Cellular phosphate metabolism in patients receiving bisphosphonate therapy.

Patients with Paget's disease of bone were treated with oral disodium dihydrogen ethylidene-1-hydroxy-1,1-bisphosphonate (EHBP), a drug that is known to stimulate renal tubular reabsorption of orthophosphate (Pi). After 2 weeks of treatment, plasma Pi rose from 1.02 to 1.67 mmol/l. No increase in Pi was observed with the related drug, dichloromethylene bisphosphonate, which also reduces bone turnover in Paget's disease. Intravenous EHBP caused a more rapid increase in plasma Pi, but maximum hyperphosphatemia was not observed until 7-11 days after treatment commenced. It is therefore unlikely that this effect is due to an immediate action of EHBP on the luminal face of the renal brush border Pi transporter. After 2 weeks of oral EHBP, the increase in the Pi concentration in patients' erythrocytes was 31% compared with 64% in plasma. In blood platelets and leukocytes, negligible changes in cellular Pi occurred. The concentrations of 2,3-diphosphoglycerate, adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) were unaltered, indicating that these organic phosphates were not offsetting a potential change in cellular Pi. The decrease in erythrocyte/plasma distribution ratio for Pi was also observed in patients receiving intravenous EHBP. However, no change occurred in cell/plasma distribution of chloride, suggesting that this apparent regulation of cellular Pi did not arise from changes in erythrocyte membrane potential, pH, or water content.

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.