Fluoride and Biological Calcification I: Effect of Fluoride on Collagen-Induced In Vitro Mineralization and Demineralization Reactions.

An in vitro system employing collagen isolated from the sheep tendons to induce mineralization and demineralization reactions was used not only to study the effect of various concentrations of fluoride on the collagen-induced mineralization and demineralization reactions but also to compare their action with the inhibitors of mineralization and/or demineralization. Studies demonstrated that under physiological conditions, at lower concentrations (5 × 10-6 to 5 × 10-5 M) fluoride inhibited while at higher concentrations (> 10-4 M), it stimulated the collagen-induced in vitro mineralization. At higher concentrations, fluoride was also found to inhibit the demineralization of the collagen bound preformed mineral phase. At low concentrations, fluoride acted like Mg2+ to inhibit mineralization while at higher concentration, it acted like crystal poisons (e.g., pyrophosphate phosphonates, citrate) to inhibit demineralization. However, unlike magnesium and pyrophosphate, fluoride at its higher concentrations was found to stimulate rather than inhibit the process of mineralization.

Role of a protein inhibitor isolated from human renal stone matrix in urolithiasis.

The role of biomolecule(s) from renal stone matrix in urolithiasis was investigated. The ability of a particular fraction (> 10 kDa fraction) isolated from the EDTA extract of powdered human renal stones to influence calcium oxalate monohydrate (COM) crystal growth was studied. The most potent inhibitor of COM crystal growth obtained from > 10 kDa fraction was purified by various chromatographic techniques and SDS-PAGE, etc. and was found to have a molecular mass of 36 kDa. The urine and serum samples obtained from normal persons were found to be more potent in inhibiting the growth of COM crystals as compared to the kidney-stone patients. Polyclonal antibodies were raised against this inhibitor and were employed to determine the concentration of 36 kDa inhibitor in urine and serum samples of normal persons and kidney-stone patients.

Effect of nucleoside-5'-phosphates on collagen-induced in vitro mineralization.

Nucleoside triphosphates (NTPs) at 4-10 microM concentrations were found to inhibit the rates of collagen-induced in vitro mineralization and ion exchange reactions. The sequential removal of the terminal phosphate groups caused a step-wise decrease in their inhibitory potency. The results suggest that NTPs inhibit the rates of ion uptake and exchange reactions at concentrations much lower than their intracellular physiological concentrations. Thus NTPs may be involved in the control of biological mineralization and the tissues which mineralize under physiological conditions develop a system to locally convert NTPs to NDPs and NMPs.

Mg2+: a potent inhibitor of collagen-induced in vitro mineralization.

At physiological concentrations, Mg2+ has been found to be a potent inhibitor of collagen-induced in vitro mineralization. Mg2+ inhibits mineralization by competing with Ca2+ for specific phosphate independent Ca2+ binding sites of the catalytic matrix. Matrix bound Mg2+ subsequently reacts with HPO4(2-) to form MgHPO4 complex which can not be further converted to the matrix bound mineral phase. The matrix, as well as the mineral phase associated with the matrix, influence the rate of mineralization.

Concentration of a potent calcium oxalate monohydrate crystal growth inhibitor in the urine of normal persons and kidney stone patients by ELISA-based assay system employing monoclonal antibodies.

Standardized calcium oxalate monohydrate (COM) crystal growth assay system was employed to study the ability of various test samples to influence growth rates of COM crystals. The inhibitory activity (IA) of various samples was expressed in terms of inhibitory units. Urine samples obtained from normal persons and kidney stone patients were found to have IA of 3.18 +/- 0.62 and 1.02 +/- 0.08, respectively. A potent inhibitor having molecular weight between 14.2 and 16.2 kDa was found to be primarily responsible for the differences observed in the urinary IAs between normal persons and kidney stone patients. The potent inhibitor was found to be tightly associated with a chromophore resembling Urobilirubin. An ELISA based assay system, using monoclonal antibodies against the above most potent inhibitor confirmed the difference observed in the urinary IA between the normal persons and kidney stone patients. This assay system has the potential to be routinely used to screen human beings for potential stone formers.

Role of biomolecules from human renal stone matrix on COM crystal growth.

