Lipoprotein-like Particles and Cholesteryl Esters in
Human Bruch’s Membrane: Initial Characterization
Chuan-Ming Li,1Byung Hong Chung,2,3J. Brett Presley,1Goldis Malek,1,4
Xueming Zhang,1Nassrin Dashti,2Ling Li,2Jianguo Chen,2Kelley Bradley,1,5
Howard S. Kruth,6and Christine A. Curcio1
PURPOSE. To isolate and characterize cholesteryl ester-contain-
ing, 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 de-
termining cholesterol, triglyceride, and phospholipid concen-
tration (by enzymatic colorimetry and fluorometry); cho-
lesteryl ester composition (by electrospray ionization mass
spectrometry, ESI/MS); and particle morphology (by negative
stain electron microscopy). Apolipoprotein (apo) gene expres-
sion was determined with RT-PCR, Western blot analysis, and
immunofluorescence of retinal–choroidal cryosections. In
paraformaldehyde-preserved eyes (20 eyes of 20 donors), cho-
lesteryl ester composition of BrM/Ch, cornea, and sclera was
determined by ESI/MS.
RESULTS. A pooled fraction of LLP released from BrM/Ch (con-
centrated 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 phospho-
lipid and unesterified cholesterol, was morphologically heter-
ogeneous. A small Peak 2 (with plasma VLDL density range),
enriched with esterified cholesterol, contained ?100 nm di-
ameter round electron-lucent particles. Both peaks contained
apoB and apoA-I, RPE and retina contained apoA-I mRNA tran-
scripts, and BrM and drusen contained apoA-I immunoreactiv-
ity. Peaks 1 and 2, native RPE, and fresh BrM/Ch were cho-
lesteryl 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 differ-
ently 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 as-
sembles and secretes a large, possibly novel, lipoprotein
particle. (Invest Ophthalmol Vis Sci. 2005;46:2576–2586)
nations.1A heterogeneous disorder, ARM’s most prominent
clinical and histopathologic lesions affect the retinal pigment
epithelium (RPE), Bruch’s membrane (BrM), and the chorio-
capillaris, ultimately affecting the function of the photorecep-
tors.2,3Early ARM is characterized by drusen and/or pigmen-
tary changes. Late ARM is characterized by geographic atrophy
and/or choroidal neovascularization and its sequelae. Limited
treatment options for ARM are directed either against choroidal
neovascularization, a sight-threatening complication in late
ARM, or entail providing antioxidant nutritional supplements
for some patients with early ARM. Valuable new information
has emerged about molecules within drusen (characteristic,
focal extracellular lesions), including inflammation-associated
proteins and advanced glycation end products.4,5Yet underly-
ing mechanisms leading to drusen and basal linear deposit
(drusenoid material in a diffusely distributed lesion),6remain
Lipoproteins are multimolecular assemblies with neutral
lipid cores of triglyceride (TG) and esterified cholesterol (EC)
surrounded by a solubilizing surface of apolipoprotein (apo),
phospholipid (PL), and unesterified cholesterol (UC).7Classi-
cally defined major lipoprotein classes include (but are not
restricted to) high-density, low-density, and very-low-density
lipoproteins and dietary chylomicrons (HDL, LDL, VLDL, and
CM, respectively). Lipoprotein particles differ in size, flotation
properties in a density gradient, electrophoretic mobility, ratio
of lipid to protein, ratio of TG to EC in the core, and major
surface apos. Lipoproteins containing apoB (hepatic VLDL and
its metabolite LDL, and intestinal CM and partly hydrolyzed CM
remnants) are considered atherogenic, as they penetrate vas-
cular intima before development of frank lesions.8,9The com-
ponent apos classify lipoprotein particles most specifically,10
and in humans, VLDL particles contain apoB-100, apoE, and
apos C-I, -II, and -III; LDL contains apoB-100 only; and CM
particles contain apoB-48, apos C-I to -III, and apoA-I.
ge-related maculopathy (ARM) is the leading cause of new,
untreatable vision loss in the elderly of industrialized
From the1Department of Ophthalmology and the2Atherosclerosis
Research Unit, Division of Geriatrics/Gerontology, Department of Med-
icine, University of Alabama School of Medicine, Birmingham, Ala-
bama; and the
Heart, Lung, and Blood Institute, National Institutes of Health, Beth-
Present affiliations:3Department of Nutrition Sciences, University
of Alabama at Birmingham, Birmingham, Alabama;
Ophthalmology, Duke University Medical Center, Durham, North Caro-
lina; and the5Center for Biophysical Sciences/Engineering, University
of Alabama at Birmingham, Birmingham, Alabama.
Supported by National Eye Institute Grants EY06109 (CAC); the
International Retinal Research Foundation; unrestricted funds to the
Department of Ophthalmology from Research to Prevent Blindness,
Inc., and EyeSight Foundation of Alabama; and Grants P01HL34343
(ND), and R01 HL60936 (BHC) from the National Heart, Lung, and
Blood Institute, and Grant P30 CA13148-32 to the UAB Mass Spectrom-
etry Center from the National Cancer Institute. CAC is a recipient of
the Lew R. Wasserman Merit Award from Research to Prevent Blind-
Submitted for publication January 10, 2005; revised February 24,
2005; accepted March 12, 2005.
Disclosure: C.-M. Li, None; B.H. Chung, None; J.B. Presley,
None; G. Malek, None; X. Zhang, None; N. Dashti, None; L. Li,
None; J. Chen, None; K. Bradley, None; H.S. Kruth, None; C.A.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Christine A. Curcio, Department of Oph-
thalmology, 700 South 18thStreet, Room H020, Callahan Eye Founda-
tion Hospital, University of Alabama School of Medicine, Birmingham,
AL 35294-0009; firstname.lastname@example.org.
