Schnepf Abigail, Yappert Marta Cecilia, Borchman Douglas
Department of Chemistry, University of Louisville, Louisville KY, USA.
Department of Chemistry, University of Louisville, Louisville KY, USA.
Exp Eye Res. 2017 Jul;160:116-125. doi: 10.1016/j.exer.2017.05.009. Epub 2017 May 25.
This project explores the regional phospholipid distribution in porcine vitreous humor, retina, and lens. Matrix-assisted laser desorption mass spectrometry has been used previously to image lipids, proteins, and other metabolites in retinas and lenses. However, the regional composition of phospholipids in vitreous humors is not known. To address this issue, we have applied this mass spectral method to explore the regional phospholipid distribution in porcine vitreous humor both ex-situ and in-vitro. To establish the possible source(s) of phospholipids in the vitreous humor, compositional studies of the lens and retina were also pursued. Due to the overall low levels of phospholipids in vitreous humor, it was necessary to optimize the experimental approaches for ex-situ and in-vitro studies. The sensitivity observed in the spectra of methanol extracts from the lens and retina was higher than that for methanol:chloroform extracts, but the compositional trends were the same. A fourfold improvement in sensitivity was observed in the analysis of vitreous humor extracts obtained with the Bligh and Dyer protocol relative to the other two extraction methods. For ex-situ studies, the 'stamp method' with para-nitroaniline as the matrix was chosen. Throughout the vitreous humor, phosphatidylcholines were the most abundant phospholipids. In-vitro results showed higher relative levels of phospholipids compared to the 'stamp' method. However, more details in the regional phospholipid distribution were provided by the ex-situ approach. Both in-vitro and ex-situ results indicated higher levels of phospholipids in the posterior vitreous region, followed by the anterior and central regions. The posterior region contained more unsaturated species whereas more saturated phospholipids were detected in the anterior region. The observed trends suggest that the phospholipids detected in the posterior vitreous humor migrate from the retina and associated vasculature while those present in the anterior regions are likely to derive from the lens. Not all species found in the lens were observed in the vitreous humor. For example, whereas cholesterol was present in lens extracts, it was not detected in the vitreous humor. Overall, the higher relative abundance of unsaturated species in the posterior vitreous humor and also present in the retina suggests that these species may be able to disrupt the water-collagen-hyaluronan network and contribute to vitreous liquefaction.
本项目探究了猪玻璃体液、视网膜和晶状体中磷脂的区域分布情况。基质辅助激光解吸质谱法此前已用于对视网膜和晶状体中的脂质、蛋白质及其他代谢物进行成像。然而，玻璃体液中磷脂的区域组成尚不清楚。为解决这一问题，我们应用此质谱方法，对猪玻璃体液中的磷脂区域分布进行了体外和离体研究。为确定玻璃体液中磷脂的可能来源，我们还对晶状体和视网膜进行了成分研究。由于玻璃体液中磷脂的总体水平较低，因此有必要优化体外和离体研究的实验方法。从晶状体和视网膜的甲醇提取物光谱中观察到的灵敏度高于甲醇：氯仿提取物，但成分趋势相同。与其他两种提取方法相比，采用布莱和戴尔方法获得的玻璃体液提取物分析灵敏度提高了四倍。对于离体研究，选择了以对硝基苯胺为基质的“印记法”。在整个玻璃体液中，磷脂酰胆碱是含量最丰富的磷脂。体外结果显示，与“印记法”相比，磷脂的相对水平更高。然而，离体方法提供了更多关于磷脂区域分布的细节。体外和离体结果均表明，玻璃体后区域的磷脂水平较高，其次是前区域和中央区域。后区域含有更多不饱和种类，而在前区域检测到更多饱和磷脂。观察到的趋势表明，在玻璃体后液中检测到的磷脂从视网膜和相关脉管系统迁移而来，而在前区域存在的磷脂可能来自晶状体。并非在晶状体中发现的所有种类都能在玻璃体液中观察到。例如，晶状体提取物中存在胆固醇，但在玻璃体液中未检测到。总体而言，玻璃体后液中不饱和种类的相对丰度较高，且视网膜中也存在这些种类，这表明这些种类可能能够破坏水 - 胶原蛋白 - 透明质酸网络，并导致玻璃体液化。
