Two millilitre of venous blood was collected from each subject, using disposable syringes, and promptly transferred to a lidded glass vial. Before clotting could occur, the reagent ethylene

diamine tetra acetic acid, which binds to lead in blood and facilitates its separation at the next stage, was added in equal volume to the blood and the mixture was shaken for 2 min10. To prevent sample contamination with exogenous lead, all laboratory glassware was cleansed with detergent and double-distilled water; they were then immersed in a 2-m HNO3 overnight and washed several times with double-distilled water before a final rinse with deionized Selleck PD-332991 water1. Each tooth was cleaned and soaked in a 3% solution of hydrogen peroxide to remove organic material, after which it was washed several times with double-distilled water and deionized water, air dried and weighed. The tooth was then dissolved in 3 mL of 70% HNO3 and 1 mL of 70% perchloric acid (HClO4) in a 50-mL beaker. The mixture was heated slowly until a clear,

colourless solution was obtained, which was then evaporated until dry. The selleck products digest was then rinsed with distilled water, filtered if cloudy, made up to 10 mL and shaken1. The lead concentration in the final digested solution was determined by using Flame Atomic Absorption Spectrophotometer (AAS) with electrothermal atomization (Varian Inc., Palo Alto, CA, USA). The specifications of the instrument were: lamp current 9.0 mA, wavelength 217.0 nm, band pass 0.5–1.0 nm, ash temperature 800°C and atomization 2300°C without temperature

control1. The blood sample was mixed thoroughly by inverting the sample container 15 times. A 3-mL aliquot of the blood sample was immediately dispensed into a centrifuge tube. Ammonium Pyrrolidine Dithio Carbamate solution (0.5 mL) was P-type ATPase added to the tube, and the tube was capped and inverted 15 times. The tube was then allowed to stand for 5 min, after which 3 mL of n-butyl acetate was added to the tube. The tube was capped again and shaken for a minimum 3 min at a rate sufficient to ensure mixing of the organic layer and blood. The tube was then centrifuged at 3000 revolutions/min for 2 min. The organic layer was aspirated into the flame of the AAS and absorbance was recorded10,11. The values obtained were subjected to statistical analysis using the Statistical Package for Social Sciences (SPSS-15) software for windows. Group comparison between males and females was carried out by using the Student’s t-test. Analysis of variance was used to assess group comparison for tooth type, age, and village. A critical value of P < 0.05 was considered statistically significant. The present study was carried out to determine and correlate the lead levels in blood and teeth of 100 children, all residents of villages located in the vicinity of a zinc–lead smelter.