6Section of Experimental Atherosclerosis, National
Investigative Ophthalmology & Visual Science, July 2005, Vol. 46, No. 7
Copyright © Association for Research in Vision and Ophthalmology
A growing body of literature indicates the involvement of
lipids and lipoproteins in the formation of the characteristic
extracellular lesions in aging and ARM eyes. Neutral lipids,
including EC and UC, are histochemically detectable in
drusen.11–16Material binding oil red O (a neutral fat stain) and
containing EC and TG accumulates in normal BrM throughout
adulthood, suggesting a large, universal, and constitutive yet
poorly understood process of lipid deposition.14,15,17–19That
lipoprotein particles are a potential means of depositing neu-
tral lipids is indicated by immunohistochemical localization of
apoB and apoE in drusen16,20,21and ultrastructural visualiza-
tion of solid 100-nm diameter particles with a surface and core
substructure in BrM- and ARM-associated lesions.14,19The
probability that a culprit particle is of intraocular origin is
increased by evidence that native human RPE expresses mRNA
transcripts for apoE, apoB, and microsomal triglyceride transfer
protein, required for assembly of an apoB-containing lipopro-
tein and the product of the abetalipoproteinemia gene (Men-
delian Inheritance in Man (MIM) 2001001; http://www.ncbi.
nlm.nih.gov/Omim/ provided in the public domain by the
National Center for Biotechnology Information, Bethesda,
MD).16,21,22Further, cultured native RPE and ARPE-19 cells
secrete apoE and neutral lipid, respectively.22,23
The potential role that lipoproteins play in ARM would be
clarified by evidence that lipoprotein-like particles (LLPs) can
be isolated from BrM/Ch, as they are from atherosclerotic
arterial intima.9,24In the current study, we showed that dou-
ble, high-salt buffer extraction9can release such particles for
initial characterization of protein and lipid composition. Spe-
cial attention was directed to EC, localized exclusively within
BrM by specific histochemistry.14Because BrM/Ch prepara-
tions unavoidably retain some plasma lipoproteins of hepatic
and intestinal origin, we compared our isolates to plasma
lipoproteins. In light of the evidence of intraocular apoB and
apoE expression, we also sought evidence of apoA-I expres-
sion. Finally, we analyzed EC composition in BrM and other
ocular regions in fresh and preserved eyes, finding evidence
that BrM EC may be uniquely susceptible to modification in
storage. These findings collectively suggest that BrM/Ch LLPs
do not resemble plasma lipoproteins in several key attributes.
Human Plasma Lipoproteins
To obtain plasma lipoproteins, fresh plasma from fasting and nonfast-
ing normolipidemic volunteers was adjusted to density (d) ? 1.24
g/mL using KBr and then centrifuged at 49,000 rpm for 36 hours at 4°C
in a swing-out rotor (SW55; Beckman Instruments, Fullerton, CA).
Lipoprotein mixtures were collected by removing the top 1-mL frac-
tion from the centrifuge tubes. Density gradients were then formed by
placing 1.0-mL aliquots of these mixtures at the bottom of a 5-mL
density gradient tube containing 2.5 mL Tris-buffered saline (TBS; 0.01
Tris and 0.15 M NaCl [pH 7.4]) at the top and 1.5 mL of a d ? 1.12
g/mL KBr solution at the bottom. After ultracentrifugation at 49,000
rpm for 150 minutes at 4°C, VLDL, banded at the top; LDL, banded at
the upper middle; and HDL, retained at the bottom of the tubes, were
collected by aspirating visible lipoprotein bands from the tube tops.
These fractionated lipoprotein samples were dialyzed against TBS for
24 hours to remove KBr. To separate any CM recovered in the VLDL
density fraction, a dialyzed VLDL density sample (1 mL) was placed
under 4 mL buffered saline in a centrifuge tube and subjected to brief
(30 minutes) ultracentrifugation at 30,000 rpm. CM floated to the tube
tops. VLDL at the tube bottoms were then collected.
Human Ocular Tissues
Human eyes were obtained from nondiabetic donors within 6 hours of
death through the Alabama Eye Bank. Eyes from only those donors ?
60 years old, with high expected BrM lipid content,14,17,18,25were
used. Globes were inspected internally with epi- and transillumina-
tion.26Maculas lacked grossly visible drusen and pigmentary change
consistent with ARM, although peripheral drusen were abundant in
some eyes.27,28Fresh eyes were used for isolating RPE and BrM/Ch
LLPs. Eyes preserved by immersion in 4% paraformaldehyde and stored
in 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C
(mean storage time, 573 ? 235 days) were used for EC compositional
analysis and immunohistochemistry. Human tissue use was approved
by institutional review (protocol X010705001), and the protocol ad-
hered to the guidelines set forth by the Declaration of Helsinki.
Particle Isolation from Human BrM
Fresh globes were incised circumferentially at the equator, penetrating
the sclera, retina, and vitreous. The neurosensory retina and vitreous
body were removed together after excision of retina at the optic nerve
head. The resultant eyecup was rinsed twice with 1 mL chilled isola-
tion buffer (0.01 M Tris, 0.15 M NaCl, 0.02 mM butylated hydroxytolu-
ene, and 0.1% EDTA, and 0.01% NaN3[pH 7.4]). Then, 500 ?L buffer
was twice added to the eyecup, and the RPE was brushed lightly from
the optic nerve outward under a dissecting microscope. Dislodged RPE
cells were collected, resuspended in 1 mL of PBS, and used for lipid
extraction and mRNA isolation. The cells in 0.1 mL volume were
digested in 0.1 N NaOH/0.1% sodium dodecyl sulfate (SDS) to deter-
mine protein content (DC Protein Assay Kit; Bio-Rad, Hercules, CA).
One milliliter buffer was added to the eyecup to clear the remaining
RPE and then discarded. Then, BrM, recognizable by its translucent
white polka-dotted appearance, and the attached choroid were re-
moved and placed in isolation buffer. After five rinses, large choroidal
vessels were removed under the dissecting microscope. Twice, blood
was milked out of the remaining large vessels by brushing gently from
the anterior margin to the optic nerve head. The resultant BrM/Ch
preparation was stored in 1 mL isolation buffer with protease inhibitor
at 4°C until particle isolation.
