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 β-chain [trimesoyl-(Lys82)β-(Lys82)β] cross-linked human hemoglobin were preincubated with haptoglobin-containing 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 (CLinflux) over the metabolic intrinsic clearance (CLint, met). The ratios of CLinflux/CLint, 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.
Footnotes
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This work was supported by grants from the National Sciences and Engineering Research Council of Canada and a Strategic Project (234768-00) and a Collaborative Health Research Program (237960-00) Grant to K.S.P. and R.H.K.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.019174.
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ABBREVIATIONS: α, hybrid constant associated with the distribution phase in compartmental modeling; β, hybrid constant associated with the elimination phase in compartmental modeling; CR, CEC, and CL, concentrations of hemoglobin in reservoir, extracellular plasma, and liver compartments, respectively; CL, hepatic clearance; CLinflux and CLefflux, influx and efflux clearances, respectively, at the sinusoidal membrane; CLint, met, hepatic metabolic intrinsic clearance; k12, micro rate constant denoting entry from central compartment to peripheral compartment; k21, micro rate constant denoting entry from peripheral compartment to central compartment; k20, micro rate constant denoting elimination from peripheral compartment; Hp, haptoglobin; HB, hemoglobin; KHB, Krebs Henseleit bicarbonate buffer; Q, total hepatic flow rate; VR, VEC, and VL, volumes of reservoir, extracellular plasma, and liver, respectively; PBPK, physiologically based pharmacokinetic; AUC, area under the curve.
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↵1 Current affiliation: Genentech Inc., South San Francisco, CA.
- Received October 9, 2007.
- Accepted February 13, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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