The free and total plasma copper and total erythrocyte copper levels have been determined by simple, yet sensitive and highly specific methods, using atomic absorption spectrophotometry.
For total copper determination, the copper was split from its protein combination in plasma or red cells by the action of hydrochloric acid at room temperature. The liberated copper was chelated by ammonium pyrrolidine dithiocarbamate and extracted into n-butyl acetate by shaking and the organic extract was aspirated into the atomic absorption spectrophotometer flame. The entire procedure was carried out in polypropylene centrifuge tubes, capped during shaking. For the free plasma copper measurement the hydrochloric acid step was omitted.
Removal of the plasma or erythrocyte proteins was found to be unnecessary, and, in addition, the presence of trichloracetic acid caused an appreciable lowering of absorption.
Using a double-beam atomic absorption spectrophotometer and scale expansion × 10, micro methods have been derived for determining the total copper of plasma or erythrocytes with 0·1 ml of sample, and the free copper of plasma with 0·5 ml. The macro plasma copper method requires 2 ml of plasma and is suitable for use with single-beam atomic absorption spectrophotometers.
With blood from 50 blood donors, normal ranges of plasma and erythrocyte copper have been determined.