Biochemical and clinical effects of ethane-1-hydroxy-1,1-diphosphonate in calcium nephrolithiasis.

1. The short- and longer-term effects of ethane-1-hydroxy-1,1-diphosphonate (EHDP), an inhibitor of crystal growth and potential preventive agent against urinary tract stones in man, have been studied. 2. Measurement of urinary excretion of EHDP was used to define the best dosage regimen. When 4.4 mmol of EHDP mmol of EHDP was given in four divided doses the murinary concentration of EHDP achieved was high enough (10-5 mol/1) to inhibit the crystallization of calcium crystals throughout the day. 3. Nine patients with recurrent calcium stones were given this dose of EHDP daily for 12 months and seven were then studied for a further 12 months under placebo. During treatment with EHDP, inhibitory activity in urine towards precipitation of calcium phosphate was restored from low values to greatly above normal. This could be accounted for by the inhibitory effect of EHDP itself, coupled with an increase in urinary inorganic pyrophosphate. After stopping EHDP the excretion of EHDP rapidly fell to undetectable levels but the excretion of pryophosphate remained elevated throughout the 12 months of placebo treatment. EHDP also induced a rise in plasma phosphate and an increase in the urinary excretion of oxalic acid and uric acid, but these changes were all fully reversible when EHDP was stopped. 4. The average rate of stone formation per patient per year decreased from 2.4 to 0.2 during treatment with EHDP and remained low during the following 24 months. However, the dose needed for this effect is known to affect bone turnover and mineralization.

Uptake by bone of pyrophosphate, diphosphonates and their technetium derivatives.

1. The uptake of inorganic pyrophosphate (PPi) from blood to bone was investigated in the rat in vivo. 2. PPi is taken up by the bone, where it appears both as PPi and as inorganic orthophosphate (Pi). The latter is due at least partly to local hydrolysis. 3. The fraction of injected PPi taken up by bone, measured as total PPi, was in the same range as that of technetium-tin-PPi, diphosphonates, technetium-tin-ethane-1-hydroxy-1,1-diphosphonate and Pi, but lower than that of calcium. 4. The plasma half-life of PPi is in the same order of magnitude as that of technetium-tin-PPi, diphosphonates, technetium-tin-ethane-1-hydroxy-1,1-diphosphonate, Pi and calcium. 5. PPi, diphosphonates and their technetium complexes are only partly ultrafiltrable in plasma. 6. It appears that the technetium complexes behave in a similar fashion to free PPi or diphosphonate.

Disaggregation of hydroxyapatite crystals.

A system has been developed to measure quantitatively the disaggregation of hydroxyapatite crystals. Disaggregation was induced by pyrophosphate, ethane-1-hydroxy-1,1-diphosphonate, dichloromethylene diphosphonate, haparin and citrate. Hyaluronic acid stimulated aggregation at low concentrations and disaggregation at high concentrations. Lactate had no effect. The possible role disaggregation might play in the resorption of calcified tissues in vivo id discussed.

Aggregation of hydroxyapatite crystals.

A system to study the aggregation of hydroxyapatite crystals was developed. The effect of several factors (Ca2+ x Pi product, Ca2+/Pi ratio, pH, and various substances) were tested. Pb2+, Zn2+, Mg2+ and methyleneblue had only small effects; citrate inhibited aggregation. Pyrophosphate was a strong inhibitor and the diphosphonates disodium ethane-1-hydroxy-1,1-diphosphonate and disodium dichloromethylene diphosphonate were even more potent. The monophosphonate pentanemonophosphonate had no effect. Potent inhibition also occurred with glycosaminoglycans: heparin greater than hyaluronic acid greater than dermatan sulfate greater than chondroitin 4-sulfate greater than chondroitin 6-sulfate. Urine also showed high inhibitory activity. The inhibition of heparin but not that of hyaluronic acid, PPi or urine was abolished by egg white lysozyme. The effects described might be relevant in the normal mineralization process as well as in the mechanisms leading to pathological calcification, such as urinary stone formation.

The influence of orthophosphate on the renal handling of inorganic pyrophosphate in man and dog.

1. The urinary excretion of inorganic pyrophosphate (PPi), a known inhibitor of the growth and aggregation of crystals of calcium phosphate and calcium oxalate, increases after ingestion of orthophosphate (Pi). This effect may contribute to the apparent ability of oral phosphate to reduce the formation of urinary stones in man. This paper is a study of the mechanism by which Pi increases PPi excretion, investigated by renal clearance techniques in man and renal arterial infusion in dogs. PPi in plasma was measured by an isotope-dilution method after ion-exchange chromatography. 2. The mean renal clearance of endogenous PPi in ten men was 7-9 +/- 1-7 (SE) ml/min, and the mean ratio of PPi clearance to creatinine clearance was 0-08 +/- 0-02 (SE). The oral ingestion of Pi increased the urinary excretion and renal clearance of PPi about threefold, without significantly changing its concentration in plasma. 3. In dogs, the infusion of Pi into one renal artery caused a greater increase in urinary PPi from the infused than from the non-infused kidney, an effect that could be accentuated by simultaneous intravenous infusion of PPi. In dogs, only 1-3% of an injected or infused dose of PPi appeared intact in the urine, regardless of whether it was infused into the systemic or renal circulation. 4. These results suggest that Pi has a direct affect on the kidney to increase the excretion of PPi. It is possible that Pi either interferes with tubular reabsorption of PPi, perhaps by competing for a common tubular transport mechanism, or that Pi diminishes the intrarenal hydrolysis of PPi.