BrM/Ch was homogenized in 3 mL isolation buffer with a glass
homogenizer until large clumps disappeared. After the homogenate
was spun at 10,000 rpm for 6 minutes, solid tissues were pelleted, and
the supernatant was transferred to a new 5-mL tube. To release any
lipoproteins bound to tissue proteoglycans, pellets were resuspended
in 1 mL of isolation buffer containing 1 M NaCl and placed in a bath
sonicator for 5 to 10 minutes. After samples were spun at 10,000 rpm
for 6 minutes, the second supernatant was pooled with the first.
Lipoproteins were isolated by adjusting the density of the pooled
buffer extract to d ? 1.24 g/mL KBr and subsequent ultracentrifuga-
tion at 49,000 rpm for 36 hours with a swing-out rotor (SW55; Beck-
man). BrM/Ch LLP, collected by withdrawing the top 1-mL fraction
from the ultracentrifuge tubes, was then subjected to density gradient
ultracentrifugation as described for plasma lipoproteins. After ultracen-
trifugation, BrM/Ch LLPs in the density gradient tubes were separated
into 19 subfractions by puncturing the tubes with a density gradient
fractionator (Beckman). Lipoprotein peaks were located in the gradi-
ent by using an enzymatic fluorometric assay to define a cholesterol
profile (described later). On the basis of this profile, density gradient
fractions containing major peaks were pooled and dialyzed with pH 7.4
Tris-HCl buffer for 4 hours. Aliquots were subjected to colorimetric
enzymatic assays for lipids using cholesterol and phospholipid enzy-
matic assay kits from Waco Diagnostic Co. (Richmond, VA), lipid
extraction, or Western blot analysis. Preliminary experiments indicated
that the fluorometric and colorimetric cholesterol assays gave a similar
three-peak distribution for plasma lipoproteins, with 50% to 80%
higher cholesterol detected by fluorometry. This higher sensitivity was
required for the 19 small subfractions but not the large pooled fraction
of BrM/Ch lipoproteins.
Concentrations of total cholesterol (TC), TG, and PL determined by
colorimetric enzymatic assays were expressed as milligrams per deca-
liter and converted to nanomoles using molecular weights for choles-
terol, cholesteryl oleate (for EC), triolein (for TG), and phosphatidyl-
IOVS, July 2005, Vol. 46, No. 7
Bruch’s Membrane Lipoproteins2577
choline (for PL). Proportions of individual lipids were expressed
relative to the total of those measured. Some lipoprotein-associated
lipid classes, notably sphingomyelin, which exceeds phosphatidylcho-
line in plaque LLP, were not measured. Therefore, the proportion of
measured lipids relative to all lipids is not known. Substituting phos-
phatidylcholine (formula weight ? 760) for sphingomyelin (formula
weight ? 703–731) does not introduce appreciable errors for our
Cholesterol Mass and EC Composition: Sample
Preparation and Assays
Lipids were extracted from tissues, cells, and lipoproteins with 7.0 mL
chloroform, 3.5 mL methanol, and 0.5 mL water.29Extracts were left to
stand for 15 minutes, and 2.5 mL water was added and left to stand for
2 hours while phases separated. Upper (aqueous) and middle (protein-
aceous) phases were discarded. After the bottom (organic) phase was
washed with methanol-chloroform-water (48:3:47), three 500-?L ali-
quots were evaporated under nitrogen and dried in a vacuum. Aliquots
were solubilized in isopropanol (50 ?L for BrM, retina, RPE, LDL, and
macrophages; 500 ?L for cornea and sclera), vortexed, and sonicated
for 15 minutes.
TC and UC in extracts of native RPE, BrM/Ch LLP, and plasma
lipoproteins was determined by an enzymatic fluorometric assay as
described14,22,30for 100-, 50-, and 2-?L aliquots of each preparation.
EC concentration was defined as the difference between TC and UC
concentrations in nanomoles. UC dissolved in isopropanol was a stan-
dard. Cholesteryl ester composition was assayed by electrospray ion-
ization mass spectrometry (ESI/MS) as described,22using isopropanol-
solubilized ?-sitosteryl acetate and 6 cholesteryl esters as internal and
external standards, respectively (Table 1). Ester concentration in nano-
moles for each specimen was determined in triplicate and normalized
to cholesteryl oleate. Three to six esters were measured in different
experiments (Table 1).
For comparison to BrM/Ch and plasma lipoproteins, cholesteryl
ester composition was also assayed in partially isolated BrM and other
parts of paraformaldehyde-preserved eyes. These tissues were used
because of availability and because EC had been extracted and assayed
from similar tissues previously.14,31,32
punches of macular retina, RPE, choroid, and sclera were obtained and
separated as described.14Examination of removed RPE by electron
microscopy indicated cell sheets with some basal laminar deposit (not
shown). For cornea, the central 8 mm was removed with a trephine,
leaving a ring of corneal periphery with arcus lipoides, a benign,
age-related EC accumulation.33The effect of paraformaldehyde fixation
was investigated by comparing ocular EC composition in two pairs of
fellow eyes. An eye fixed for 7 days was compared to a fellow eye that
was dissected fresh and extracted immediately. In another pair, one
eye was preserved and stored in paraformaldehyde, and the fellow eye
was frozen fresh and stored at ?80°C. Both eyes were dissected and
extracted after 3 months. Cholesteryl ester composition was also
assayed in LDL (20 ?L, 5 mg/mL) isolated from healthy subjects by
ultracentrifugation and acetylated LDL (50 ?L, 2.5 mg/mL) used to load
cultured macrophages.34Results from LDL and acetylated LDL were
similar and therefore combined. Human monocyte macrophages were
isolated, cultured, exposed to acetylated LDL to induce large intracel-
lular EC-rich droplets,34and extracted as described earlier.
ARPE-19 and HepG2 cell lines were obtained from the American Type
Culture Collection (Manassas, VA). ARPE-19 cells of passage 22 were
grown for 4 weeks in T-75 flasks or six-well plates in DMEM/F12 (1:1)
containing 10% fetal calf serum (FCS) as described.35Medium was
changed twice weekly. HepG2 cells were grown for 5 days in MEM
containing 10% FCS in six-well plates with medium changed every
other day. All cells were cultured at 37°C and 5% CO2.
