Skrtic D, Eanes E D
Bone Research Branch Research Associate Program, Natioanl Institute of Dental Research, Natioanl Institute of Standards and Technology, Gaithersburg, Maryland 20899.
Calcif Tissue Int. 1992 Jan;50(1):55-60. doi: 10.1007/BF00297298.
The present study examined the effect of membrane cholesterol on liposome-mediated calcium phosphate precipitation in metastable aqueous solutions (2.25 mM Ca2+ and 1.5 mM inorganic phosphate) at 22 degrees C, pH 7.4 and 240 mOsm. The liposomes were prepared from 7:2:X molar mixtures of phosphatidylcholine, dicetylphosphate, and cholesterol (x = 0, 1, 5, or 9) and contained either 0 or 50 mM encapsulated phosphate. The membranes were made permeable to Ca2+ by addition of the cationophore, X-537A. Changes in external Ca2+ concentration were used as the principal monitor of the course of precipitation. Without encapsulated phosphate, 7:2:X liposomes (with or without ionophore) induced no precipitation. With 50 mM encapsulated phosphate and in the presence of ionophore, precipitation significantly depended on the cholesterol level in the membrane. At 0 and 10 mole% cholesterol, precipitate developed rapidly both within and outside the liposomes. At 35 and 50 mole% cholesterol, no observable intraliposomal precipitation occurred, and extraliposomal precipitation started only after an induction period of 24 hours. Delayed extraliposomal precipitation also took place in PO4-containing liposomes without added ionophore. In this latter case, however, cholesterol was essential for this precipitation to occur with the optimum level being around 10 mole%. Suppression of ionophore-mediated intraliposomal precipitation at higher cholesterol levels could be related to the inflexible cholesterol molecules making the membrane more rigid, thereby restricting Ca-ionophore transport. This restriction could be reversed with ethanol. Delayed extraliposomal precipitation in the absence of added ionophore (or at higher cholesterol levels in its presence) could be explained by seeding from low, unobserved levels of intraliposomal precipitate formed during slow, unfacilitated Ca2+ leakage into the liposomal interior.
本研究考察了膜胆固醇对脂质体介导的磷酸钙沉淀的影响，该沉淀发生于22℃、pH 7.4和240 mOsm的亚稳水溶液（2.25 mM Ca2+和1.5 mM无机磷酸盐）中。脂质体由磷脂酰胆碱、磷酸二鲸蜡酯和胆固醇按7:2:X摩尔比混合制备（X = 0、1、5或9），并含有0或50 mM包封的磷酸盐。通过添加阳离子载体X-537A使膜对Ca2+具有通透性。外部Ca2+浓度的变化用作沉淀过程的主要监测指标。没有包封磷酸盐时，7:2:X脂质体（有或没有离子载体）不会诱导沉淀。有50 mM包封磷酸盐且存在离子载体时，沉淀显著依赖于膜中的胆固醇水平。胆固醇含量为0和10摩尔%时，脂质体内外均迅速形成沉淀。胆固醇含量为35和50摩尔%时，脂质体内未观察到沉淀，脂质体外沉淀仅在24小时的诱导期后开始。在未添加离子载体的含PO4脂质体中也发生了延迟的脂质体外沉淀。然而，在后一种情况下，胆固醇对于这种沉淀的发生至关重要，最佳水平约为10摩尔%。较高胆固醇水平下离子载体介导的脂质体内沉淀的抑制可能与刚性的胆固醇分子使膜更僵硬有关，从而限制了Ca-离子载体的运输。这种限制可用乙醇逆转。在未添加离子载体的情况下（或在其存在时较高胆固醇水平下）延迟的脂质体外沉淀可以通过缓慢、无促进的Ca2+泄漏到脂质体内部过程中形成的低水平、未观察到的脂质体内沉淀的晶种作用来解释。
