Abstract
Purpose::
To evaluate age-related accumulation of lipoprotein-like particles (LLPs) for extractability with known lipid solvents; to estimate the mass of total lipid content of human macular BrM.
Methods::
The Folch reagent (chloroform : methanol = 2 : 1) was applied to extract lipids from human macular BrM (donor age 86 years). The resulting ultrastructure was examined using the quick-freeze/deep etch (QFDE) method and compared to the same eye without lipid extraction. Cross-sections of macular BrM of preserved, grossly normal eyes (donor age 27 to 78 years, n = 12) were also visualized using thin-section TEM. The thickness of each layer of BrM was measured on prints (magnification: * 34200) of TEM images. Combining these data with the volume fraction of each layer in the same eyes (Huang et al., ARVO 2006), we are able to estimate the age-related changes of accumulated LLPs in BrM.
Results::
By QFDE, BrM treated with Folch reagent showed very few LLPs, containing instead numerous holes of size and locations similar to LLPs in BrM without lipid extraction. By TEM, both inner and outer collageneous layer thickness increased, but the elastic layer thickness decreased, with donor age. We combined these data with LLP volume fractions of these layers and assumed the LLP density of 1 g/cm3 to find that the LLP accumulation in the central 7 mm of macular BrM increased from approximately 1 µg to 11 µg during five decades of human life.
Conclusions::
This study shows that the LLPs can be extracted from BrM using the Folch method, and thus the LLPs are composed primarily of lipids. We find that the age-related increase in LLPs in our study is very similar to the age-related changes in total lipids (1 - 9 µg) found by Holz et al. (1994) in a BrM/choroid preparation. Therefore, our results suggest that LLPs represent a major portion of lipids found in BrM.
Keywords: Bruch's membrane • lipids • aging