Precipitation Of Amorphous Calcium Phosphate Research Articles

Effect of solvent and temperature on the size distribution of casein micelles measured by dynamic light scattering

The objectives of this study were to investigate the effect of the solvent on the accuracy of casein micelle particle size determination by dynamic light scattering (DLS) at different temperatures and to establish a clear protocol for these measurements. Dynamic light scattering analyses were performed at 6, 20, and 50°C using a 90Plus Nanoparticle Size Analyzer (Brookhaven Instruments, Holtsville, NY). Raw and pasteurized skim milk were used as sources of casein micelles. Simulated milk ultrafiltrate, ultrafiltered water, and permeate obtained by ultrafiltration of skim milk using a 10-kDa cutoff membrane were used as solvents. The pH, ionic concentration, refractive index, and viscosity of all solvents were determined. The solvents were evaluated by DLS to ensure that they did not have a significant influence on the results of the particle size measurements. Experimental protocols were developed for accurate measurement of particle sizes in all solvents and experimental conditions. All measurements had good reproducibility, with coefficients of variation below 5%. Both the solvent and the temperature had a significant effect on the measured effective diameter of the casein micelles. When ultrafiltered permeate was used as a solvent, the particle size and polydispersity of casein micelles decreased as temperature increased. The effective diameter of casein micelles from raw skim milk diluted with ultrafiltered permeate was 176.4±5.3nm at 6°C, 177.4±1.9nm at 20°C, and 137.3±2.7nm at 50°C. This trend was justified by the increased strength of hydrophobic bonds with increasing temperature. Overall, the results of this study suggest that the most suitable solvent for the DLS analyses of casein micelles was casein-depleted ultrafiltered permeate. Dilution with water led to micelle dissociation, which significantly affected the DLS measurements, especially at 6 and 20°C. Simulated milk ultrafiltrate seemed to give accurate results only at 20°C. Results obtained in simulated milk ultrafiltrate at 6°C could not be explained based on the known effects of temperature on the casein micelle, whereas at 50°C, precipitation of amorphous calcium phosphate affected the DLS measurement.

An in vitro study of the effect of some dietary components on calculus formation: regulation of calcium phosphate precipitation

We studied the effects of food components on the in vitro formation of calcium phosphate precipitates. The effects of food components, such as starch, soybean flour, fish meal, rapeseed oil, and coconut oil, on calcium phosphate precipitation were studied using a pH drop method. Although the addition of starch had no effect on the rate of precipitation of amorphous calcium phosphate (ACP), it increased both the rate of transformation of ACP to hydroxyapatite (HAP) and the induction time (i.e. time for the initiation of transformation of ACP to HAP to occur); this was irrespective of the heat treatment of the starch. Amylopectin (insoluble constituent of starch) was effective in increasing the rate of HAP transformation, but amylose (soluble constituent of starch) was not. Oil specimen obtained from rapeseed (400 microl ml(-1)) increased the entire reaction of calcium phosphate precipitation, but that from coconut did not. Protein food, such as soybean flour and fish meal, decreased the rate of transformation of ACP to HAP and increased the induction time, while they had no effect on the rate of ACP precipitation. These results suggest that carbohydrate and oil (both are staple diets for the humans) enhance oral calcification (dental calculus formation or re-mineralization of tooth enamel), while side dishes of protein food would decrease it.

The Influence of Polyelectrolytes on the Formation and Phase Transformation of Amorphous Calcium Phosphate

A promising route to the fabrication of biomimetic coatings for artificial implants is the deposition of organic/inorganic composite materials consisting of polyelectrolyte multilayers alternating with layers of “in situ” grown calcium phosphate crystals. To facilitate understanding of the underlying mechanisms, in this paper we discuss the influence of polyelectrolytes (PEs), polystyrene sulfonate (PSS), poly-l-lysine (PLL), and poly-l-glutamic acid (PGA) on the formation and properties of amorphous calcium phosphate (ACP) and on the nucleation and growth morphology of the crystalline phase. pH vs time curves revealed three distinct precipitation events, i.e., (I) precipitation of ACP, (II) secondary precipitation of a crystalline phase upon the amorphous precursor, and (III) solution-mediated phase transformation and crystal growth. Finally, crystalline mixtures with low Ca/P molar ratios (1.39), consisting of octacalcium phosphate crystals and small amounts of apatite, were obtained. From the pH vs time curves, the induction time, ti, preceding crystal nucleation was determined. All PEs at low concentrations induced and at high concentrations inhibited nucleation. The efficiency of induction increased in the order: LMw PGA ≅ HMw PGA < LMw PLL < HMw PLL < PSS, while the inhibition efficiency increased as LMw PLL ≅ HMw PLL < PSS < LMw PGA < HMw PGA. ACP particles formed in the presence of PE were smaller and less aggregated and had a higher surface charge than in the controls. All investigated PEs also inhibited growth of the crystalline phase in a nonspecific way.

