人晶状体膜中未知磷脂的分离与鉴定

Separation and characterization of the unknown phospholipid in human lens membranes.

作者信息

Byrdwell W C, Borchman D, Porter R A, Taylor K G, Yappert M C

机构信息

Department of Chemistry, University of Louisville, KY 40292.

出版信息

Invest Ophthalmol Vis Sci. 1994 Dec;35(13):4333-43.

PMID:8002253

Abstract

PURPOSE

The major component of human lens membranes was thought to be sphingomyelin until 1991, when a study by phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy revealed the presence of an unknown phospholipid that constituted approximately half the human lens phospholipids. The objective of this work was to isolate this phospholipid and to elucidate its identity.

METHODS

The separation of sphingomyelin from the unknown was accomplished using high-performance liquid chromatography (HPLC) and an amino-bound column. Sphingomyelin standard and the membranes from human lenses were chromatographed. Chromatographic fractions were collected and spectrally characterized by proton (1H) NMR and 31P NMR spectroscopy.

RESULTS

The chromatographic method did not affect the integrity of the sphingomyelin. Besides the bands corresponding to the unknown components, the chromatogram of the human lens membranes showed three large peaks, the central one with a shoulder, with elution times similar to that for sphingomyelin. The 1H NMR spectra for the fractions collected during the elution of these peaks showed differences. The study by 31P NMR indicated that the first peak contained the unknown phospholipid. The subsequent fractions showed the presence, in different relative levels, of both the unknown and sphingomyelin. By comparison and interpretation of the two-dimensional 1H NMR spectra for sphingomyelin and for the fraction containing the unknown, the unknown phospholipid is proposed to be 4,5 dihydrosphingomyelin, in which the site of unsaturation present in the sphingosine moiety is no longer present.

CONCLUSIONS

The ability to separate the unknown from sphingomyelin and the power of 1H NMR spectroscopy allowed the proposition of the identity of the major component of human lens membranes as 4,5-dihydrosphingomyelin. Although the synthetic compound is known to be involved in the formation of extended hydrogen-bonding networks, its biologic and physicochemical properties need further study.

摘要

目的

直到1991年，人们一直认为人晶状体膜的主要成分是鞘磷脂，当时一项利用磷-31（31P）核磁共振（NMR）光谱进行的研究揭示，存在一种未知的磷脂，其构成了人晶状体磷脂的大约一半。这项工作的目的是分离这种磷脂并阐明其身份。

方法

使用高效液相色谱（HPLC）和氨基键合柱将鞘磷脂与未知物分离。对鞘磷脂标准品和人晶状体膜进行色谱分析。收集色谱馏分，并通过质子（1H）NMR和31P NMR光谱进行光谱表征。

结果

色谱方法未影响鞘磷脂的完整性。除了对应未知成分的条带外，人晶状体膜的色谱图显示出三个大峰，中间的峰带有一个肩峰，其洗脱时间与鞘磷脂的相似。在这些峰洗脱过程中收集的馏分的1H NMR光谱显示出差异。31P NMR研究表明，第一个峰含有未知磷脂。随后的馏分显示出未知物和鞘磷脂以不同的相对水平存在。通过对鞘磷脂和含有未知物的馏分的二维1H NMR光谱进行比较和解释，提出未知磷脂为4,5-二氢鞘磷脂，其中鞘氨醇部分中存在的不饱和位点不再存在。