The comparative effects of vitamin d deficiency and ethane-1-hydroxy-1,1-diphosphonate administration on the histology and glycolysis of chick epiphyseal and articular cartilage.

A comparison has been made between the effect of a vitamin D--deficient diet and treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) on the morphology of chick epiphyseal cartilage and on the production of lactate in vitro by epiphyseal and articular cartilage. The cell populations in the growth plate were different following the two treatments. Vitamin D deficiency was characterized by an increase in proliferating cells, with a relative paucity of hypertrophic cells; EHDP treatment was characterized by an increase in hypertrophic cells. When similar cell types were compared, neither treatment changed lactate production from the control value. This stresses the need to correlate the morphology of cell types with their metabolic function. The present results indicate that the major effect of vitamin D deficiency in the chick is to block the differentiation of proliferating to hypertrophic cells. In contrast, EHDP may act by inhibiting calcification directly. Even though EHDP at the doses used is known to interfere with the production of 1,25-dihydroxycholecalciferol there is no block to cell differentiation under EHDP similar to that seen in dietary deficiency of vitamin D.

Quantitative determination of ethane-1-hydroxy-1,1-diphosphonate in urine and plasma.

A technique has been developed to measure the diphosphonate ethane-1-hydroxy-1, 1-diphosphonate (EHDP) quantitatively in 5 ml of urine or 2 ml of plasma. The procedure is based on a coprecipitation of EHDP with calcium phosphate, elimination of inorganic phosphate as an insoluble triethylamine-phosphomolybdate complex, decomposition of the P-C-P bond with ultraviolet light and spectrophotometric determination of the inorganic phosphate released. A trace amount of [14C] EHDP is used to correct for losses. The method appears specific for the diphosphonate, exhibits quantitative recoveries, and has a mean coefficient of variation of 3.7% for urine and 7.3% for plasma. The limit of detection is in the order of 2.5 mumol/1 in 5 ml urine and 0.5 mumol/1 in 2 ml of plasma.

The uptake and metabolism of (32-P)pyrophosphate by mouse calvaria in vitro.

In order to study the uptake and metabolism of PP(i) by bone, (32)PP(i) was added to the medium surrounding explanted mouse calvaria maintained in organ culture. Most of the PP(i) was hydrolysed during incubation, but there was a measurable entry of intact PP(i) into bone. When (32)P(i) was added to the medium, synthesis of PP(i) and organic phosphates from P(i) was observed in bone. There was no detectable passage of PP(i) from bone into the medium. These results are discussed in terms of two models of pyrophosphate hydrolysis and exchange. Some quantitative estimates about the fate of PP(i) in bone were made.

Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and other disorders of bone.

An isotope dilution method, using (32)P-labeled pyrophosphate, has been developed for the measurement of inorganic pyrophosphate (PP(1)) in human plasma. The specificity of the method was better than 90% as assessed by elution patterns during ion-exchange chromatography, by paper chromatography, and by incubation with inorganic pyrophosphatase. The 99% confidence limits for a single estimation of plasma PP(1) was +/-13%. There were no differences in plasma PP(1) between men and women, but the values in young people (0-15 yr) were slightly higher than in older people. The mean concentration (+/-SE) of PP(1) in the plasma of 73 men and women was 3.50 +/-0.11 mumoles/liter (0.217 +/-0.007 mug P/ml) and the normal range (99% limits) was 1.19-5.65 mumoles/liter (0.074-0.350 mug P/ml). It has been suggested that PP(1) may be important in calcium metabolism because PP(1) can prevent the precipitation of calcium phosphates in vitro and in vivo, and can slow the rates at which hydroxyapatite crystals grow and dissolve. Plasma PP(1) was therefore measured in several disorders of bone. Normal values were found in osteogenesis imperfecta, osteopetrosis, "acute" osteoporosis, and primary hyperparathyroidism. Plasma PP(1) was invariably raised in hypophosphatasia. The excess of PP(1) in plasma might be the cause of the defective mineralization in hypophosphatasia and the function of alkaline phosphatase in bone may be to act as a pyrophosphatase at sites of calcium deposition.