用于测定大鼠肝细胞膜磷脂组成的新型高效液相色谱法。

Novel high-performance liquid chromatography for determination of membrane phospholipid composition of rat hepatocytes.

作者信息

Kurumi Y, Adachi Y, Itoh T, Kobayashi H, Nanno T, Yamamoto T

机构信息

Second Department of Internal Medicine, Kinki University School of Medicine, Osakasayama, Japan.

出版信息

Gastroenterol Jpn. 1991 Oct;26(5):628-32. doi: 10.1007/BF02781680.

DOI:10.1007/BF02781680

PMID:1752394

Abstract

We have developed a new, quick and efficient method of high-performance liquid chromatography (HPLC) for the isolation and quantitative determination of phospholipids in hepatocyte membranes. A silica gel column was used for the isolation and determination, and an isocratic mixture of acetonitrile, methanol and 85% phosphoric acid (130:5:1.7, v/v/v) was used as a mobile phase. Six kinds of phospholipids, i.e. phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphinogomyelin (SPH), in this order, were completely isolated within 45 min. The phospholipid composition of sinusoidal membrane vesicles (SMV) and canalicular membrane vesicles (CMV) obtained from rat liver, as well as of human erythrocyte ghosts were determined by this HPLC method. The level of SPH in CMV was significantly higher than that in SMV, and the level of PC in CMV was significantly lower than that in SMV. These results were considered attributable to the low fluidity of CMV. The phospholipid composition of human erythrocyte membrane was different from that of rat SMV and CMV. The present technique is suitable for quantitative determination of phospholipids in cell membranes.

摘要

我们开发了一种新的、快速高效的高效液相色谱（HPLC）方法，用于分离和定量测定肝细胞膜中的磷脂。使用硅胶柱进行分离和测定，以乙腈、甲醇和85%磷酸的等度混合物（130:5:1.7，v/v/v）作为流动相。六种磷脂，即磷脂酰肌醇（PI）、磷脂酰丝氨酸（PS）、磷脂酰乙醇胺（PE）、磷脂酰胆碱（PC）、溶血磷脂酰胆碱（LPC）和鞘磷脂（SPH），按此顺序在45分钟内完全分离。采用该HPLC方法测定了大鼠肝脏来源的窦状膜囊泡（SMV）和胆小管膜囊泡（CMV）以及人红细胞血影的磷脂组成。CMV中SPH的水平显著高于SMV，CMV中PC的水平显著低于SMV。这些结果被认为归因于CMV的低流动性。人红细胞膜的磷脂组成与大鼠SMV和CMV不同。本技术适用于细胞膜中磷脂的定量测定。