睑板腺功能障碍患者睑脂磷脂成分的LC-MS/MS分析

目的用液相色谱-串联质谱联用技术（LC-MS/MS）检测睑板腺功能障碍（meibomian gland dysfunction，MGD）患者睑板腺分泌物磷脂的3种主要成分卵磷脂（溶血磷脂酰胆碱）、脑磷脂（磷脂酰乙醇胺）、神经鞘磷脂与正常人的区别；并分析其相对浓度水平与MGD临床客观体征检测指标的相关性。方法前瞻性配伍组设计。MGD患者22例（22眼）为MGD组，年龄相匹配的正常人30例（30眼）为对照组。MGD组患者选择症状体征较重的眼作为观察对象。用无菌棉签棒按压睑缘后，用无菌的不锈钢刮匙收集睑缘的睑板腺分泌物。所有样本使用LC-MS/MS定性定量分析MGD组睑脂磷脂的主要成分（卵磷脂、脑磷脂、神经鞘磷脂）与正常对照组的区别。2组之间比较采用独立样本t检验，Pearson相关分析MGD组3种磷脂相对浓度水平与睑板腺分泌物性状评分、泪膜破裂时间（BUT）、角膜荧光素染色评分（CFS）、基础泪液分泌试验（SⅠT）、裂隙灯显微镜下睑缘评分5项指标的关系。结果①睑板腺分泌物3种主要磷脂成分在MGD组与正常对照组间有明显差异，MGD组的睑脂卵磷脂上调（t=-2.45，P<0.05），脑磷脂、神经鞘磷脂下调（t=16.73、18.59，P<0.05）。②卵磷脂相对浓度水平与睑板腺分泌物性状评分、CFS、裂隙灯显微镜下睑缘评分呈正相关（r=0.602、0.716、0.710，P<0.01），与BUT、SⅠT 呈负相关（r=-0.628、-0.477，P<0.05）。脑磷脂与睑板腺分泌物性状评分、CFS、裂隙灯显微镜下睑缘评分呈负相关（r=-0.565、-0.724、-0.702，P<0.01），与BUT、SⅠT呈正相关（r=0.659、0.498，P<0.05）。神经鞘磷脂相对浓度水平与睑板腺分泌物性状评分、CFS、裂隙灯显微镜下睑缘评分呈负相关（r=-0.597、-0.719、-0.698，P<0.01）；与BUT、SⅠT 呈正相关（r=0.631、0.468，P<0.05）。结论MGD患者较正常人有较高水平的卵磷脂，较低水平的脑磷脂和神经鞘磷脂。睑脂磷脂成分的变化可能是MGD发生发展的一个重要标志。

Identification of meibomian phospholipids in meibomian gland dysfunction patients using liquid chromatography/tandem mass spectrometry

ObjectiveTo evaluate the differences in three kinds of meibomian phospholipid composition in healthy subjects and in patients suffering from meibomian gland dysfunction （MGD）. MethodsIn this prospective study， 22 MGD patients （MGD group） and 30 age and sex matched healthy individuals （control group） were enrolled. One eye of each subject was included in the study. MGD patients underwent sequential examinations as follows： the property of meibomian secretion， tear film break-up time （BUT）， corneal fluorescein staining （CFS）， Schirmer Ⅰ test and lid margin and ocular surface examinations by slit lamp. Meibomian gland secretions were collected using sterile stainless steel curettes in the healthy control group and MGD group after gentle massage of the lid margin with sterile cotton swabs. The meibomian phospholipids， mainly lysophosphatidylcholine， phosphatidylethanolamine， and sphingomyelin， were detected using liquid chromatography/tandem mass spectrometry （LC-MS/MS）， and comparisons were made between the MGD group and the healthy control group. The data from the two groups were compared by an independent-samples t test and a Pearson correlation analysis was used to assess the correlation between the relative composition concentrations of the three kinds of phospholipids with the clinical parameters of the objective signs of MGD. ResultsThe relative concentration level of lysophosphatidylcholine in the MGD group was significantly higher than that in the healthy controls （t=-2.45， P<0.05）， while phosphatidylethanolamine and sphingomyelin levels were lower in the MGD group than in healthy controls （t=16.73， P<0.01 and t=18.59， P<0.01， respectively）. The relative concentration of lysophosphatidylcholine was positively correlated with the property of meibomian secretion， CFS， lid margin and ocular surface examination by slit lamp （r=0.602， 0.716， 0.710， P<0.01， respectively）； while a negative correlation was found with the BUT and Schirmer Ⅰ test （r=-0.628， -0.477， P<0.05， respectively）. The relative concentrations of phosphatidylethanolamine and sphingomyelin were negatively correlated with the properties of the meibomian secretion， CFS， lid margin and ocular surface examination （r=-0.565， -0.724， -0.702； r=-0.597， -0.719， -0.698， P<0.01， respectively）. However， a positive correlation was detected with the BUT and Schirmer Ⅰ test （r=0.659， 0.498， P<0.05； r=0.631， 0.468， P<0.05， respectively）. ConclusionThe profile of the three kinds of meibomian phospholipids （lysophosphatidylcholine， phosphatidylethanolamine and sphingomyelin） in MGD patients is dramatically different from healthy subjects. Its significant correlation with the clinical parameters of the signs of meibomian gland dysfunction suggest that a change in phospholipids composition in meibomian gland secretions is a probable reason for the development of MGD.