Measurement of intracellular inorganic phosphate in human blood red cells, leucocytes and platelets.

The measurement of Pi in red blood cells, leucocytes, platelets and plasma by this method is both highly selective and sensitive for Pi. Whereas the ratio of intracellular to extracellular Pi is less than 1 in red cells, it is about 4 in platelets and about 2 in leucocytes. In platelets, where there are storage granules, the nucleotide levels are higher than in red cells or leucocytes. Cell lysis by digitonin proved to be a reliable technique for the rapid and efficient separation of cytosol from the particulate fraction. The isolation of cytosol with minimal contamination by lysosomes, electron dense granules and mitochondrial matrix depended on the use of appropriate concentration of digitonin. There was a linear relationship between platelet number and digitonin concentration which induced leakage of 90% of the LDH. In platelets about half of the Pi and 50% of the total ATP + ADP were found in the particulate fractions. In leucocytes most of the Pi and the bulk of nucleotides are present in the cytosol. These findings indicate the presence of at least two pools of nucleotides in platelets and leucocytes, and suggests that the inorganic phosphate in cytosol is at a higher concentration than outside the cells, implying a concentration gradient across the plasma membrane.