The Influence of Polyelectrotytes on the Formation and Phase Transformation of Amorphous Calcium Phosphate

A promising route to the fabrication of biomimetic coatings for artificial implants is the deposition of organic/inorganic composite materials consisting of polyelectrolyte multilayers alternating with layers of “in situ” grown calcium phosphate crystals. To facilitate understanding of the underlying mechanisms, in this paper we discuss the influence of polyelectrolytes (PEs), polystyrene sulfonate (PSS), poly-l-lysine (PLL), and poly-l-glutamic acid (PGA) on the formation and properties of amorphous calcium phosphate (ACP) and on the nucleation and growth morphology of the crystalline phase. pH vs time curves revealed three distinct precipitation events, i.e., (I) precipitation of ACP, (II) secondary precipitation of a crystalline phase upon the amorphous precursor, and (III) solution-mediated phase transformation and crystal growth. Finally, crystalline mixtures with low Ca/P molar ratios (1.39), consisting of octacalcium phosphate crystals and small amounts of apatite, were obtained. From the pH vs tim...

THE INFLUENCE OF POLYMER ARCHITECTURE ON INHIBITION OF AMORPHOUS CALCIUM PHOSPHATE PRECIPITATION

The influence of polymer architecture on the precipitation of amorphous calcium phosphate from supersaturated solutions has been investigated using the pH-stat technique. It has been found that low concentration of acrylic acid based copolymers and terpolymers markedly inhibit the precipitation of calcium phosphate. Homopolymers containing anionic, non-ionic, and cationic groups under similar experimental conditions are inefficient as calcium phosphate inhibitors. The inhibition data of several polymers indicate that the performance of polymers depends upon the functional group, ionic charge, and the molecular weight. In addition, it has been found that cationic flocculants and biocides when present at low concentrations exhibit strong antagonistic effect on the performance of calcium phosphate inhibiting polymers. INTRODUCTION In recent years many studies have been undertaken concerning the precipitation of sparingly soluble alkaline earth metal phosphates from their supersaturated solutions. Precipitation of calcium phosphates is of particular interest because of its importance in many areas. Common examples include dental calculus, calcified plaques of arteriosclerosis, kidney calcinosis, etc. Calcium phosphates deposits have been encountered during pasteurization of milk. In industrial water systems (i.e., desalination, boiler and cooling) precipitation and deposition of calcium phosphates on membrane and heat exchanger surfaces continue to pose serious operational challenges. In addition, calcium phosphates are widely produced in industry, in such forms as, ceramics, nutrient supplements, medicines, dentifrices, and stabilizers for plastic. They are utilized in solid state chemistry as fluorescent lamp phosphors and play a role in waste water treatment processes. It is generally agreed that initially precipitated calcium phosphate from aqueous solution of high supersaturation and at pH >7, is an amorphous calcium phosphate, ACP. The formation and stability of ACP phase in aqueous solution has been the subject of numerous investigations. It has been suggested that if ACP is allowed to remain in contact with the solution, it transforms to the thermodynamically stable hydroxyapatite, HAP, perhaps via the hydrolysis of octacalcium phosphate. The metastability of the precursor is very sensitive to the conditions of precipitation such as pH, temperature, and degree of supersaturation. The presence of additives and impurities, ranging from simple ions to small molecules and polymers, including proteins at crystal surfaces, is frequently found to have a profound effect on crystallization properties. Some impurities can suppress growth entirely; others may actually enhance nucleation and crystal growth. An important aspect in industrial and biological systems is the role that various macromolecules play as precipitation and crystal growth inhibitors for a variety of sparingly soluble salts such as calcium oxalate, calcium carbonate, calcium fluoride, barium sulfate, etc. It has been reported that the presence of both dissolved and suspended impurities in supersaturated solution exhibit marked influence on the performance of polymers as precipitation inhibitors. In the present work, we have investigated the effect of polymer architecture (i.e., polymer composition, ionic charge, molecular weight, etc.) on the precipitation of calcium phosphate from supersaturated solution. This study also reports the impact of cationic polymeric coagulants/flocculants and biocides on the performance of calcium phosphate precipitation inhibitors.

