Graduate Thesis Or Dissertation
 

The binding of pentobarbital to hemoglobin and its possible involvement in affecting oxygen dissociation

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  • Pentobarbital binding to hemoglobin, evidence of an interaction between pentobarbital and 2,3-DPG in binding to hemoglobin, and the effect of altered oxygen dissociation on the outcome of barbiturate poisoning were studied. Ultrafiltration and equilibrium dialysis techniques were used to determine the binding of pentobarbital to solutions of purified hemoglobin, diluted whole blood, diluted suspended red blood cells, diluted plasma and to hemoglobin obtained from suspended red blood cells. Ultrafiltration was used to study whether the binding of pentobarbital to hemoglobin affected the binding of 2,3-DPG and if there is evidence of competition between these two molecules for a common binding site. Mice were dosed with 10 ml/kg of 0.1 M inosine, 0.1 M pyruvate, and 0.25 M inorganic phosphate which produced an elevation of 2,3-DPG levels and subsequently received either 75 mg/kg or 100 mg/kg of sodium pentobarbital. The recovery times and mortality rates were compared to a control population which received pentobarbital alone. The percent of pentobarbital bound to purified hemoglobin and to hemoglobin obtained from fresh red blood cells was 8-12% and 9-13%, respectively. The percent of pentobarbital bound to the diluted whole blood was 22-27%, to the diluted blood cell suspension was 7-8%, and to the diluted plasma was 10-13%. There was no change in the percent of pentobarbital bound to hemoglobin in the presence of varying concentrations of 2,3-DPG ranging from zero to twice physiological. The effect of pentobarbital on the binding of 2,3-DPG to hemoglobin could not be determined due to an unreliable assay method. There was no significant effect of altered oxygen dissociation produced by an elevated 2,3-DPG concentration on the outcome of pentobarbital poisoning in terms of survival rate or revival time. The study suggests that pentobarbital binds to the hemoglobin molecule in whole blood. However, whether this binding interferes with the binding of 2,3-DPG to the central cavity of hemoglobin could not be concluded. It appears that an elevated 2,3-DPG concentration does not affect the outcome of barbiturate poisoning. Since suicidal doses of barbiturates have been shown to significantly affect the oxygen dissociation curve, this study suggests the need for determining the plasma concentration at which a significant effect is observed.
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