Assessment of Hemoglobin-Dependent Neurotoxicity: Alpha-Alpha Crosslinked Hemoglobin

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Abstract
Adult human hemoglobin A0 (HbA0) has been shown to be neurotoxic, and we wish to report on similar studies conducted using a modified hemoglobin, which has been crosslinked between the alpha subunits (α-αHb). Cortical cell cultures were prepared from fetal Swiss-Webster mice at 15–16 days gestation. Mature cultures (days in vitro, 12–16) were exposed to α-αHb in a defined medium for 24–48 hours at 37° C. Low micromolar amounts of α-αHb were neurotoxic in a concentration-dependent fashion. This toxicity was attenuated by the antioxidants Trolox and U-74500A and by the iron chelator deferoxamine. The hemoglobin-binding protein, haptoglobin, also completely blocked α-αHb-dependent neurotoxicity. The latter result was unexpected because complex formation between α-αHb and haptoglobin was not detected using assays of haptoglobin fluorescence and hemoglobin peroxidase activity.