Figure 2: Heme transfer to Hpx and lipid oxidation in heme rich biological samples.

(A) Shows heme binding in citrated plasma obtained at the end of exchange transfusions, before and after the addition of Hpx (n=6 samples). Light grey - no Hb or holo-Hpx, red - holo-Hpx and dark grey cell-free Hb. Prior to exogenous Hpx addition, holo-Hpx concentration was 6.20 ± 2.5 mmol/L (Mean ± SD). After Hpx addition, holo-Hpx increased to 34.0 ± 9.6 mmol/L (Mean ± SD) (p <0.001), suggesting a 5-fold increase in heme transfer from non-Hpx proteins and lipids. (B) Shows differences in healthy control donor plasma (390 μg/ml ± 88.7, n=10) and SCD (77.3 ± 55.0, n=10) (p <0.001), data from the same sample cohort shown for quick reference (PMID: 30047285). (C) Shows citrated plasma in the presence of reconstituted lipoprotein (rLP) as a source for heme partitioning and oxidation. Box plots with highest to lowest individual values (n=12-24 per group) show the accumulation of lipid peroxidation products (thiobarbituric acid reactive substances, TBARS). SCD samples + saline + rLP show the greatest generation of TBARS (left), which is significantly reduced (p=0.004) with the addition of Hpx. Alone, rLP does not increase TBARS generation (right). (D) Dose finding for the primary murine study is based on the efficiency of Hpx dosing (50, 160 and 500 mg/kg/day) to bind heme in SCD mice (n=4) after five daily doses followed by blood collections at 0, 2, 6, 24 hours after the final dose. Red circles represent total Hpx, blue circles represent heme bound Hpx, solid blue and red lines indicate mean values and bars indicate SD. Data indicates that doses equal to 50 and 160 mg/kg/day x 5 days consume Hpx within a heme-Hpx complex, while dosing at 500 mg/kg/day exceeds transferable heme availability and suggests a steady state transferable heme concentration of approximately 40 - 80 uM.