Total RNA was isolated from neurosensory retina, native RPE, ARPE-19,
and HepG2, as described.16The following primer sequences for RT-
PCR were designed: apoA-I (sense, 5?-AAG ATG AAC CCC CCC AGA
G-3?; antisense, 5?-TTG AAG CTC TCC AGC ACG G-3?), lecithin cho-
lesterol acyl transferase (LCAT, sense, 5?-CCT CAA TGT GCT CTT CCC
C-3?; antisense, 5- GCT TGC GGT AGT ACT CCT C-3?). To distinguish
between amplified mRNA and genomic DNA, all primers were de-
signed to span intron boundaries. One-step RT-PCR was used with kits
(Qiagen, Valencia, CA). Reverse transcription was performed at 52°C
for 30 minutes followed by 15 minutes at 95°C to inactivate the reverse
transcriptase and activate the DNA polymerase (HotStarTaq; Bio-Rad).
The reaction was amplified through 30 cycles, each consisting of 30
seconds at 94°C (denaturing), 30 seconds at 60°C (annealing), and 1
minute at 72°C (extension). The reaction was incubated at 72°C for
another 10 minutes. RT-PCR products were resolved on a 1% agarose
gel, stained with ethidium bromide, and visualized by ultraviolet tran-
sillumination. Expected sizes of RT-PCR products are 679 bp (apoA-I)
and 470 bp (LCAT).
Western Blot Analysis
ApoB. Aliquots of BrM/Ch LLP were concentrated approximately
15-fold (Centricon YM-10; Millipore, Bedford, MA) and separated by 4%
to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE).36After
electrophoresis, proteins were transferred onto polyvinylidene fluo-
ride membranes by Laemmli’s transfer buffer system36and detected by
Western blot analysis with biotinylated antibody to human apoB-100,
ApoA-I. Aliquots of BrM/Ch LLP were subjected to SDS-PAGE on
12% or 16% Tris-glycine gels. Proteins were transferred to a nitrocel-
lulose membrane. The membrane was blocked in 3% gelatin in TBS for
1 hour at room temperature and incubated with biotinylated affinity-
purified polyclonal goat anti-human apoA-I antibody (Brookwood Bio-
medical, Birmingham AL) overnight at 4°C. After a thorough TBS wash,
the membrane was incubated with streptavidin-alkaline phosphatase
conjugate (Bio-Rad) for 1 hour at room temperature. ApoA-I immuno-
reactivity was developed by an alkaline phosphatase conjugate sub-
strate kit (Bio-Rad) according to the manufacturer’s protocol. Purified
human plasma apoA-I38served as a positive control.
TABLE 1. Samples Assayed for Cholesteryl Ester Composition by ESI/MS
SamplePrep Donors (n) Eyes/Donor (n) 16:0 18:0 18:118:2 20:4 22:6
BrM/ Ch lipoproteins
BrM, cornea, sclera
BrM, RPE, retina
BrM, cornea, sclera, retina
?, ester was assayed; —, not done; F, fresh; P, preserved in 4% paraformaldehyde; 16:0, cholesteryl palmitate; 18:0, cholesteryl stearate; 18:1,
cholesteryl oleate; 18:2, cholesteryl linoleate; 20:4, cholesteryl arachidonate; 22:6, cholesteryl docosahexaenoate.
2578 Li et al.
IOVS, July 2005, Vol. 46, No. 7
Negative Stain Electron Microscopy
LLPs were visualized as described.39Samples were dialyzed in 0.26 mM
EDTA, 2.6 mM ammonium carbonate, and 0.125 M ammonium acetate
at 4°C for 12 hours (Slide-A-Lizer Dialysis Cassettes; Pierce, Rockford,
IL). Two microliters of dialyzed sample was placed on 200-mesh
copper grids with polyvinyl formal (Formvar; SPI, West Chester, PA)
carbon support film freshly decharged with 100% ethyl alcohol. Grids
were stained for 30 seconds with 2 ?L of 2% potassium phosphotung-
stic acid solution extruded through a filter with 0.2-?m-diameter pores.
Excess moisture was wicked with filter paper wedges and the grids
allowed to dry. Comparison grids contained only dialyzing buffer.
Samples were viewed on a microscope (JEM-1200EX II; JEOL, Tokyo,
Japan; accelerating voltage 60 KV) and photographed (4489 film;
Kodak. Rochester, NY). Negatives were scanned (model 1100 Power-
Look scanner and Umax Magiscan 4.5; Umax Technology, Milton
Keynes, UK) at 600 ppi, printed at 2.87? magnification, and imported
into image-manipulation software (Photoshop 7.0; Adobe Systems,
Mountain View, CA) for contrast enhancement and compositing. Elec-
tron microscopy supplies came from EMS (Fort Washington, PA).
Indirect Immunofluorescence of Apos
Cryosections of human retina and choroid were processed for apoA-I
immunoreactivity as described,16with a polyclonal antibody to apoA-I
(catalog number K45252G; Biodesign, Saco, ME), and nonimmune
rabbit immunoglobulin at an equivalent protein concentration was
used for the control. Sections were viewed by microscope (Optiphot
2; Nikon, Melville, NY), epifluorescence optics (excitation, 546 nm;
emission, 590 nm), and a dry 40? planapochromat objective (numeric
aperture [NA] ? 0.95). Photographs were then taken (Delta 100 film;
Ilford Photograph Corp., Paramus, NJ) with matched exposure times
for experimental and control sections. Images were scanned from
negatives (SprintScan 4000; Polaroid Corp., Cambridge, MA) and as-
sembled into composite photomicrographs (Photoshop; Adobe Sys-
Cholesterol-Containing Particles in BrM/Choroid
To define the cholesterol profile of BrM/Ch LLP, we subjected
concentrated buffer extracts containing all lipoproteins (d ?
