High- and low-affinity PEGylated hemoglobin-based oxygen carriers: Differential oxidative stress in a Guinea pig transfusion model

Abstract

Hemoglobin-based oxygen carriers (HBOCs) are an investigational replacement for blood transfusions and are known to cause oxidative damage to tissues. To investigate the correlation between their oxygen binding properties and these detrimental effects, we investigated two PEGylated HBOCs endowed with different oxygen binding properties - but otherwise chemically identical - in a Guinea pig transfusion model. Plasma samples were analyzed for biochemical markers of inflammation, tissue damage and organ dysfunction; proteins and lipids of heart and kidney extracts were analyzed for markers of oxidative damage. Overall, both HBOCs produced higher oxidative stress in comparison to an auto-transfusion control group. Particularly, tissue 4-hydroxynonenal adducts, tissue malondialdehyde adducts and plasma 8-oxo-2'-deoxyguanosine exhibited significantly higher levels in comparison with the control group. For malondialdehyde adducts, a higher level in the renal tissue was observed for animals treated with the high-affinity HBOC, hinting at a correlation between the HBOCs oxygen binding properties and the oxidative stress they produce. Moreover, we found that the high-affinity HBOC produced greater tissue oxygenation in comparison with the low affinity one, possibly correlating with the higher oxidative stress it induced.

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality

“…20 Maleimide-PEG conjugation at non cysteine residues is possible via the use of reagents such as 2-iminothiolane to introduce thiol reactivity at terminal amino residues and surface lysines. Such methods of PEGylation have been used successfully in the design of HBOC products tested in animal models (and clinical trials) such as Hemospan, 21 Euro-PEG-Hb 22 and Sanguinate®. 23 However, two issues have arisen.…”

Protein carbonylation detection methods: A comparison

“…Heart and kidney tissue samples were obtained from 14 Hartley Guinea pigs that underwent either autotransfusion or transfusion with PEG-Hb oxy or PEG-Hb deoxy , two PEGylated hemoglobin-based oxygen carriers [1] , [2] , [3] , [4] , [5] , [6] endowed with different oxygen affinity [2] , as described in FRBM [11] . The aim of the work was to assess the degree of oxidative stress in the treatment groups in comparison with the control (autotransfused) group by determining the level of carbonylated proteins.…”

“…The total carbonyl content as detected in heart tissue with the Levine method and the ELISA method (OxiSelect™ Protein Carbonyl ELISA Kit, Cell Biolabs, San Diego, CA, USA) is reported in Fig. 1 for the three animal groups described in FRBM [11] . No significant difference was observed between the two methods, with the ELISA method being more expensive.…”

“… The data reported here are a comparison among four different methods for the detection of carbonylated proteins, a validated biomarker of oxidative stress. The reference samples were heart and kidney extracts of Guinea pigs transfused with hemoglobin-based oxygen carriers (Alomari et al FRBM, [11] ). We measured the carbonyl content of organ extracts by using i) the Levine spectrophotometric method, which takes advantage of the chromogenic reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH), ii) a commercially available ELISA assay based on an anti-DNPH antibodies, iii) a commercially available Western blot method based on anti-DNPH antibodies and iv) an in-gel detection approach with the fluorophoric reagent fluorescein-5-thiosemicarbazide.…”

Künstliche Sauerstofftransporter können mehr als Sauerstoff liefern

“…Um die Zirkulationsdauer der LEH zu verlängern, werden die Liposomen mit Polyethylenglykol (PEG) dekoriert [74]. Diese PEGylierung ist weit verbreitet, weil PEG durch die Bildung einer Hydrathülle um die Partikel deren Opsonierung verhindern kann.…”