Huang Y, von Eckardstein A, Assmann G
Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität, Münster, FRG.
Arterioscler Thromb. 1993 Mar;13(3):445-58. doi: 10.1161/01.atv.13.3.445.
Pulse-chase incubations of human plasma with [3H]cholesterol-laden skin fibroblasts or low density lipoproteins (LDL) and nondenaturing two-dimensional electrophoresis were used to study the transfer and esterification of cell-derived unesterified cholesterol (UC) in human plasma lipoproteins. Specific radioactivities ([3H]UC per microgram of UC) were calculated, and net cholesterol mass transfer was quantified using a fluoro-enzymatic assay to validate productive transfers of UC between high density lipoprotein (HDL) and LDL. Cellular UC was initially taken up by pre-beta 1-HDL and subsequently transferred in the sequence pre-beta 2-HDL-->pre-beta 3-HDL-->alpha-HDL-->LDL. During the first 5 minutes of this process, only 5% of cellular cholesterol was esterified in pre-beta 3-HDL and alpha-HDL; the remainder reached LDL as UC. Cellular UC accumulating in LDL was then redistributed to various HDL particles via two pathways: 1) the partially LDL receptor-mediated uptake and re-secretion of UC by cells and 2) the direct transfer of UC to HDL, mostly to alpha-HDL and a small amount to pre-beta-HDL. UC was not transferred from LDL to HDL after inhibition of lecithin:cholesterol acyltransferase (LCAT). The esterification of cellular [3H]cholesterol in plasma was competitively inhibited by the addition of excess unlabeled LDL but not of excess HDL. However, both excess LDL and excess HDL prevented the esterification of cell-derived cholesterol in apolipoprotein B-free plasma. This demonstrated that LDL is the major source of UC to the LCAT reaction and that the transfer of UC from LDL to HDL is LCAT dependent. In conclusion, the effective esterification of cell-derived cholesterol in plasma involves a rapid transfer of UC via HDL particles to LDL, from which it is distributed to pre-beta-HDL and alpha-HDL. Furthermore, we hypothesize that the transfer per se of cellular UC to LDL forms a cholesterol concentration gradient between cell membranes and HDL and thus a second, reverse cholesterol transport mechanism in addition to the esterification of cholesterol by LCAT.
用人血浆与载有[3H]胆固醇的皮肤成纤维细胞或低密度脂蛋白（LDL）进行脉冲追踪孵育，并采用非变性二维电泳来研究人血浆脂蛋白中细胞源性游离胆固醇（UC）的转运和酯化。计算了比放射性（每微克UC的[3H]UC），并使用荧光酶法对净胆固醇质量转移进行定量，以验证UC在高密度脂蛋白（HDL）和LDL之间的有效转移。细胞UC最初被前β1-HDL摄取，随后按前β2-HDL→前β3-HDL→α-HDL→LDL的顺序转移。在此过程的前5分钟内，只有5%的细胞胆固醇在前β3-HDL和α-HDL中被酯化；其余部分以UC的形式到达LDL。LDL中积累的细胞UC随后通过两条途径重新分布到各种HDL颗粒：1）细胞对UC的部分LDL受体介导的摄取和再分泌；2）UC直接转移到HDL，主要是转移到α-HDL，少量转移到前β-HDL。卵磷脂：胆固醇酰基转移酶（LCAT）被抑制后，UC不会从LDL转移到HDL。血浆中细胞[3H]胆固醇的酯化受到过量未标记LDL的竞争性抑制，但不受过量HDL的抑制。然而，过量的LDL和过量的HDL都阻止了无载脂蛋白B血浆中细胞源性胆固醇的酯化。这表明LDL是LCAT反应中UC的主要来源，并且UC从LDL到HDL的转移依赖于LCAT。总之，血浆中细胞源性胆固醇的有效酯化涉及UC通过HDL颗粒快速转移到LDL，然后从LDL分布到前β-HDL和α-HDL。此外，我们假设细胞UC向LDL的转移本身在细胞膜和HDL之间形成了胆固醇浓度梯度，因此除了LCAT对胆固醇的酯化作用外，还形成了第二种反向胆固醇转运机制。
