Role of Haptoglobin on the Uptake of Native and β-Chain [Trimesoyl-(Lys82)β-(Lys82)β] Cross-Linked Human Hemoglobins in Isolated Perfused Rat Livers

Abstract

ABSTRACT:The role of haptoglobin in liver cell entry of acellular native hemoglobin, and cross-linked human hemoglobin, a potentially useful oxygen-carrier alternative in transfusion medicine, was examined in the recirculating, perfused rat liver preparation. Doses of tritiated native human or ␤-chain [trimesoyl-(Lys82)␤-(Lys82)␤] crosslinked human hemoglobin were preincubated with haptoglobincontaining rat plasma or Krebs Henseleit bicarbonate buffer for 30 min and used for perfusion. Concentrations (dpm/ml) in reservoir, before and after separation of the hemoglobins and metabolites by gel filtration fast protein liquid chromatography column chromatography, were similar, showing mostly the presence of intact hemoglobin. Each hemoglobin species underwent a rapid distribution phase, followed by a protracted elimination phase. The radioactivity in bile at 3 h consisted of low molecular weight metabolites, and cumulative excretion was slightly higher when rat plasma was present: for native hemoglobin, 7.1 ؎ 1.6% versus 9.2 ؎ 2.1% dose; for cross-linked hemoglobin, 5.0 ؎ 1.7% versus 7.2 ؎ 0.8% dose. Data fit to a two-compartment model and physiologically based model revealed a significantly faster influx clearance (CL influx ) over the metabolic intrinsic clearance (CL int, met ). The ratios of CL influx /CL int, met were 125 and 535 for native hemoglobin in the absence and presence of rat haptoglobin, respectively, according to compartmental analyses; the ratios were 25 and 53, respectively, according to physiological modeling. The corresponding ratios for cross-linked hemoglobin in the absence and presence of rat haptoglobin were 55 and 81, respectively, and 24 and 70 for compartmental and physiological modeling. Although haptoglobin enhanced the hepatic internalization of the hemoglobins, the impact on the net clearance was lessened since degradation was the rate-limiting step.Hemoglobin is a tetrameric heme protein that is found within red blood cells. This protein is composed of a pair of ␣␤ dimers to form its tetrameric structure (Greer et al., 1981;Voet and Voet, 1995;Chang, 1999). In the tetrameric format, hemoglobin plays an important role in the binding to oxygen and the supply of oxygenated blood to tissues and organs [for review, see Winslow (1992)]. During intravascular hemolysis, hemoglobin is released from the red blood cells and is distributed into the plasma where free hemoglobin (HB) is rapidly bound to haptoglobin (Hp), a specific plasma binding protein of high affinity (K d Ϸ 1 ϫ 10 Ϫ15 M) toward the alpha chain on the hemoglobin (Putnam, 1976;McCormick and Atassi, 1990;Lim et al., 1998). The complex is then taken up by an unknown receptor on hepatocytes and by the CD163 hemoglobin scavenger receptor on macrophages that are present in the spleen, thymus, Kupffer cells, and bone marrow (Graversen et al., 2002;Polfliet et al., 2006).In cases of transfusion of acellular hemoglobin or severe hemolysis, high levels of hemoglobin are present and will easily saturate the haptoglobin present in plasma (...

Recognition mechanisms of hemoglobin particles by monocytes – CD163 may just be one

“…It can be speculated that the same could be true for HBOCs. This fits with a case report by Drieghe et al, which suggests that hemopexin (Hpx) may also play a role in the elimination of HBOCs [ 25 , 29 ]. Hpx was depleted before a change in Hp levels could be observed when catecholamine-requiring patients were treated with an HBOC for intentional NO scavenging and consecutive increase in peripheral vascular resistance.…”

“…The corresponding binding site at the receptor appears to be the same as for the binding of Hp–Hb complexes, according to Schaer et al [ 27 ]; whereas within Hb, the binding site for direct interaction with CD163 is probably within the β chain of Hb (binding of Hb to Hp via a binding site within the Hb α chain) [ 27 ]. Furthermore, not only cells of the monocyte/macrophage lineage appear to be involved in sequestering Hb, but also hepatocytes [ 25 – 26 ]. It can be speculated that the same could be true for HBOCs.…”

Pharmacokinetics and mechanisms of plasma removal of hemoglobin-based oxygen carriers

“…Supporting this is the fact that the disappearance of radioactively labeled HBOCs from plasma corresponds closely to their rate of disappearance as determined by a spectrophotometric assay , Keipert et al 1994). In addition, when low doses of tritiated HBOC were recirculated through isolated, perfused rat liver, most of the radioactivity in the perfusate remained associated with intact Hb (Chow et al 2008). On the other hand, low molecular weight metabolites rapidly appeared in the bile.…”

“…HBOC clearance cannot be quantitatively explained by • catabolism in the liver and monocytes/macrophages (Schaer et al 2007, Chow et al 2008, Etzerodt et al 2013, Boretti et al 2014. Kidney fi ltration of crosslinked and polymerized • HBOCs is minimal (Berbers et al 1991, Keipert 1992, Keipert et al 1992, Hsia et al 1993, Lee et al 1995, Conover et al 1997a, Wicks et al 2003, Baek et al 2012).…”

“…Macromolecule extravasation is dependent on size, shape, charge, and hydrophilic/lipophilic balance (Gandhi and Bell 1992, Takakura et al 1998, El-Sayed et al 2001, Lin 2009). Although the mechanisms of extravasation have been debated for decades, caveolae-mediated transcytosis through the endothelial cell interior of capillaries is believed to be an important pathway for albumin, along (Chow et al 2008). Hp modestly facilitated the uptake of either Hb type, and the removal of the crosslinked Hb was slower than that of the unmodifi ed counterpart.…”