Herbert Fleisch

The Inhibitory Effect of Phosphonates on the Formation of Calcium Phosphate Crystals in vitro and on Aortic and Kidney Calcification in vivo

Abstract 1. Various phosphonates, which are compounds containing C‐P bonds, have been studied to see whether they are able to inhibit, in a manner similar to that of pyrophosphate and the condensed phosphates, the crystallization of calcium phosphate in vitro and the pathological calcification of the aorta and the kidneys of rats given large amounts of vitamin D3.

Bone-resorbing cytokines enhance release of macrophage colony-stimulating activity by the osteoblastic cell MC3T3-E1.

SummaryIt has been observed that bone resorption in response to interleukin 1 (IL 1) or tumor necrosis factor (TNF) is accompanied by an increase in osteoclast number. Because the osteoclast is of hemopoietic lineage, recruitment could be regulated by colony-stimulating factors, one of which may be macrophage colony-stimulating factor (MCSF). In this study, we show that the constitutive release of M-CSF activity by the osteoblastic cell MC3T3-E1 is enhanced by the presence of recombinant IL 1α, recombinant TNFα, or by the concurrent presence of purified transforming growth factorβ (TGFβ) and epidermal growth factor (EGF). Increased release of CSF by the osteoblast in response to these agents may provide a signal for the growth and maturation of osteoclast precursors leading to subsequent bone resorption.

The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes.

Diphosphonates are known to inhibit bone resorption in tissue culture and in experimental animals. This effect may be due to their ability to inhibit the dissolution of hydroxyapatite crystals, but other mechanisms may be important. Since lysosomal enzymes have implicated in the process of bone resorption, we have examined the effect of several phosphonates and of a polyphosphate (P20,2) on lysosomal hydrolases derived from rat liver and rat bone. Dichloromethylene diphosphonate strongly inhibited acid beta-glycerophosphatase (EC 3.1.3.2) and acid p-nitrophenyl phosphatase (EC 3.1.3.2) and to a lesser degree (in descending order) acid pyrophosphatase (EC 3.1.3.-), arylsulfatase A (EC 3.1.6.1), deoxyribonuclease II(EC 3.1.4.6) and phosphoprotein phosphatase (EC 3.1.3.16) of rat liver. Inhibition of acid p-nitrophenyl phosphatase and arylsulfatase A was competitive. Ethane-1-hydroxy-1, 1-diphosphonate did not inhibit any of these enzymes, except at high concentrations. Neither dichloromethylene diphosphonate nor ethane-1-hydroxy-1, 1-diphosphonate had any effect on beta-glucuronidase (EC 3.2.1.31), arylesterase (EC 3.1.1.2) and cathepsin D (EC 3.4.23.5). Of several other phosphonates tested only undec-10-ene-1-hydroxy-1, 1-diphosphonic acid inhibited acid p-nitrophenyl phosphatase strongly, the polyphosphate (P20, I) had little effect. Acid p-nitrophenyl phosphatase in rat calvaria extract behaved in the same way as the liver enzyme and was also strongly inhibited by dichloromethylene diphosphonate, but not by ethane-1-hydroxy-1, 1-diphosphonate. It is suggested that the inhibition of bone resorption by dichloromethylene diphosphonate might be due in part to a direct effect of this diphosphonate on lysosomal hydrolases.

Aggregation 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.

Pyrophosphatase and ATPase of isolated cartilage matrix vesicles

Some of the characteristics of the pyrophosphatase and ATPase activities studied in isolated cartilage matrix vesicles were found to be similar to those already reported for the solubilized and purified bone alkaline phosphatase. Thus, the pH optimum of the pyrophosphatase activity responded similarly to changes in the concentration of Mg2+, Ca2+, and PPi. Further, the ATPase activity was not activated by Ca2+ in the presence of an optimal Mg2+ concentration. It is proposed that a function of the alkaline phosphatase of matrix vesicles in vivo is to hydrolyze the substrates PPi, ADP, and ATP, which are known inhibitors of calcium phosphate precipitation.

Isolation of inorganic pyrophosphate from bovine and human teeth.

Abstract Inorganic pyrophosphate has been detected in enamel and dentine from human and bovine teeth. The techniques used were ion exchange chromatography and paper chromatography with 32P-pyrophosphate as marker, and precipitation as the manganese salt with subsequent infrared spectroscopy. During extraction of enamel, a part of the pyrophosphate was found to bind to an unidentified substance that altered its chromatographic behaviour. Using an isotope dilution technique, pyrophosphate was measured quantitatively in human teeth. Dentine contained 6.9 and enamel 1.3 mg pyrophosphate-P/g total-P. The possible origin and significance of pyrophosphate in teeth is discussed.

Effect of diphosphonates on the synthesis of prostaglandins in cultured calvaria cells.

SummaryIn calvaria cells cultured for 6 days in the presence of dichloromethanediphosphonate (Cl2MDP) (0.025 – 250 µM), the synthesis of prostaglandin E2 (PGE2) was inhibited by up to 90 %. Inhibition of PGE2 synthesis might be one mechanism whereby this diphosphonate inhibits bone resorption. This effect is not common to all diphosphonates since 25 µM 3-amino-1-hydroxypropane-1,1-diphosphonate (AHPDP) stimulated the synthesis of PGE2.