Human renal calculi surgically removed from kidney stone patients were obtained and chemically analysed. Stones with CaOx (calcium oxalate) as the major component were washed in 0.15 M NaCl with gentle stirring for 48 h and then pulverised to a fine powder. The powder was extracted with 0.05 M EGTA, 1 mM PMSF and 1% beta-mercaptoethanol for 4 days at 4 degrees C, the suspensions and the supernatants obtained were filtered through an Amicon Model 200 apparatus (mol. wt. cut off of 10,000 daltons) under nitrogen at 40 p.s.i. and concentrated to a known volume. The method of Nakagawa et al. [7] was employed to study the ability of > 10 kDa fractions to influence COM growth using metastable solution of CaCl2 and Na2C2O4 containing traces of 14C-oxalic acid. Potent biomolecules having the ability to influence CaOx precipitation were subjected to isolation, purification and characterization. Standard biochemical procedures, e.g. ultracentrifugation, ion-exchange chromatography, molecular sieve chromatography and SDS-PAGE, etc., were employed. Results revealed that human renal calculi extract contains biomolecules that can inhibit as well as stimulate the growth of preformed COM (calcium oxalate monohydrate) crystals. Most potent stimulator of CaOx growth was found to have a molecular weight of 66 kDa.

Inhibitors of in vitro mineralization from rabbit aorta and their role in biomineralization.

Mineralization of aorta is known to occur late in life and appears to be a pathological phenomenon. In vitro studies revealed that the matrix prepared from the thoracic aorta pieces after their extraction with 3% Na2HPO4 and 0.1 mM CaCl2 were mineralized under physiological conditions of temperature, pH, and ionic strength of the media to form matrix-bound mineral phase resembling hydroxyapatite in nature. However, the matrix identically prepared from the unextracted rabbits aortae failed to mineralize under identical assay conditions. The addition of the aorta extract in the assay system inhibited the above mineralization process. Standard biochemical techniques, e.g., dialysis, ion exchange, and molecular sieve chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis by high-performance liquid chromatography were employed to isolate, purify, and characterize the potent inhibitory biomolecules from the aorta extract. The inhibitory activity of the aorta extract was found to be primarily due to the presence of three biomolecules having molecular weights of 66, 45, and 27-29 kDa. The above inhibitory biomolecules loosely associated with aorta may be involved in the control of calcification associated with arteriosclerosis.

Inhibitors of in vitro mineralization from flexor tendons of rabbits and their role in biological mineralization.

Studies demonstrate that flexor tendons contain loosely associated biomolecules which inhibit its mineralization under physiological conditions. Based upon their molecular weights, these inhibitory biomolecules, could be classified into two categories, having molecular weights less than and greater than 13,000 daltons. The main inhibitory biomolecule was found to be an acidic polypeptide having molecular weight of 12,400 daltons.

Calcium and oxalate uptake by the renal brush border membrane vesicles in magnesium-deficient rats.

Calcium and oxalate uptake by renal brush border membrane vesicles (BBMV) was examined in magnesium-deficient and pair-fed control rats. Uptake studies were carried out by rapid filtration technique and rate of influx of calcium and oxalate as a function of extravesicular concentration (0.1 nM--1.0 mM) examined. Calcium uptake by renal BBMV exhibited saturable kinetics while oxalate uptake followed a biphasic transport mechanism showing saturable kinetics at low oxalate concentrations and passive diffusion at higher concentrations. In magnesium deficiency the kinetics of calcium and oxalate uptake by renal BBMV remained unaltered but the rate of uptake was significantly enhanced at all the extravesicular concentrations studied. Double reciprocal plot for calcium uptake showed no change in Vmax but a decrease in Km (2.08 mM) in magnesium--deficient rats as compared to pair-fed controls (Km = 5.00 mM). Similar plot for oxalate uptake showed an increase in Vmax (7.69 nmoles/8 min/mg protein) in magnesium deficient group as compared to pair-fed controls (5.55 nmoles/8 min/mg protein), while Km remained unchanged. The results of the present study indicate high risk of calcium oxalate stone formation in magnesium--deficient rats due to hyperabsorption and retention of calcium and oxalate by the renal tubular brush border membrane.

Oxalate metabolism in magnesium-deficient rats.

