Development and Validation of a Lead-Induced Hemolysis Assay in Human Erythrocyte Using Calcium Disodium EDTA

*Edwin Maurice Idagu and Okafor Azubuike Ikechukwu

Department of Medical Biochemistry, University of Cross River State, Calabar, Nigeria ^*Corresponding author: edwinmaurice1998@gmail.com; +2348131114954

ABSTRACT

The present study reports the development and validation of a lead-induced hemolysis assay in human erythrocytes and the evaluation of calcium disodium ethylenediaminetetraacetate (CaNa₂EDTA) as a chelating inhibitor of lead-induced membrane damage. Erythrocyte exposure to increasing concentrations of lead acetate trihydrate (0–200 µg/assay) produced a clear concentration-dependent increase in absorbance at 540 nm and percentage hemolysis, with minimal hemolysis observed in the control group (0.0 ± 0.00%) and a marked elevation at the highest concentration (90.0 ± 0.50%), indicating severe membrane disruption and near complete erythrocyte lysis. Co-treatment with CaNa₂EDTA significantly attenuated lead-induced hemolysis in a concentration-dependent manner. At Lead:CaNa₂EDTA ratios of 1.0:0.5, 1.0:1.0, and 1.0:2.0, hemolysis was reduced to 66.7 ± 0.32%, 50.0 ± 0.26%, and 25.9 ± 0.15%, respectively, corresponding to progressive inhibition of 25.9 ± 0.16%, 44.4 ± 0.25%, and 71.2 ± 0.35%. Nonlinear regression analysis using a variable slope sigmoidal dose-response model revealed an IC₅₀ value of 242 µg/mL for CaNa₂EDTA, with a high coefficient of determination (R² = 0.975), indicating a strong model fit. The Hill slope (0.173) suggested a gradual, concentration-dependent chelation interaction between CaNa₂EDTA and lead ions. The findings demonstrate that lead acetate induces significant, dose-dependent erythrocyte membrane destabilization, and that CaNa₂EDTA effectively mitigates this effect through a chelation-mediated mechanism. The validated assay provides a reliable in vitro model for assessing lead-induced hemolysis and screening potential chelating agents.

Keywords: Lead acetate; Hemolysis assay; Human erythrocytes; Calcium disodium-EDTA; Chelation; IC₅₀

CITE AS: Edwin Maurice Idagu and Okafor Azubuike Ikechukwu (2026). Development and Validation of a Lead-Induced Hemolysis Assay in Human Erythrocyte Using Calcium Disodium EDTA. IAA Journal of Biological Sciences 14(2):12-17. https://doi.org/10.59298/IAAJB/2026/1421217