1.24 g/mL fraction, herein called the pooled fraction) from
seven pairs of eyes to density gradient ultracentrifugation and
obtained 19 subfractions. In contrast to the three clearly de-
fined plasma peaks (Fig. 1A), the d ? 1.24 g/mL fraction of all
BrM/Ch preparations contained two peaks (Fig. 1B). A large
Peak 1 located in the plasma LDL-HDL range (fractions 3–13)
was low in EC (EC/TC molar ratio ? 0.14). A small Peak 2
located in the plasma VLDL range (fractions 16–19) was rich in
EC (EC/TC molar ratio ? 0.54). In different eyes, the ratio of
TC recovered from Peak 1 to TC recovered from Peak 2 ranged
from 2.63 (donor 4) to 25.96 (donor 6). Thus, cholesterol from
Peak 1 dominated the pooled fraction.
A more detailed characterization of lipids in the pooled
fraction and in Peaks 1 and 2 is shown in Table 2. Regarding
the pooled fraction, the total amount of cholesterol recovered
was highly variable among eyes. The coefficient of variation
(SD/mean) ranged from 39% to 53% for each of the measured
lipids. However, each lipid’s proportion of the total was re-
markably consistent across the same eyes, with coefficients of
variations ranging from 4.5% to 18.5%. Mean proportions of PL,
EC, UC, and TG mass measured in eight donors was 0.38, 0.20,
0.37, and 0.06, respectively. Overall, the proportion of TC that
was esterified was 0.35 ? 0.04. Because a different number of
subfractions comprised Peaks 1 and 2, more weight should be
given to molar ratios of lipids within peaks than measured
concentrations. Regarding Peak 1, sufficient material was avail-
able for analysis by colorimetric assay in seven donors. In these
samples, PL, EC, UC, and TG represented mean mass propor-
tions of 0.43, 0.10, 0.41, and 0.05, respectively, of the lipids
measured in Peak 1. The proportion of TC that was esterified
was 0.20 ? 0.07, and the ratio of UC to PL, which solubilizes
UC, was 0.95 ? 0.14. Regarding Peak 2, no lipids except
cholesterol could be assayed due to the small sample size. At
0.59 ? 0.15, the proportion of TC that was esterified in Peak 2
was two times higher than Peak 1. TG was measurable but not
abundant in the pooled fraction and in Peak 1 (pooled fraction,
TG/EC ? 0.31 ? 0.06; Peak 1 ? 0.64 ? 0.31). For comparison,
plasma TG/EC for the HDL, LDL, and VLDL peaks of a normo-
lipemic subject are 0.09, 0.26, and 14.2, respectively.
To characterize the EC composition of BrM/Ch LLP, ex-
tracts of the pooled fraction, Peak 1, and Peak 2 were assayed
by ESI/MS and the concentrations normalized to cholesteryl
oleate (18:1; Fig. 2). In these fractions, cholesteryl linoleate
(18:2) was the predominant ester, being 2.1-, 2.7-, and 2.5-fold
more abundant, respectively, than cholesteryl oleate. The cho-
lesteryl palmitate (16:0) concentration was similar to that of
cholesteryl oleate. In contrast, little cholesteryl stearate (18:0),
cholesteryl arachidonate (20:4), or cholesteryl docosahexaeno-
ate (22:6) was found in the pooled fraction (normalized con-
Concentrations determined by enzymatic fluorometry were normal-
ized to peak concentration of UC. High densities were located in
low-number fractions and low densities in high-number fractions. (A)
Plasma HDL, LDL, and VLDL are denoted. (B) LLPs from a representa-
tive BrM/Ch isolate segregated in a large peak (Peak 1) spanning the
high- and low-density ranges and in a small peak in the very-low-density
range (Peak 2).
Cholesterol profiles of plasma and human BrM/Ch LLPs.
IOVS, July 2005, Vol. 46, No. 7
Bruch’s Membrane Lipoproteins 2579
centrations of 0.56, 0.14, and 0.12, respectively), Peak 1
(0.092, 0.157, 0.008), or Peak 2 (0.099, 0.149, 0.012). These
proportions were not dissimilar from plasma lipoproteins as-
sayed by this method. For example, LDL from a normolipemic
subject has 2.2-fold more cholesteryl linoleate than cholesteryl
oleate and normalized cholesterol docosahexaenoate concen-
tration of only 0.01 (Fig. 2A). Of the three plasma lipoproteins
examined, the EC composition of Peaks 1 and 2 resembled LDL
most closely and CM least (Fig. 2B).
To determine whether BrM/Ch cholesterol is associated
with particles resembling lipoproteins, Peaks 1 and 2 were
examined by negative stain electron microscopy to reveal elec-
tron-lucent material on a dense background. Peak 1 material
was heterogeneous in size and morphology, exhibiting solid,
round lucent particles in the 100-nm diameter range (Fig. 3A,
arrow) and larger and more irregular assemblies with an elec-
tron-lucent surface and moderately electron-dense core (Fig.
3A, arrowhead). In contrast, Peak 2 material was sparse, con-
sistent with the peak’s low cholesterol concentration, but the
available material resembled the large lucent particles in Peak
1. For comparison, similarly prepared plasma LDL, VLDL, and
CM appeared as 22- to 23-, 55- to 65-, and 200- to 400-nm
diameter uniformly electron-lucent spherical particles (Figs.
3C–E, respectively). A distinctive lipid-containing BrM compo-
nent, the coated membrane-bound body (Fig. 3F, arrow), was
mirrored in the isolates by occasional complex structures with
internal particles (Fig. 3G, arrow), indicating that isolates con-
tained material from BrM itself.