Biomineral-inspired fabrication of semi-permeable calcium phosphate–polysaccharide microcapsules

Mineral–polysaccharide microcapsules are produced by a one-step method in which the deposition of a semi-permeable alginate/chitosan membrane around droplets of sodium alginate is coupled with in situ precipitation of amorphous calcium phosphate. Nucleation of calcium phosphate occurs within the membrane by counter-diffusion of Ca2+ and HPO42− ions and can be controlled by the phosphate concentration in the alginate droplets to produce hybrid microcapsules with enhanced mechanical strength and reduced permeability to encapsulated molecules such as haemoglobin and ibuprofen.

Influence of bile salt conjugation and species on amorphous calcium phosphate precipitation in an agar gel system

Influence of bile salt conjugation and species on amorphous calcium phosphate precipitation in an agar gel system

Effects of silicate ions on the formation and transformation of calcium phosphates in neutral aqueous solutions

The effects of silicate ions on calcification reactions have been investigated in neutral solutions containing calcium and phosphate ions. The results have shown that the silicate ions enhanced the precipitation of amorphous calcium phosphate (ACP) and dihydrated dicalcium phosphate (DCPD). Transformation of these precursors to hydroxyapatite (HAP), However, was inhibited by silicate ions. X-Ray photoelectron spectroscopy (XPS) results have shown that a type of silicate was formed at the surface of the precursors in the early stage of the calcification process, resulting in inhibition of the transformation. On the contrary, silica, investigated for comparison with silicate ions, was found to provide favourable sites for ACP and DCPD nucleation. Thus, silica played the role merely of substrate for the nucleation, and as a result silica did not inhibit the conversion of the precursors to HAP.

Inhibition of calcium phosphate precipitation by bile salts: a test of the Ca(2+)-buffering hypothesis.

The ability of bile salts to inhibit the precipitation of either calcium hydroxyapatite or its precursor, amorphous calcium phosphate, by reducing Ca2+ activity or poisoning nascent crystals was determined. When apatite precipitated rapidly (1-4 h), glycocholate and taurine-conjugated bile salts (up to 100 mM) had little effect on apatite formation, but prevented amorphous calcium phosphate precipitation by lowering Ca2+ activity. In contrast, glycodeoxycholate and glycochenodeoxycholate (2-3 mM) inhibited apatite formation for at least 24 h by poisoning embryonic apatite. When apatite precipitated slowly (> 24 h), all the dihydroxy bile salts prevented apatite formation for at least 4 days. At constant initial supersaturation, the phosphate concentration determined the degree of inhibition caused by the six bile salts mixed together in physiologic proportion. At low phosphate concentrations (1.2 mM) total inhibition was achieved by poisoning embryos (approximately -5 mM total bile salt), but with 4.0 mM phosphate only approximately 60% inhibition was attained (150 mM bile salt) by a combination of poisoning and Ca(2+)-buffering. Thus, at low supersaturation all dihydroxy bile salts can prevent apatite formation by reducing free Ca2+ (taurine and glycine conjugates) or poisoning embryos (glycine conjugates). With mixtures of bile salts at higher supersaturation, inhibition of apatite depends on a combination of poisoning and reduction of free Ca2+, mainly caused by glycodeoxycholate and glycochenodeoxycholate.

Apparent solubilities of two amorphous calcium phosphates and of octacalcium phosphate in the temperature range 30–42°C

Precipitation of amorphous calcium phosphate and its transformation into crystalline materials are investigated. The variation in pH during the total process can be explained by initial formation of one amorphous phase, ACP1, followed by formation and growth of another amorphous phase, ACP2, causing dissolution of the initially formed phase. The amorphous phase ACP2 similarly dissolves due to formation and growth of octacalcium phosphate, OCP, which finally dissolves due to formation and growth of deficient hydroxyapatite. Apparent solubility products are determined for ACP1, ACP2 and OCP. From the variation of the solubility products with temperature, H ⊝ and S ⊝ for the dissolution of ACP1, ACP2 and OCP are determined.

