Quantitation of blood protein adducts of acrolein by tritiated sodium borohydride reduction

The present study describes a rapid and sensitive method for the estimation of adducts of albumin and hemoglobin after in vitro and in vivo treatment with acrolein. The in vitro study was conducted by incubating purified human albumin, human hemoglobin, human plasma, or human blood with increasing concentrations of acrolein (0-10 mM) in 100 mM phosphate buffer, pH 7.2, for 2 h at 37°C. Albumin was also incubated with crotonaldehyde under the same conditions as with acrolein. Blood was separated by centrifugation into red cells and plasma. Red cells were washed. lysed, and centrifuged to obtain hemolysate. The hemolysate and the plasma were exhaustively dialyzed and the protein samples were treated with NaB³H₄. The radioactivity measured was expressed as nmol of carbonyl function/mg of protein. The response was found to be dose dependent in the range studied. This method is about four times more sensitive than amino acid analysis in detecting covalent binding of acrolein to albumin. Although not as reactive, crotonaldehyde (2-butanal, a metabolite of butadiene) also binds covalently with albumin. For the in vivo study. rats were treated with acrolein by gavage (13 mg/kg) and killed at 1,2.4. and 6 h. Blood samples were collected and analyzed for adducts of plasma proteins and hemoglobin as described above. The adducts of both plasma proteins and hemoglobin were found to be significantly higher compared with the controls. The efficacy of this method needs to be established in humans before it can be used for molecular dosimetry. Key Words: Acrolein-Protein adducts-Blood-Tritiated borohydride reduction.