Apo Gene Expression
To determine whether BrM/Ch fractions contained apos asso-
ciated with classic plasma lipoprotein particles, pooled frac-
tions, Peak 1, and Peak 2 from seven eyes were subjected to
Western blot analysis. ApoB-100 bands and a lower molecular
weight band appeared in Peaks 1 and 2 (Fig. 4A). A strong band
at 28 kDa for apoA-I appeared in the individual subfractions
comprising Peaks 1 and 2 (e.g., Fig. 4B; lanes 4–7). Other
bands were detected at 56 kDa in lanes 2, 3, 4, and 5 and at 84
kDa in lane 5 only. Of the seven eyes analyzed, apoB-100 was
detected in four, and apoA-I was detected in seven. ApoA-I was
also detected in human retina and native RPE (Fig. 4C, lanes
To determine whether RPE and neurosensory retina express
mRNA transcripts for apoA-I, we performed RT-PCR using total
mRNA isolated from retina, native RPE, ARPE-19 cells, and
HepG2 hepatoma cells. The expected 679-bp PCR product was
detected in each sample (Fig. 5A). In plasma, apoA-I activates
the cholesterol-esterifying enzyme lecithin cholesterol acyl
transferase (EC 220.127.116.11, LCAT40). LCAT is bound to HDL and
converts UC and phosphatidylcholines in HDL to EC, which is
transferred to apoB-containing lipoproteins by cholesterol es-
ter transfer protein. Therefore, we determined whether retina
and RPE also express mRNA for LCAT. Once again, the
expected PCR product (470 bp) appeared in each sample
TABLE 2. Characterization of Lipids in Pooled d ? 1.24 g/mL Fraction, Peak 1, and Peak 2
TCUC TG PL ECUC TG PLEC EC/TC TG/EC
d ?1.24 g/ml
Except TC and UC of Peak 2 detected by enzymatic fluorimetry, all others were detected by enzymatic colorimetry. —, not done.
* Molecular weight used: UC, 386.7; TG, 885.4; PL, 760.1; EC, 651.1.
† Expressed relative to (TC?TG?PL).
2580Li et al.
IOVS, July 2005, Vol. 46, No. 7
To localize apoA-I in normal human retina and choroid, we
probed cryosections with polyclonal antibodies to human
apoA-I and detected immunoreactivity by indirect immunoflu-
orescence. Sections of normal peripheral retina contain intense
labeling in BrM and drusen and less intense, diffuse labeling
within the choroidal stroma (Fig. 6A, 6C). This labeling is
specific, as its pattern differs from control sections processed
with equal concentrations of nonimmune immunoglobulin
(Fig. 6B, 6D). ApoA-I immunoreactivity was also detected in
plasma retained in retinal vessels (not shown).
EC Composition of BrM, Lipoproteins,
and Ocular Tissues
The results so far indicate that LLP can be isolated from BrM/
Ch. They contain EC, among other lipids, and apos that are
detectable in situ within BrM and deposits.16Although lending
credence to the notion of one or more species of BrM lipopro-
tein particles, these data do not elucidate the potential source
of these lipoproteins. The particles differ from plasma lipopro-
teins in a key property, behavior in a density gradient, but they
resemble plasma lipoproteins, especially LDL, in EC composi-
tion. Here, we sought insight by investigating BrM EC compo-
sition further, for two reasons. First, hot-stage polarizing mi-
croscopy studies have shown birefringence with EC-like
crystalline morphology and thermal behavior in BrM, drusen,
and sclera.15That EC-related birefringence disappears at higher
temperatures from BrM and drusen than sclera was attributed
to differences in the relative saturation of long-chain fatty acids
in EC. Second, the high proportion of cholesteryl linoleate
relative to cholesteryl oleate in human connective tissues was
an important clue implicating insudation of plasma lipopro-
teins as a process initiator in both atherosclerosis and xan-
The EC mass in native RPE ranged from 1.9 to 24.2 ?g/mg
cell protein, with the proportion of TC that is esterified ranging
from 0.09 to 0.40 (Table 3). Then, by ESI/MS assay, the EC
composition in RPE and partially isolated BrM from fresh tis-
sues (Fig. 7) was similar to that in the isolated BrM/Ch LLP
fractions (compare with Fig. 2). That is, cholesteryl linoleate
was more than two times more abundant than cholesterol
oleate, and little cholesterol docosahexaenoate was detectable.
However, a different picture emerged from analysis of EC
composition of BrM, cornea, and sclera of paraformaldehyde-
preserved eyes. Cornea and sclera, like LDL, are relatively
enriched in cholesteryl linoleate, but preserved BrM, like intra-
cellular droplets of activated macrophages, is enriched in cho-
lesteryl oleate (Fig. 8A). We also compared the EC composition
of preserved BrM to RPE and retina of the same eyes (Fig. 8C).
All three tissues were enriched in cholesteryl oleate, although
the total mass of cholesteryl oleate differed substantially
tron microscopy (A–D, F). (A) Particles in Peak 1 were heterogeneous
in size and electron density. Some particles were large and solidly
electron lucent (arrow). Most had empty interiors with electron-lucent
surfaces (arrowhead). (B) Particles in Peak 2 were mostly large and
solidly electron lucent (arrowhead). (C) Plasma LDL. (D) Plasma
VLDL. (E) Dietary CM. (F) A coated membrane-bounded body in BrM
in situ (arrow) in a thin section transmission electron micrograph. RPE
is at the top of the image. Arrowheads: basal lamina. (G) Membrane-
bound aggregate containing small electron-lucent particles, Peak 1.
Bars: (A, B, E, G) 100 nm; (C, D) 50 nm; (F) 500 nm.
Lipoproteins from BrM/Ch and plasma. Negative stain elec-
isolated and centrifuged, and lipids were extracted from the pooled
d ? 1.24 g/mL fraction and from Peaks 1 and 2. The concentrations of
six cholesteryl esters were assayed by ESI/MS and normalized to cho-
lesteryl oleate (18:1, ✱). See Table 1 for carbon saturation formulas.
Error bar, standard deviations (three aliquots for each sample). (A)
BrM/Ch particles (d ? 1.24 g/mL, n ? 3 donors; Peaks 1 and 2, n ? 4
donors). (B) EC composition from LDL, VLDL, and CM of normoli-
pemic human plasma.
