An evaluation of serum osteocalcin in Paget's disease of bone and its response to diphosphonate treatment.

We found that serum bone gamma-carboxyglutamic acid-containing protein (BGP) (osteocalcin) had lower sensitivity and specificity for measurement of disease activity in Paget's disease of bone than other biochemical measures of disease activity. The administration of diphosphonates induced suppression of urinary hydroxyproline excretion and a subsequent decrease in alkaline phosphatase values, but no consistent change in BGP values. Serum BGP measurements have limited value as a screening test for Paget's disease or for monitoring treatment of the disorder.

Duration of effect of oral diphosphonate therapy in Paget's disease of bone.

The effects of the diphosphonates etidronate and clodronate were studied in 144 patients with Paget's disease. All five programmes of treatment tested induced a similar suppression of disease activity as judged by serum alkaline phosphatase concentrations, but the proportion of patients responding and the duration of responses differed significantly between programmes. The proportion responding to etidronate 5-10 mg/kg/day for six months was less than for other regimens, and the most sustained response was after treatment with clodronate 1600 mg daily for six months. More complete biochemical suppression was associated with the more prolonged responses irrespective of the regimen used.

Effective short term treatment of Paget's disease with oral etidronate.

Twelve patients with Paget's disease of bone were treated with high doses of disodium etidronate for one month and compared with patients given treatments for longer periods. The effects of treatment for one month with etidronate 20 mg/kg daily were indistinguishable from six months' continuous treatment with the same dose but significantly better than treatment with 5 mg/kg daily in suppressing biochemical indices of disease activity. Treatment for one month was associated with transient osteomalacia but sustained suppression of bone resorption. Short term treatments with high doses of disodium etidronate may maximise suppression of disease activity but decrease exposure to unwanted effects.

A technique for the measurement of orthophosphate in human erythrocytes, and some studies of its determinants.

A technique is described for the determination of orthophosphate (Pi) in human erythrocytes. The advantages of the technique are that it uses whole blood rather than separated erythrocytes, that it avoids major hydrolysis of organic phosphates, that it takes account of incomplete recovery of Pi and that it minimizes the effects of chilling the cells. In chilled samples from 46 patients in an intensive care unit, the cellular concentration of Pi was proportional to that in plasma. Blood samples from nine normal subjects were incubated at 37 degrees C. The cellular Pi was 0.79 mmol/litre of cells using an external standardization and 0.67 using an internal standardization. When the same cell samples were chilled on ice for 30 min, the internally standardized value decreased further to 0.57 mmol/litre of cells. These results suggest that differences in recovery, and the extent of chilling, contribute to the variability in the previously reported values for erythrocyte Pi. If Pi, like chloride, had distributed passively between cells and plasma in these samples, the cell to plasma molar concentration ratio for Pi should have been 0.29, compared with the measured value of 0.64. This difference suggests that some factor, in addition to passive diffusion, determined the distribution of Pi.

Phosphate metabolism in erythrocytes of critically ill patients.

Orthophosphate (Pi), adenosine 5'-diphosphate (ADP), adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) were measured in the erythrocytes of patients in an intensive care unit. The patients' plasma concentration of Pi varied from 0.1 to 4.2 mmol/l, and the corresponding concentration in erythrocytes varied from 0.1 to 2.0 mmol/litre of cells. Marked ATP depletion (less than 1 mmol/litre of cells) was only observed when erythrocyte Pi was less than 0.3 mmol/litre of cells and plasma Pi was less than 0.35 mmol/l. No dependence of 2,3-DPG concentration on the cellular concentration of Pi was detected. The phosphorylation potential [ATP]/([ADP] X [Pi]) varied inversely with the erythrocyte concentration of Pi. Hence the calculated free energy of hydrolysis of ATP in the cell increased from -58 kJ/mol in the most hypophosphataemic samples to -51 kJ/mol in the most hyperphosphataemic. Such changes may adversely affect cell function by altering the steady state mass-action ratios of ATPase reactions. When erythrocytes from normal donors were incubated in solutions containing 1 or 5 mmol/l Pi, the cellular concentrations of Pi stabilized at 1.09 and 2.85 mmol/litre of cells respectively. The corresponding rates of lactate production were 2.09 and 3.11 mmol h-1 litre-1 of cells. In spite of this stimulation of glycolysis (and hence of the flux through ATP synthesizing steps of the Embden-Meyerhof pathway), no significant change in ATP concentration was observed. As in the patients' cells, this indicates that, when extracellular Pi concentrations are perturbed, the concentrations, in erythrocytes, of organic phosphates are more closely regulated than the concentration of Pi.

Intravenous clodronate in the treatment and retreatment of Paget's disease of bone.

