate was then maintained for 35 minutes before being ramped down to 0.2 mLPM. The circuit was maintained at this flow rate for 15 minutes, allowing the animal and the circuit to equilibrate before the circuit flow was stopped. While mean arterial pressure (MAP, Figure 1a) showed differences at baseline (BL) between HSA and both other groups, there was no significance observed after hemodilution and the ramp up phase of the experiment. This difference in MAP between HSA and the two other solutions returned at all timepoints after the ramp up phase. Hb oxygen saturation (sO2, Figure 2) within the microvasculature showed a drop for both arterioles and venules in both the protein cocktail and ApoHb-Hp groups at the full VV-ECC timepoint. The plasma Hb (Figure 3) for the protein cocktail and ApoHb-Hp groups were significantly lower than the HSA control from the start of VV-ECC to the equilibration stage when compared to the HSA control. These results confirm that the ApoHb and protein cocktail solutions have the capability to bind and sequester free Hb, which has the potential to reduce the extravasation of Hb from the circulatory compartment into tissues, preserves the vascular nitric oxide (NO) signaling pathway, and reduce Hb and heme toxicity. Future work is needed to determine impact on organs, especially the kidneys, and to quantify the binding efficiency of these proteins to free hemoglobin.Figure 1. Hemodynamic parameters measured via the carotid artery: a) Mean arterial pressure (MAP) and b) heart rate (HR). bl = baseline, dil = hemodilution, up = ramp up, ecc = VV-ECC at max flow rate (50% of cardiac output), down = ramp down, equ = equilibration, off = off pump. * P < 0.05, ** P < 0.01, *** P < 0.001 between groups. N = 6 for all groups.Figure 2. Changes in microvascular hemoglobin oxygen saturation. The top row shows arterioles, while the bottom row shows venules. Arterioles and venules were analyzed for 4 hamsters per group. bl = baseline, dil = hemodilution, up = ramp up, ecc = VV-ECC at max flow rate (50% of cardiac output), down = ramp down, equ = equilibration, off = off pump. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 between groups. † P<0.05 compared to baseline (BL).Figure 3. a) Hematocrit (Hct) and b) plasma hemoglobin (pHb). 0 = baseline (BL), 25 = hemodilution, 45 = VV-ECC (start), 75 = VV-ECC (end), 105 = equilibrate, 120 = off pump. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 between groups. † P<0.05 compared to BL. Table 1. - Experimental timeline highlight the blood flow through the circuit. Baseline Hemodilution Ramp Up VV-ECC Ramp Down Equilibrate Off Pump Duration 15 min 15 min 15 min 35 min 15 min 15 min 10 min Blood Flow 0 mLPM 0.2 mLPM 0.2 mLPM → 50% CO 50% CO 50% CO → 0.2 mLPM 0.2 mLPM 0 mLPM Hyperspectral images were taken every 5 minutes. mLPM = milliliters per minute, CO = cardiac output, VV-ECC = full venovenous extracorporeal circulation.