Effects of LDL, Cholesterol, and Their Oxidized Forms on the Precipitation Kinetics of Calcium Phosphates

Abstract

AbstractBackground: LDL, cholesterol, and their oxidized forms are known cardiovascular risk factors and are often found in atherosclerotic lesions of various stages. Little is known, however, about whether they are directly involved in the formation of calcium phosphate compounds.Methods: We used the pH-stat technique to follow the kinetics of calcium phosphate precipitation at pH 7.4, 37 °C, and ionic strength 0.150 mol/L, in the presence or absence of LDL, oxidized LDL, cholesterol, cholestane-3β,5α,6β-triol, and cholesteryl linoleate. The precipitates were characterized by x-ray diffraction, scanning and transmission electronic microscopy coupled with energy-dispersion x-ray analysis, and inductively coupled plasma atomic emission spectroscopy.Results: Under the experimental conditions, LDL (14.8 and 43.1 mg/L protein) had no significant effect on the precipitation kinetics. Oxidized LDL (14.8 and 43.1 mg/L protein) prolonged the nucleation phase and diminished the amount of total precipitate, and both the extent of oxidation and the concentration of the protein affected the kinetics. Cholesterol microcrystals (71.4 and 143 mg/L) made the nucleation phase shorter (300 min vs 390 min for the control), and the precipitated particles had an organic core and a shell composed of calcium phosphates. l-α-Phosphatidylcholine vesicles (143 mg/L), cholesterol (71.4 mg/L)/phospholipid (143 mg/L) mixed vesicles, cholesteryl linoleate (143 mg/L), and cholestane-3β,5α,6β-triol (71.4 mg/L) prolonged the nucleation phase.Conclusions: LDL is not involved directly in the precipitation of calcium phosphates. Oxidized LDL inhibits both nucleation and crystal growth, possibly by attracting calcium ions in the solution and thus reducing supersaturation. Cholesterol microcrystals serve as seeds for the precipitation of hydroxyapatite, whereas l-α-phosphatidylcholine, cholesteryl linoleate, and cholestane-3β,5α,6β-triol exhibit inhibitive effects on the nucleation of calcium phosphates.