Influence of polyelectrolytes on the precipitation of amorphous calcium phosphate

The pH-stat technique has been used to study the influence of polyelectrolytes on the precipitation of amorphous calcium phosphate (ACP) from moderately supersaturated solutions at pH 7.5 and temperatures ranging from 25 to 37°C. The polyelectrolytes studied were: homopolymers of acrylic acid, methacrylic acid, acrylamide, diallyldimethylammonium chloride, benzene polycarboxylic acids, tricarballylic acid and citric acid. The results indicate that the precipitation of ACP is proceeded by an induction period. Temperature changes, polyelectrolyte concentration, molecular weight and polyelectrolyte composition greatly affect the duration of the induction period. A mechanism based upon a surface adsorption model has been proposed to explain the marked effect on the initial induction period by the polyelectrolytes.

Clinical significance of phosphaturia

From September 1986 to August 1987, heavy precipitation of amorphous calcium phosphate, that is phosphaturia, was found at our outpatient clinic in 153 postprandial urine specimens from 115 patients, in 1.5% of all the specimens examined during this period. One patient was excluded because he had both urolithiasis and urinary tract infection. The remaining 152 specimens with phosphaturia were then divided into 3 groups; Group I from patients with urolithiasis, Group II from patients with urinary tract infection and Group III from patients without either urolithiasis or urinary tract infection (Phosphaturia Group, Table 1). It is evident from this table that phosphaturia is repeated more frequently in patients with urolithiasis than in patients without urolithiasis (p less than 0.01). 200 urine specimens examined during this period were selected randomly. Three bloody or purulent specimens were excluded. The remaining 197 specimens from 189 patients were divided into 3 groups, as in the phosphaturia group (Control Group, Table 2). When the proportion of the number of specimens to the total is compared between the phosphaturia group and the control group, it is clear that phosphaturia is found more frequently in specimens from patients with urolithiasis than in specimens from patients without urolithiasis (p less than 0.01). Since phosphaturia was almost always found in the specimens with urine pH greater than or equal to 7, 486 patients, in whom the pH of the first urine specimen was equal to or above 7, were selected from among 1434 patients undergoing urinalyses during this period and divided into 3 groups as has been described above (Table 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of gelatin on the precipitation of amorphous calcium phosphate

The influence of gelatin on the formation, stability and transformation of amorphous calcium phosphate, ACP, at 298 K and initial pH 7.4 was investigated. Precipitation of ACP was brought about from equimolar solutions (3×10 −3 mol dm −3) of calcium chloride and sodium phosphate while the concentration of gelatin varied from 0.0001 to 1.12 g dm −3. The interaction between gelatin and ACP was characterized by particle electrophoretic mobility, pH and turbidity measurements. The ACP electrophoretic mobility during the precipitation in the presence of gelatin indicated gelatin adsorption onto ACP as the dominating interaction mode. By adsorbing on the surface of primary particles gelatin slows down or even prevents ACP aggregation thus stabilizing its colloidal state. Besides, the presence of gelatin promotes the transformation of ACP into a crystalline phase.

In vitro precipitation of calcium phosphate under intracellular conditions: formation of brushite from an amorphous precursor in the absence of ATP.

Release of mitochondrial calcium has been shown to occur concomitant with mineral ion loading of matrix vesicles at the onset of mineralization in epiphyseal growth plate cartilage. Matrix vesicles contain amorphous calcium phosphate (ACP), a mineral form that usually results from rapid precipitation at high initial levels of Ca2+ and/or inorganic P (Pi). Since the cytosol of growth plate chondrocytes has been found to contain high levels of Pi, rapid release of mitochondrial Ca2+ into the cytosol may cause local precipitation of calcium phosphate and thus be coupled with matrix vesicle formation. Studies were carried out to determine the kinetics and nature of mineral formation that occur when small amounts of Ca2+ are added under various conditions to a Pi buffer composed of electrolytes matched in concentrations and pH to that of the cytosol of epiphyseal chondrocytes. Depending on the manner in which Ca2+ was added, ACP, dicalcium phosphate dihydrate (DCPD), or apatite (HA) first formed. In the presence of ATP, ACP was the only solid phase detected, being stable for at least 24 h. However, in its absence, ACP rapidly transformed into DCPD. Increasing the pH of the reaction buffer from 6.9 to 7.5 increased the amount of ACP initially formed, but DCPD was consistently found upon ACP transformation. Yet at pH 8.0, ACP persisted for at least 24 h. The amount of precipitate formed was proportional to the level of added Ca2+; precipitates formed when as little as 1.0 mmole was added per liter of buffer. Our findings support the possibility that rapid release of mitochondrial Ca2+ may cause localized intracellular precipitation of ACP. Since nascent ACP is known to stimulate membrane fusion and blebbing of vesicles, these findings may explain the presence of ACP in matrix vesicles. The rapid conversion of ACP to DCPD in the absence of ATP under these conditions may also explain the reported occurrence of DCPD in samples of early mineralizing tissue.