EC composition of BrM/Ch LLPs. After BrM/Ch LLPs were
IOVS, July 2005, Vol. 46, No. 7
Bruch’s Membrane Lipoproteins 2581
among them (6700 ? 1156, 1190 ? 850, and 180 ? 67
nanomoles/g dry weight, respectively), consistent with the
previously reported measurable but low EC mass in the neu-
rosensory retina.14Finally, in fellow eyes in which BrM, retina,
sclera, and cornea were preserved for 7 days or processed
fresh, all ocular regions of both eyes were cholesteryl linolea-
te–enriched (not shown). In fellow eyes preserved or frozen
for 3 months, tissues from only the frozen eye was cholesterol
linoleate–enriched (not shown). These results raise the possi-
bility that BrM EC composition is affected by long-term storage
differently than are esters of plasma lipoprotein origin that
accumulate in sclera and cornea.
Our main finding was that particles resembling lipoproteins
isolated from BrM/Ch did not resemble plasma lipoproteins in
taining BrM/Ch LLP. After BrM/Ch particle isolation and density ultra-
centrifugation, Peaks 1 and 2 were subjected to Western blot analysis.
(A) Stained with anti-apoB antibody, apoB-100 bands were found in
both Peaks 1 and 2. Lane 1: Peak 1 from donor 9; lane 2: Peak 2 from
donor 9; lane 3: Peak 1 from donor 10; lane 4: Peak 2 from donor 10;
lane 5: human plasma. Lane 5 was developed simultaneously with
other lanes and scanned separately due to its higher density. (B)
Stained with anti-apoA-I antibody, apoA-I was found in both Peaks 1
and 2. Lane 1: 50 ng apoA-I standard; lane 2: 40 ?L of Peak 1 from
donor 9; lane 3: 40 ?L of Peak 2 from donor 9; lanes 4 to 7: 10 ?L each
of fractions 6, 7, 9, 12 of Peak 1 from donor 9; lane 8: 10 ?L of fraction
17 of Peak 2 from donor 9. (C) ApoA-I expressed in human RPE and
retina. Lane 1: 10 ng of plasma apoA-I; lane 2: 50 ng of plasma apoA-I;
lane 3: 100 ?g of native RPE protein; lane 4: 100 ?g of retina protein.
Western blot analysis of apoB and apoA-I in fractions con-
(lane P), ARPE-19 (lane 19), and HepG2 (lane H) cells. Total RNA was
isolated from human retina, RPE, ARPE-19, and HepG2 cells, and
one-step RT-PCR was performed. Expected RT-PCR products of apoA-I
and LCAT were 679 and 470 bp, respectively. Lane M: 100-bp DNA
ladder; lengths in bp are indicated.
ApoA-I and LCAT expressed in human retina (lane R), RPE
sections of BrM/Ch and drusen. Sections from the peripheral retina of
a 63-year-old woman were probed with polyclonal anti-apoA-I (A, C) or
nonimmune immunoglobulin (B, D). Primary antibodies were detected
with rhodamine-conjugated secondary antibody. (A) ApoA-I immuno-
reactivity in BrM (arrows). Arrowheads: autofluorescent RPE. (B)
Autofluorescence only in RPE and BM. (C) ApoA-I immunoreactivity in
drusen (d), enveloped by autofluorescent RPE (arrowheads). (D)
Autofluorescence in RPE and BrM, not in drusen. Bar, 40 ?m.
ApoA-I immunofluorescence and autofluorescence in cryo-
2582Li et al.
IOVS, July 2005, Vol. 46, No. 7
density profile, cholesterol distribution, and morphology. Par-
ticles were heterogeneous and distributed into two density
peaks, including a large particle in a EC-rich Peak 2 that resem-
bles the 80- to 100-nm particles appearing in situ.14,19Particles
in Peak 1 were more heterogeneous, including some Peak
2-type particles, liposome-like structures, and intact coated
membrane-bound bodies.43Eyes differed substantially in TC
yield, probably because of differences in drusen load28,44or
BrM cholesterol content,14but the proportions of measured
lipids were remarkably consistent across eyes. Although it
cannot yet be excluded that some BrM particles are transcy-
tosed from the plasma compartment, it appears that BrM/Ch
LLPs differ in important ways from plasma lipoproteins.
An important unanswered question is the number of parti-
cle classes present in native BrM. Further, if there are multiple
classes, they may arise either independently or by interconver-
sion among them by hydrolysis or lipid transfer, inter alia.
Given the abundant Peak 2-type particles seen in situ by con-
ventional and lipid-preserving postfixation methods, intercon-
version seems likely. However, EM studies have described
70-nm-diameter vesicle-like bodies (now known to represent
solid particles) within coated membrane-bound bodies and 70-
to 110-nm-diameter vesicle-like bodies elsewhere within
BrM.43A range of particles at different stages of maturation or
metabolic and/or degradative modification in BrM may be pos-
Our ultrastructural data suggest some BrM particles are
remnant lipoproteins modified in the sub-RPE space by cur-
rently undefined processes. Early studies indicated that plasma
lipoproteins ?70 nm in diameter were unlikely to enter arterial
intima because of size-restricted transcytosis at the vascular
endothelium.45,46However, larger remnants of VLDL and CM
are now known to enter after the hydrolyzing action of endo-
thelial and lipoprotein lipases that leave UC-enriched surface
components intact.9Among particles isolated from arterial
intima,9,24,47,48heterogeneity in morphology (pitting, cluster-
ing, or loss of internal lucency) has been interpreted as evi-
dence for enzymatic, oxidative, or other modifications in the
extracellular space. Although no modifying mechanism can be
excluded for BrM currently, the loss of internal lucency with
retention of external surface (see also Ref. 19) resembles VLDL
particles exposed to lipase activity in vitro9and therefore
could represent modification in the extracellular space before
TABLE 3. Total Cholesterol and Esterified Cholesterol Mass in Native
TCUC EC EC/TC
Data are expressed as micrograms per milligram protein.
centration of cholesterol esterified to long-chain fatty acids denoted by
the key was determined by ESI/MS and normalized to that of choles-
terol oleate (18:1, ✱). See Table 1 for carbon saturation formulas.