Male weanling rats were maintained on magnesium-deficient diet for 30 d and compared with pair-fed control rats fed magnesium-supplemented diet. Magnesium deficiency led to slow growth and finally to a significant decrease in body weight (P < 0.001) accompanied by a significant hypomagnesaemia, hypomagnesuria and hyperoxaluria (P < 0.001 in each case) in experimental rats as compared to the control rats. Magnesium deficiency altered the glyoxylate metabolism in the liver and kidney mitochondria by significantly decreasing glyoxylate oxidation (by 26 per cent in liver and 17 per cent in kidney) and activity of alpha-ketoglutarate:glyoxylate carboligase enzyme (by 35 per cent in liver and 27 per cent in kidney) in the experimental animals. A significant increase in the specific activities of glycolic acid oxidase (P < 0.001) and glycolic acid dehydrogenase (P < 0.01) and a significant decrease in alanine transaminase (P < 0.01) was also observed in magnesium-deficient rats. No change in liver and kidney lactate dehydrogenase was observed. Thus magnesium deficiency in rats leads to accumulation of glyoxylate in the tissues, a part of which is converted into oxalate, thereby promoting hyperoxaluria.

Intestinal absorption of calcium and oxalate in magnesium-deficient rats.

To examine the contribution of exogenous calcium and oxalate in magnesium deficiency, intestinal absorption of both calcium and oxalate was studied by preparing brush border membrane vesicles (BBMV). Purity of the BBMV was ascertained biochemically by enrichment of the marker enzyme alkaline phosphatase by 14-fold with a concomitant 90 per cent decrease in the basolateral marker enzyme Na+/K(+)-ATPase in the purified membrane preparation as compared to the respective homogenate in both the groups. Uptake studies were carried out by a rapid filtration technique. The kinetics were studied by measuring the rate of influx as a function of concentration (0.1-1.0 mM). BBMV from both the groups showed a linear positive relationship between the uptake rate and the concentration for both calcium and oxalate, thereby demonstrating that calcium and oxalate are transported through intestinal microvillus membrane by a simple passive diffusion process. However, the rate of uptake of calcium and oxalate was significantly higher in the magnesium-deficient group as compared to the pair-fed control group, as shown by the increase in the slope line for both calcium and oxalate (for calcium, control = 3.88, deficient = 5.86; for oxalate, control = 4.41, deficient = 7.20). Analysis of the lipid composition of the BBM revealed a significant decrease in the cholesterol content (P < 0.01) with a concomitant increase in the triglycerides (P < 0.01) and total fatty acid content (P < 0.001) in the magnesium-deficient group. Thus the results indicate that, although the mechanism of translocation of calcium and oxalate in the intestine is similar in the two groups, magnesium deficiency leads to hyperabsorption of both the ligands through alterations in the lipid composition of the membrane, thereby increasing the risk of stone formation.

Electrical conductivity of kidney stones.

Electret behaviour of surgically removed kidney stones through thermally stimulated polarization and thermally stimulated depolarization has already been reported by the authors. To develop an understanding of the conduction mechanism of such samples, d.c. conductivity was studied as a function of temperature and electric field. Temperatures from 308 to 448 K and fields from 0.5 X 10(6) to 2.5 X 10(6) V/m were used on the compressed powder pellet of a kidney stone. Conductivity was found to be 6 X 10(-11) s/m to 1.0 X 10(-11) s/m in the studied temperature range. Possibility of semiconduction is shown and it is suggested that kidney stone is a partially compensated semiconductor of n-type. The field independence of conductivity is observed at higher temperatures. The slopes of current density versus field curves suggest a warm electron effect.

Effect of an Ayurvedic compound preparation on the ability of rat urine to influence mineral phase formation.

Rat urine could inhibit not only the in vitro initial precipitation of calcium and phosphate/oxalate ions as mineral phase but also the subsequent growth of the preformed mineral phase. Oral administration of the aqueous extract of a commercially available Ayurvedic compound preparation to rats, was found to significantly increase the ability of the urine samples to inhibit both the initial mineral phase formation and its subsequent growth.

The electret effect of kidney stones.

Surgically removed kidney stones were powdered and samples made from them were subjected to a d.c. field of 20-50 kV/cm whilst being heated at 1 degree C/min. The thermally stimulated polarization (TSP) spectrum obtained showed the dipolar nature of the material. Dipolar polarization in the stones was confirmed by other experiments.