Inhibition of vitamin D metabolism by ethane-1-hydroxy-1, 1-diphosphonate☆

Abstract The administration of disodium-ethane-1-hydroxy-1,1-diphosphonate (20 mg/kg body weight subcutaneously) to chicks given adequate amounts of vitamin D 3 causes a hypercalcemia, inhibits bone mineralization, and inhibits intestinal calcium transport. The administration of 1,25-dihydroxyvitamin D 3 , a metabolically active form of vitamin D 3 , restores intestinal calcium absorption to normal but does not restore bone mineralization in disodium-ethane-1-hydroxy-1,1-diphosphonate-treated chicks. In rachitic chicks, the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment does not further reduce the low intestinal calcium transport values while it nevertheless further reduces bone ash levels and increases serum calcium concentration. These observations prompted a more detailed study of the relationship between disodium-ethane-1-hydroxy-1,1-diphosphonate treatment and vitamin D metabolism. A study of the hydroxylation of 25-hydroxyvitamin D 3 in an in vitro system employing kidney mitochondria from chicks receiving disodium-ethane-1-hydroxy-1,1-diphosphonate treatment demonstrates a marked decrease in 1,25-dihydroxyvitamin D 3 production and a marked increase in the 24,25-dihydroxyvitamin D 3 production. In addition, the in vivo metabolism of 25-hydroxy-[26,27- 3 H]vitamin D 3 in disodium-ethane-1-hydroxy-1,1-diphosphonate treated chicks supports the in vitro observations. In rachitic chicks the disodium-ethane-1-hydroxy-1,1-diphosphonate treatment markedly reduces the 25-hydroxyvitamin D 3 -1-hydroxylase activity of kidney, but does not increase the 25-hydroxyvitamin D 3 -24-hydroxylase. These results provide strong evidence that large doses of disodium-ethane-1-hydroxy-1,1-diphosphonate produce a marked effect on calcium metabolism via alterations in the metabolism of vitamin D as well as the expected direct effect on the bone.

Sodium EDTA enhances intestinal absorption of two bisphosphonates

SummaryBisphosphonates are poorly absorbed when given orally and their absorption is subject to a large inter-and intraindividual variability. This poor absorbability is thought to result, at least in part, from formation of unabsorbable complexes with calcium. It was therefore investigated whether the calcium chelator EDTA could improve intestinal absorption of two bisphosphonates, 4-amino-1-hydroxybutylidene-1, 1-bisphosphonate (AHBuBP), and dichloromethylenebisphosphonate (Cl2MBP). Absorption was assessed indirectly by measuring the suppression of hypercalcemia induced in thyroparathyroidectomized rats by a retinoid. The absorption of AHBuBP was in the range of 1–3%. EDTA increased absorption about tenfold at a AHBuBP dose of 0.6 mg P/kg and about twofold at lower doses, with the minimal effective dose of EDTA being 10 mg/kg. The absorption of Cl2MBP was also increased by EDTA, although to a smaller extent, the lowest effective dose being 100 mg/kg EDTA. Thus, EDTA can, in certain circumstances, increase the intestinal absorption of bisphosphonates. The mechanism might involve an increase in available bisphosphonate and a change in mucosal permeability. The amount of EDTA required is, however, too high for use clinically.

Synthesis of membrane‐ and matrix‐bound colony‐stimulating factor‐1 by cultured osteoblasts

Colony‐stimulating factor‐1 (CSF‐1) is synthesized as a secreted or membrane‐bound molecule. We investigated whether osteoblastic cells produce these forms of CSF‐1. Glutaraldehyde‐fixed cell layers supported proliferation of the macrophage cell line BAC1.2F5, suggesting the presence of membrane‐ or/and matrix‐associated CSF‐1. Furthermore, CSF‐1 activity could be either extracted from the matrix or released from the cell membrane. A neutralizing antiserum against CSF‐1 inhibited these activities. After labeling the cellular proteins with [35S] met/cys or [35S] SO2−4, CSF‐1 was immunoprecipitated and analyzed by SDS‐PAGE. Under nonreducing conditions, bands with MW more than 200, 200, 100, and 50 kd were detected. These bands shifted to lower MW under reducing conditions. Treatment with chondroitin lyase ABC decreased the MW of the 200 kd monomer, proving the proteoglycan structure. Much smaller quantities of CSF‐1 were found in the matrix extract than in the conditioned medium. Transforming growth factor β (TGF‐β) increased both the synthesis of CSF‐1 and its accumulation in the matrix. CSF‐1 released with trypsin from the membrane fraction yielded on SDS‐PAGE a band with MW of 60 and 30 kd under nonreducing and reducing conditions, respectively. Transcripts encoding both the secreted and the membrane‐associated forms of the cytokine were detected in osteoblasts by reverse transcription polymerase chain reaction. These data indicate that osteoblastic cells produce the secreted forms, either remaining in the culture supernatant, or being associated to the matrix, and the membrane associated form of CSF‐1.