The effects of short courses (5 days) of intravenous clodronate 300 mg daily were studied in 31 patients with active Paget's disease of bone. The diphosphonate induced a striking reduction in biochemical indices of disease activity, which was sustained for at least 6 months after withdrawal of treatment. Apparent resistance to further treatment in patients previously treated for Paget's disease was an artefact due to incomplete relapse before retreatment. There was no significant difference in the degree of suppression of alkaline phosphatase activity between patients given intravenous clodronate and 45 patients given clodronate 1.6 g daily by mouth for 6 months. Short-term intravenous clodronate provides a useful alternative strategy for the treatment of patients with Paget's disease.

Comparative effects of an antiviral drug, inosiplex, and diphosphonates in Paget's disease of bone.

The effects of the antiviral agent, inosiplex, were assessed in four patients with Paget's disease of bone. Treatment for 6 months did not suppress disease activity as judged by serum alkaline phosphatase and hydroxyprolinuria, and viral inclusions persisted in the one patient from whom a bone biopsy was taken. These results contrasted markedly with the suppressive effects of diphosphonate treatment in these same patients.

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.

Biochemical markers of bone turnover in Paget's disease.

The two most commonly used biochemical markers of bone turnover are the serum alkaline phosphatase and the urinary excretion of peptide-bound hydroxyproline, both of which are increased in Paget's disease. Serum alkaline phosphatase is assumed to be derived from osteoblasts during the process of bone formation, whereas small peptides containing hydroxyproline are excreted in the urine following the degradation of bone collagen. The alkaline phosphatase is probably the more useful measurement for diagnosis and for following response to treatment, whereas hydroxyproline, although very sensitive, presents technical difficulties in collection and measurement. Several other biochemical changes in Paget's disease indicate abnormal bone metabolism. These include increased urinary excretion of hydroxylysine and its glycosides derived from collagen, as well as the release into the circulation and subsequent urinary excretion of fragments of pro-collagen indicative of increased collagen formation. Proteins specific to bone, such as osteocalcin, are increased in serum, bone, such as osteocalcin, are increased in serum, as are various enzymes possibly derived from bone cells, including acid phosphatase and proline imino-peptidase. Treatment of Paget's disease results in a fall in urinary hydroxyproline before alkaline phosphatase. This indicates that drug treatment, whether with diphosphonates, calcitonin or mithramycin, has a primary action to inhibit bone resorption, with a subsequent adaptive reduction in bone formation rate.

Effect of dichloromethylene diphosphonate in Paget's disease of bone and in hypercalcaemia due to primary hyperparathyroidism or malignant disease.

30 patients with disorders of calcium metabolism were treated with dichloromethylene diphosphonate (C1(2)MDP, or clodronate disodium), an inhibitor of bone resorption. 13 patients with Paget's disease of bone were given C1(2)MDP by mouth (1.6 g/day). Serum-alkaline-phosphatase and urinary hydroxyproline fell to normal or near-normal within 3-7 months, and there was a clinical improvement in all but 1 patient. C1(2)MDP (0.8-3.2 g/day) also reduced plasma-calcium and urinary calcium in 17 patients with hypercalcaemia due to primary hyperparathyroidism or secondary to malignant disease. C1(2)MDP seems to be an effective oral drug for inhibiting excessive bone resorption in man.

High affinity calcium binding peptides from the gills, gut and kidneys of the freshwater eel (Anguilla anguilla).

We report the isolation of high affinity calcium-binding peptides from homogenates of gills, gut and kidneys of the freshwater eel Anguilla anguilla. These peptides show identical gel-filtration profiles on Biogel P-6, from which a molecular weight of 3500 was estimated. The average binding constant (Kf) for calcium is 7 . 10(7)M-1, and MgCl2 up to 5 mM does not displace calcium.

Biochemical measurements in Paget's disease of bone.

In a group of eighty-three patients with untreated Paget's disease of bone, plasma alkaline phosphatase activity (AP), plasma non-protein-bound hydroxyproline concentration (PHP) and urinary excretion rate of total hydroxyproline (THP) were closely correlated with each other but not with fasting plasma concentrations of calcium or inorganic phosphate. Probit plots of AP and THP showed log-normal distributions overlapping the normal ranges.

An estimate of the turnover rate of bone-derived plasma alkaline phosphatase in Paget's disease.

A through-knee amputation for suspected osteogenic sarcoma was performed on a patient with Paget's disease of the tibia. Plasma alkaline phosphatase (AP) activity fell to normal values, apparently as a single exponential function of time, with a half-life of 1.7 days. The daily turnover of bone-derived plasma AP was estimated to be approximately 200 I.U. from the Pagetic tibia and 20 I.U. from the rest of the skeleton.