Lipoprotein-like particles and cholesteryl esters in human Bruch's membrane: initial characterization.
OBJECTIVE
To isolate and characterize cholesteryl ester-containing, lipoprotein-like particles (LLPs) from normal aged human Bruch's membrane (BrM)/choroid (Ch).


METHODS
From BrM/Ch of 20 eyes of 10 donors aged >60 years, LLPs were released by high-salt buffer, fractionated by density gradient ultracentrifugation, and characterized by determining cholesterol, triglyceride, and phospholipid concentration (by enzymatic colorimetry and fluorometry); cholesteryl ester composition (by electrospray ionization mass spectrometry, ESI/MS); and particle morphology (by negative stain electron microscopy). Apolipoprotein (apo) gene expression was determined with RT-PCR, Western blot analysis, and immunofluorescence of retinal-choroidal cryosections. In paraformaldehyde-preserved eyes (20 eyes of 20 donors), cholesteryl ester composition of BrM/Ch, cornea, and sclera was determined by ESI/MS.


RESULTS
A pooled fraction of LLP released from BrM/Ch (concentrated total LLP, density [d] < 1.24 g/mL fraction) was fractionated into two peaks. A large Peak 1 (with plasma LDL-HDL density range), containing predominantly phospholipid and unesterified cholesterol, was morphologically heterogeneous. A small Peak 2 (with plasma VLDL density range), enriched with esterified cholesterol, contained approximately 100 nm diameter round electron-lucent particles. Both peaks contained apoB and apoA-I, RPE and retina contained apoA-I mRNA transcripts, and BrM and drusen contained apoA-I immunoreactivity. Peaks 1 and 2, native RPE, and fresh BrM/Ch were cholesteryl linoleate enriched and contained little cholesteryl docosahexaenoate. Preserved BrM/Ch was cholesteryl oleate-enriched, unlike sclera and cornea.


CONCLUSIONS
BrM/Ch LLP do not resemble plasma lipoproteins in density profile, cholesterol distribution, or morphology. Peak 2 contains EC-rich LLP resembling BrM particles in situ. BrM/Ch cholesteryl esters respond to long-term storage differently than esters of plasma lipoprotein origin accumulated in other ocular tissues. Evidence of intraocular apoB and apoA-I expression supports an emerging hypothesis that the RPE assembles and secretes a large, possibly novel, lipoprotein particle.
Department of Ophthalmology, University of Alabama School of Medicine, Birmingham, AL 35294-009, USA.
Chuan-Ming
CM
Li
Byung Hong
BH
Chung
J Brett
JB
Presley
Goldis
G
Malek
Xueming
X
Zhang
Nassrin
N
Dashti
Ling
L
Li
Jianguo
J
Chen
Kelley
K
Bradley
Howard S
HS
Kruth
Christine A
CA
Curcio
D016632:Apolipoprotein A-I / Q000235:genetics / Q000378:metabolism; D001055:Apolipoproteins B / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D016570:Bruch Membrane / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism; D002788:Cholesterol Esters / Q000302:isolation & purification / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D008074:Lipoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008854:Microscopy, Electron; D008875:Middle Aged; D010743:Phospholipids / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization; D014280:Triglycerides / Q000378:metabolism
D016632:Apolipoprotein A-I; D001055:Apolipoproteins B; D002788:Cholesterol Esters; D008074:Lipoproteins; D010743:Phospholipids; D012333:RNA, Messenger; D014280:Triglycerides; D002784:Cholesterol