Native RPE was obtained from four donors ?60 years of age with
grossly normal maculas. BrM/Ch was isolated from a fresh donor eye.
Error bars, standard deviation.
EC composition in native human RPE and BrM. The con-
served ocular tissues. EC concentrations in extracts were determined
by ESI/MS and normalized to the concentration of cholesteryl oleate
(18:1, ✱). See Table 1 for carbon saturation formulas. Error bars,
standard deviations. (A) Cornea, sclera, and BrM (n ? 10 eyes), com-
pared to LDL. (B) BrM (n ? 4 eyes) and cholesterol-loaded monocyte-
macrophages. (C) BrM, RPE, and neurosensory retina (n ? 6 eyes).
EC composition in LDL, monocyte-macrophages, and pre-
IOVS, July 2005, Vol. 46, No. 7
Bruch’s Membrane Lipoproteins 2583
the donor’s death (e.g., hydrolysis by lipases resident in the
choroid49). It is also possible that some Peak 1 heterogeneity
represents vesicles released from cellular membranes during
homogenization and sonication inherent in sample prepara-
With regard to an intraocular atherogenic apoB-containing
lipoprotein, our data do not fully resolve the crucial question of
apo identity. Western blot analysis convincingly demonstrated
apoB-100 as well as a lower-molecular-weight band consistent
with either apoB-48 or an apoB-100 degradation product. Such
bands frequently appear in apoB Western blots, because apoB
is readily proteolyzed at predictable sites during isolation, as an
intracellular regulatory mechanism, and as an indicator of trans-
location arrest in the absence of microsomal triglyceride trans-
fer protein.50–52Our data also do not resolve the crucial ques-
tion of whether BrM particles are TG-rich, as predicted by their
size and the chromatographic evidence that the TG/EC molar
ratio in intact BrM/Ch is 1.77.18Perhaps a more sensitive
technique for TG detection (e.g., Ref. 53) would provide a
different answer. Alternatively, low TG levels may reflect the
results of lipolytic enzyme activity in the sub-RPE space.
The conclusions of prior compositional studies on the
source of BrM lipids were based on lipids not exclusively
localized to BrM (i.e., PL25or UC14), which could derive from
membranes of incompletely removed choroidal cells. Despite
the expectation that fatty acyl residues in BrM EC should reflect
composition of potential sources, such sources are not conclu-
sively identified by our study. An attractive hypothesis is that
these residues represent direct or indirect degradative prod-
ucts emanating from outer segment phagocytosis. However,
because BrM EC has little cholesteryl docosahexaenoate or
cholesteryl stearate, the two most abundant fatty acids in outer
segment PL,54it does not resemble outer segment PL, in either
preserved or fresh eyes (Fig. 9). These results are consistent
with an intermediary mechanism with specific substrate pref-
erences that repackages fatty acids for neutral lipid secretion.
Leading candidate mechanisms are diacylglycerol acyl trans-
ferase and acyl cholesterol acyl transferase, which catalyze the
final committed steps in TG and EC synthesis, respectively, and
stearoyl-coA-desaturase, which supplies oleate to these en-
zymes. Genes encoding the latter two proteins are both ex-
pressed in RPE.22,55Evidence for expression of LCAT, which
prefers linoleate as a fatty acid substrate,56adds to the potential
means for ocular cholesterol esterification. In addition to its
presence on apoA-I-containing plasma HDL, LCAT is expressed
in the brain, where it may esterify cholesterol on UC-rich
lipoprotein particles secreted by astrocytes.57It will be inter-
esting to determine whether RPE LCAT gene expression signi-
fies a capacity to synthesize linoleate-enriched cholesteryl es-
ters in the sub-RPE space.
The finding that BrM EC is cholesteryl oleate enriched in
preserved eyes and cholesteryl linoleate enriched in fresh eyes
and isolated BrM particles was unexpected, yet consistent with
the work of Haimovici et al.,15applying a physicochemical
technique to cryosections of paraformaldehyde-preserved nor-
mal aged eyes. The basis of this paradoxical finding is un-
known, because all preserved eye parts were stored together,
but it underscores that BrM and retinal ECs differ from that in
connective tissue. Selective modification (e.g., oxidation of
cholesteryl linoleate) during storage is a possible explanation
for this concordant finding in two laboratories.
Detection of apoA-I in RPE and neurosensory retina, extend-
ing proteomics results,58,59expands the retinal apo gene ex-
pression repertoire. Weak bands detected at 56 and 84 kDa
with an affinity-purified antibody are most likely the dimeric
and trimeric forms of apoA-I, respectively, as apoA-I self-asso-
ciates in solution.60With these data, drusen are now known to
contain apoB, apoE, and apoA-I. With regard to RPE, it remains
to be determined whether apoA-I protein is secreted on a small
HDL-like particle, or more intriguingly, on a novel, large CM-
like particle. This notion is appealing, because RPE lipoprotein
release after outer segment phagocytosis could be conceptual-
ized as a postprandial event, similar to CM release. Whether all
three apos occupy the same or different particles in BrM can be
answered indirectly, by immunogold electron microscopy of
particles in situ, or directly, by isolating and characterizing the
full range of particles from appropriately lipid-loaded RPE cells
In summary, LLP in fractions containing apoB, apoA-I, and
cholesterol can be isolated from normal human BrM/Ch. Cho-
lesteryl ester composition and interocular expression of genes
encoding apos augment circumstantial evidence that particles
are formed within the eye. There, they may contribute to age-
and ARM-related drusen and basal linear deposits in a process
analogous to atherosclerosis initiation in arterial intima by
accumulation of plasma apoB-containing lipoproteins.61–63
The authors thank the Alabama Eye Bank for timely retrieval of donor
eyes, Landon Wilson and Ray Moore of the UAB Mass Spectrometry
Center for ESI/MS analysis of cholesteryl esters, and helpful discussions
with Steven J. Fliesler, PhD (Dept. of Ophthalmology, Saint Louis
University Eye Institute, St. Louis, MO), at the beginning of this study.
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