31P NMR as a spectroscopic monitor of the spontaneous precipitation of calcium phosphates.

High-resolution 31P NMR spectroscopy is shown to be a potentially valuable new method for monitoring the spontaneous precipitation of calcium phosphates from metastable supersaturated solutions. An apparatus capable of pH-statting the sample in a spinning 20 mm NMR sample tube is briefly described. The spontaneous precipitation of dicalcium phosphate dihydrate, CaHPO4 X 2H2O, pH-statted at pH 5, is characterized by a base-uptake curve which follows the decrease in the intensity of the solution 31P resonance. The precipitation of amorphous calcium phosphate at neutral pH, which exhibited an induction period of approximately 10 min, was also studied. No evidence of NMR peaks from transient clusters or the initial colloidal solid phase has been seen.

A thermodynamic analysis of the amorphous to crystalline calcium phosphate transformation.

A thermodynamic analysis of the precipitation of amorphous calcium phosphate (ACP) and its transformation to crystalline apatite had been made. A nearly constant ion product, over a wide variety of conditions, was obtained for a tricalcium phosphate (TCP)-like phase suggesting that the molecular unit which governs the solubility of ACP may be similar in composition to TCP. The introduction of 10% acid phosphate into the formula for the TCP ion product improves the fit of experimental data and results in an invariant ion product. The stability of ACP in solution was found to be dependent upon its thermodynamic instability with respect to an octacalcium phosphate (OCP)-like phase. The dependence of the induction period for the amorphous to crystalline transformation upon the pH and the Ca/P ratio of the solution is best explained by the assumption that an OCP-like phase is initially nucleated on the surfaces of the ACP particles. The events that occur in the immediate post-transition period suggest the hydrolysis of this OCP-like material to an apatitic phase.

Effect of phospholipids on the transformation of amorphous calcium phosphate to hydroxapatite in vitro.

The conversion of amorphous calcium phosphate (ACP) to crystalline hydroxyapatite (HA) was studied in vitro in the presence or absence of phosphatidyl serine (PS) and other phospholipids. ACP transformation and HA crystal growth were monitored by electron microscopy, selected-area electron diffraction and X-ray diffraction, and by measuring supernatant calcium and phosphate. PS, and other acidic phospholipids, had a significant stablizing effect on ACP. With performed ACP at ratios of only 1 lipid molecule per 30-50 Ca atoms, PS markedly delayed HA crystal formation. When PS was present during ACP precipitation, inhibition of conversion to HA was less pronounced, but crystal habit and aggregation were greatly altered resulting in stacks of thin, membrane-like sheets approximately 38-42 A thick. PS appeared to be most effective in blocking ACP to HA conversion when oriented primarily on the surface; it most affected subsequent crystal formation when distributed throughout the amorphous precursor. Phospholipids possessing anionic, and hence Ca-binding properties, were effective in stabilizing ACP; neutral zwitterion lipids, which have amphipathic properties but do not bind Ca, were not. In view of the presence of anionic lipids in matrix vesicles and their association with early mineral deposits, the current findings add further evidence that lipids may play a role in the control of normal mineralization in vivo.

Relationship between pyrophosphate, amorphous calcium phosphate and other factors in the sequence of calcification in vivo.

The amounts of inorganic pyrophosphate (PPi), orthophosphate and calcium have been measured in resting, proliferating, hypertrophic and calcified cartilage from foetal calf epiphyses and also in cancellous, periosteal and compact bone. In the cartilage samples, the content of PPi increased progressively in the order named above, from values of 8.59 μg P/g dry weight in resting cartilage to 236 μg P/g dry weight in calcified cartilage. However, the ratio of PPi to orthophosphate followed the reverse relationship and was highest in the resting zone and fell dramatically as the tissue calcified. The possible role of PPi in inhibiting the precipitation of amorphous calcium phosphate (ACP) and in the slowing the transformation of ACP to hydroxyapatite (HA) in calcifying tissues is discussed in relation to other factors, such as collagen, magnesium, phospholipids and proteinpolysaccharides, which might also influence the processin vivo. At present, no single factor can be identified as a proven physiological regulator.